The Friedman Lab Chronicles

Behind the cover

2011-07-12T21:16:00.002+03:00

Getting a cover image for a journal was a first for me. As such I thought it would be interesting to revisit the behind the scenes. I am going for a short recap.

Initially when the paper was accepted the editor encouraged us to sugget a cover image. We (actually the Broad people, Manfred, Brian, and Aviv) recruited Sigrid Knemeyer who collected ideas and came up with some concept images. We (the three mentioned above, Moran, and me) then had several rounds of emails with Sigrid where each of us made comments, said what we liked or not about the options. Sigrid came up with a more detailed concept, which we sent to the editor. The editor wasn't too excited about it, and so we went back to the drawing board.

This time Sigrid, came up with four initial concepts.

We really liked some elements of each, but thought that the vortex of letters does not capture the idea of short reads. The stacks and stacks of papers was also nice, but didn't make it clear that the reads are short.

The next concept was to replace stacks of papers with short strips, like fortunes (as in fortune cookies), and combine it with the vortex.

We really liked this, but the final touch was adding the assembled sequence coming out of the vortex. This and change of color tone resulted in the concept that we sent to the journal.

They really liked it, and sent it to their graphic editors who came up with the final version.

As you can see, they dispensed with the fortune-cookie strips, but kept the letters in "words" that capture the idea of short fragments and made other visual changes.

In conclusion, this was an interesting process. Sigrid was great in making this happen, and deserve a lot of credit. Thanks!

We made the cover!

2011-07-12T00:17:00.000+03:00

Our paper on transcript assembly appeared online few weeks ago. Now the actual issue came out, and we made the cover, with a striking image.

The idea came out of iterations between all of us (Manfred Grabher, Brian Haas, Moran, Aviv and me) and Sigrid Knemeyer who is an artist/illustrator working at the Broad Institute. Sigrid was great in developing the concept with our input of rough concepts, and this whirlwind was the final outcome.

Party: One Farewell + Two Welcome

2011-07-01T15:14:00.001+03:00

Recently it seems that we have many farewell party. Another one today, to celebrate Noa's graduation and to say farewell on her departure to the Weizmann Institute. At the same time we also welcomed two new additions to the extended lab family: Oren and Daniela who born recently.

We had a nice picnic in a park near Jerusalem, and enjoyed the presence of current and recent lab members and the many young children of the next generation. As noted by few participants, the lab has been extremely productive on that front...

Substantial Histone Reduction Modulates Genome-wide Nucleosomal Occupancy and Global Transcriptional Output

2011-06-29T18:07:00.000+03:00

The recent wave of publications seem to continue, and another paper just appeared in PLoS Biology. Like the previous one, this one was in the reviewing rounds for very long, and so we are very happy that it finally appeared.

This project is a collaboration with the group of Alessandra Agresti and Marco Bianchi from San Raffaele University in Milan. They are part of a European consortium that we are also members of, and they approached us to get help on dealing with nucleosome organization. Assaf got involved, into what was dubbed "The Italian Job" in lab meeting. After almost two years, the result is an impressive article.

To summarize the basic idea, the experiments by Barbara Celona, the main author, and other member's of the Milan group show that deletion in a key gene reduces the number of nucleosomes in the genome. As a result the DNA strands are packed by smaller number of nucleosomes. By mapping the locations of these nucleosomes we can see if nucleosome reduction leads to uniform change in density, or preferential reduction at particular locations, and what is the effect on transcription.

Source: PLoS Biology Synopsis by Robin Mejia

Our results show that nucleosome loss is mostly in locations that are less packed in normal cells, and moreover, that this loss correlates with increased transcription.

Cool!

Presidential Conference

2011-06-23T22:28:00.008+03:00

This week we participated in the Israeli President's annual conference. This is a the place where important people from Israel and abroad come to talk about the future of the country, the region, and the world.

The Hebrew University was a co-sponsor of the event, and had its own space to presents research highlight. The target audience was not scientifically minded, so the message was to be kept at high level. Each participating lab had a small space where a large poster was presented on a table. We had TV monitors showing a movie about our lab.

Assaf and Avital participated as guides, and got to see the important people and even to talk with few. We did manage to get to hear some of the sessions, although I suspect the main action at these events in beyond the scenes.

The Comeback + Visitors

2011-06-21T14:25:00.001+03:00

About three months ago Moran went off on a maternity leave. Today was the official first step on returning to academic work. To make the transition easier she was accompanied by her family. We got a chance to meet the family and the joy young ones bring.

Another weekend diving

2011-06-11T22:42:00.002+03:00

Two weekend days to enjoy diving in Eilat. Many good sights and nice company of local people and fellow divers.

Patterns and Mechanisms of Ancestral Histone Protein Inheritance in Budding Yeast

2011-06-07T22:02:00.002+03:00

Hot from the (virtual) press! A paper that we have been working on for almost a year appeared today in PLoS Biology. This is three way collaboration between Fred van Leeuwen's group at the Netherlands Cancer Institute, Ollie Rando's group at UMass Medical School and our group. The work on our end was mostly Assaf's who is a co-first author.

The interesting aspect of this paper is that using a trick developed in Fred's group, they can switch a "tag" on a histone H3 protein. This means that up to a certain point all H3 proteins are tagged by one tag, and after the switch they are all tagged with a different tag. Using this switch we can find out where these "old" histones are several generations after the switch. This gives us insight on how the cells perserve old histones during several cycles of replication.

To quote ourselves:

To our surprise, ancestral histones accumulate near the 5′ end of long, relatively inactive genes. Using a mathematical model, we show that our results can be explained by the combined effects of histone replacement, histone movement along genes from 3′ towards 5′ ends, and histone spreading during replication. Our results show that old histones do move but stay relatively close to their original location (within around 400 base-pairs), which places important constraints on how chromatin could potentially carry epigenetic information. Our findings also suggest that accumulation of the ancestral histones that are inherited can influence histone modification patterns.

To get to these conclusions Assaf implemented a mathematical model that takes into account the processes that affect nucleosome positions, and then showed that this model can provide a good fit to the data if we consider turnover, passback of nucleosomes (from 3' to 5'), and localized dispersion during replication.

Freiburg/Heidelberg

2011-06-06T23:08:00.004+03:00

Just returned from a week long trip to Germany. It started by a visit to Max Planck Institute for Immunobiology and Epigenetics at Freiburg to attend a one day workshop on Chromatin. After the talks the hosts took us to a nice resto-bar where there was a long and lively scientific discussion over beer, schnitzel and white asparagus.

The next morning, it was raining, but I went on a brief walk in Frieburg's old town.

Afterwards, I continued to Heidelberg to attend the EMBL Chromatin and Epigenetics meeting. This was a four and half day meeting with a lot of interesting talks and discussions with people.

The meeting was held in an impressive new training center at EMBL's campus on the hills above Heidelberg. It is built as a double helix, and so there are no floors, but rather helical ramp ways all around.

I also took a half day off to go bike riding in the area and had few hours to visit the famous Schloss.

Another graduation and a new lab website

2011-06-02T13:27:00.002+03:00

Last week Noa officially graduated. She turned in her dissertation on "Transcription Regulation Models and Their Application to Human Disease Research". She is moving on to the Weizmann Institute to work with Yaqub Hanna.

As a parting gift before leaving us to the Weizmann, Noa created a new website for the lab (we were missing one for too long).

We will have a proper sendoff for Noa soon, although the scheduling of the event seem to be a serious challenge.

New furniture

2011-05-22T19:31:00.000+03:00

The remaining open item in our lab renovation was furniture for the common kitchen/meeting room and for the offices. Since we were not sure about the amount of surplus budget, we left these for later stages of the renovation.

Once the major experimental areas were finished, we started on this mini-project. As usual, the planning stage took longer than expected, and then we had to get quotes, issue a purchase order, and wait for the carpenter to make the furniture.

Last week this process ended, and Benny our carpenter showed up to install the furniture. As part of the renovation of the kitchen he also had help in installing new kitchen top and sink.

The end result was a brand new room with much nicer table to sit around, areas for storage, and kitchen top for the coffee machine and water dispenser. Below the counter there is a place for several refrigerators so that each group can keep its own stuff separately.

On his next visit Benny installed the office (when I was away for the retreat). The new office design looks much better and provides area to sit and work.

CSLS Retreat

2011-05-19T20:08:00.001+03:00

I am one of the heads of the Computer Science and Computational Biology dual-major program at the Hebrew University. The unofficial name more often used is Computer Science - Life Science (CSLS). As part of our activities we hold a yearly retreat with all the undergraduate students in the program, teachers, and some of the program graduates.

During the retreat the third year students present their senior year project, we have some social activities, discuss the program and issues raised by students at different stages, and have a guest lecturer.

Our guest lecturer this year was Roy Kishony from Harvard Medical Center who talked about bacteria, anti-biotics, and forces that shape they evolution of anti-bacterial resistance.

The location of the retreat is on the beach in Hof Dor (also known as Tantura, see a wikipeida article on the complex history of the place), south of Haifa, which hosts a very beautiful settings for activities. The location is northen border of the sandy beaches of southern Israel, which are formed by sand from the Nile. From here to the north there are hills of sandstone that form ragged beaches. The location was the southern-most Phoenician city of Dor, which served as a port for various sea-routes of the Phoenician traders. They also had an active industry for collecting sea shells from which the "Royal Purple" color was manufactured (a very rare and expensive dye in the ancient world).

Today, the natural lagons are a harbour for fishing boats and a public beach.

We finished the retreat with a short trip in Ramat Hanadiv, a park established by the Baron Edmund de Rothschild Foundation in his memory at the southern part of the Carmel mountain. This includes very nice gardens and a large area for nature conservation

Full-length transcriptome assembly from RNA-Seq data without a reference genome

2011-05-15T23:17:00.003+03:00

Just hot off the press - a new paper appeared in advanced online publication in Nature Biotechnology. This paper describe a new computational pipeline to recover RNA transcript sequence and abundance (aka the "transcriptome") from RNA-seq data without using the genome as a reference.

This paper is a joint work of Moran from our group and Brian Haas and Manfred Grabher, both from the Broad Institute. The three of them (Brian, Manfred and Moran) developed a three-part tool to handle massive number of sequencing reads and assembling them to accurate account of the transcriptome. They also applied a very thorough evaluation of our method and how it compares to most of the state of the art methods in the field. This evaluation by itself is of interest to the this emerging area of analysis.

The basic problem is that RNA-seq returns many (millions) of sequences of different fragements of the original RNA molecules from the sample. To make sense of it, we need to assumble it (like puzzle) into longer pieces. The two general strategies for doing so are nicely illustrated in this figure (from a review by Haas and Zodie):

The "straightforward" approach is to align reads to the reference genome, and then use this mapping to guide reconstruction. The less obvious approach, that we took here, is to first assemble the puzzle, and then map to the genome. This turns out to be often as accurate (or even more), since it is less suceptibles to problems in mapping to the genome, differences between the reference genome and the actual sample, and partial/fragmented references.

Our strategy is based on three steps, each processing the data very efficiently while maintaining information and dealing with sequence errors and rare events.

London and Oxford

2011-05-15T12:56:00.001+03:00

I just returned from a quick three day visit to the UK. I was invited to participate in a symposium on "Integrative Network Biology and Cancer" at Institute for Cancer Research in London. This was an excuse to visit collaborators at the Wellcome Trust Centre for Human Genetics in Oxford and at the Imperial College in London. As such, I found myself giving three different talks in three days :-) Nonetheless it was fun and interesting meeting with multiple colleagues and friends, and also hearing some very interesting talks.

Although I didn't have time for serious touristic activities, I did get few chances to take pictures.

Oxford, in particular, was very picturesque.

In London I had fewer chances to go around, but the I still managed to catch few images that I like.

Tuneups and a small (but important) improvement

2011-05-08T17:40:00.004+03:00

The observant readers of the blog would have notice that conspicuous pink block of hard foam showed up at some point. We used it to stabilize the handoff position of the incubator. The problem was that the incubator is shaking (to aerate the plates inside). These vibrations are then transferred to the attached rail. Since that rail is long, it amplifies the vibrations, leading to unstable pickup by the robotic arm.

Sticking a block of foam beneath the rail damped the vibrations. However, the good people at Neotec, where not happy with it as a long term solution. So today they replaced it with a proper support, that is both sturdy and professional.

More than that, Yoram and Moshe also realigned the "LiHa" arm that has individual pipettes. We have been using the four fixed pipettes a lot, but haven't used the four disposable ones that much. These were haven't been properly tuned yet, and now that we started working with 384 well plates this has become an issue.

It turned out that this tuneup required disassembling part of the fixed tips. Aligning the disposable ones, and then realigning the fixed ones relative to the alignment of the disposable ones.

The alignment involved a special pin that replaces the tip and has to align to a specific point on the work table.

Once the head is aligned, the actual tips are aligned to be straight down. This requires examining from all possible angles and adjusting the small screws at their base.

Hotels in actions

2011-05-04T18:02:00.000+03:00

Last week we installed a new external hotel to hold plates. Yesterday Shai Kaplan and Assaf programmed the KiNEDx to recognize the hotel coordinates. Today Assaf put the new device to use by building a program to perform cherry-picking from individual wells from the whole yeast library. Thus, saving himself the need to go into specific wells in 16 plates, each with 384 wells.

The program worked nicely, and we can see a short part of it in this movie.

We did learn few important lessons about hotels. First, manually loading places onto it can be tricky. If you are not careful it is easy to drop a plate. Second, the installation is a bit too close to the KiNEDx rail, and so we might try to move the hotel back a bit.

In addition, Assaf will try to parallelize the program so it brings the next plate while working with the previous one, and so on.

Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells

2011-05-03T17:47:00.000+03:00

An "online" version of a paper appeared in Nature Biotechnology. This work of Michal Rabani, a Hebrew University graduate who is now a PhD student in Aviv Regev's lab at MIT. Michal is also a member of our group, spending several months a year here. This work also included Ido Amit, a postdoc at Aviv's lab who is starting a new lab at the Weizmann later this year.

In this work Michal and Ido used a new method to label newly synthesized RNA using a modified version of uridine that can be later used to separate it from the total RNA pool. This way they measured and sequenced "new" RNA. Michal then used these measurements to estimate the production rate of RNA in cells that respond to an external stimuli. Moreover, by contrasting the production rate of RNA and total RNA she could reconstruct what are the degradation rates through the process.

The main takeaway message from the paper is that RNA degradation rate (or stability) differs between different RNA species. However, for most genes, it did not change dramatically during the response. Thus, most of the shape of RNA level was regulated by production. We did find that for some genes changes in degradation rate was important to modulate the response.

Weekend getaway

2011-05-02T19:28:00.006+03:00

After a break of almost three months I went for a weekend of diving in the Red Sea with Matan, my ex-student and dive buddy. Few pictures.

Hotels!

2011-04-27T12:07:00.002+03:00

One of the big issues in running large experiments on the robot is managing plasticware. As the experiment progresses we need to use new plates, and these have to be stored somewhere until then.

To solve this issue, we decided to introduce hotels -- racks that can store multiple plates. Since we have an external arm (the KiNEDx), we ordered a hotel that can be accessed by the arm. This allows us to have a larger hotel and make it also more accessible for humans.

We decided to start with a single hotel to see how it works. Several weeks ago we received it, and then had a meeting with the nice people from the mechanical workshop to discuss how to mount it. Today they showed up with the "stage". It is large enough to hold five hotels in the end.

After installation, we have a nice hotel standing up. Next, we need to teach the robotic arm to access it.

Comparative Functional Genomics of the Fission Yeasts

2011-04-24T12:57:00.003+03:00

Fresh off the press: An online version appeared on the Science Express website (pre-publication online release of Science articles). This is a genome paper where the main result is the sequencing of a genome. In this case, actually two new species, both related to the fission yeast, S. Pombe.

(figure from Nick Rhind's website)

The fission yeast is well studied model organsim. As its name suggests, it divides in half during growth (unlike the budding yeast). It is also closer to humans than the budding yeast (aka Baker's yeast) in terms of many mechanisms. And so, many important discoveries in fission yeast in the areas of cell cycle, silenced genomic regions (heterochromatin) and other regions, were more directly related to mammalian cells.

(figure from http://physiology.uvm.edu/lord )

The new world of genomes allows to compare related genomes and use these comparisons to understand what part of the genome are functional. Such comparative genomics has proved immensely fruitful. However, so far very few related species of S. pombe were sequenced. The sequencing of the new genomes, and functional annotations of the genes in the these two genomes provide fresh insights into S. pombe genetics.

As you can see in this evolutionary tree that contains most of the currently sequenced yeast genomes, the "neighborhood" of S. cervisiae (bakers' yeast) and C. albicans (a species of Candida, a human pathogen) are well populated (horizontal branch lengths correspond to evolutionary distance). With the addition of S. japanicus and S. octosporus, the branch leading to S. pombe is now better explored.

(figure courtesy of Naomi and Ilan)

This project was spearheaded by Nick Rhind (UMass Medical School) and Chad Nusbaum (Broad Institute). Our contribution was in helping make sense of functional data and comparative analysis. Moran helped in identifying transcribed genes in the two new species based on samples collected at Aviv Regev's lab. She discovered many antisense transcripts that regulate some of the key genes in developmental decision making in these yeasts. Naomi and Ilan used comparative methods to find regulatory elements in these genomes, and showed how regulatory elements that are conserved throughout the tree of ascomycota yeasts change their function in these yeasts to match their life style.

Spring time in Boston

2011-04-12T20:34:00.000+03:00

I got invited by the students of The Computational and Systems Biology program (CSBi) at MIT to give a seminar talk and meet with them. It is a great honor to be chosen by the students. This involved two days of meetings with many interesting people at MIT, especially many of the graduate students in the program.

We had a roundtable lunch discussion where Michelle Chan, who coordinated my visit, led with a series of questions about how to choose career paths, scientific problems, and such. I was somewhat unsure as to my qualifications to answer such questions, but the discussion did raise interesting points.

This was also a chance to catch up with many people at the Boston area. I want to thank Michelle for making intensive effort to get the visit perfectly organized, and making sure that everything works smoothly.

Unlike my previous visit to Boston, the weather was definitely show that spring has arrived, and many of the trees were starting to bloom.

The new courtyard between Biology, CS (the (in)famous Stata Center) and the new Koch Institute (cancer research center)

I had a chance to take an afternoon walk toward Boston

A somewhat curious squirrel in the Botanical gardens

Multi-Color FACS

2011-04-05T13:05:00.031+03:00

As you may remember, we are using the Flow Cytometer (FACS) to measure the fluorescence levels of individual cells. Our FACS is an older BD FACSCalibur that has gone through several updates. Almost exactly a year ago, we had all the electronic boards refurbished. At the same time we already had plans to replace the old 488nm laser with two lasers at different colors. These lasers arrived with the electronics, but we decided to hold off on their installation that until we stop using GFP markers (that need 488nm excitation).

After few months we realized that this will not happen so fast, and ordered a third laser with 488nm capabilities. Due to many reasons the delivery of this upgrade was postponed for a long while. Finally, last week, all the necessary parts arrived. Last week Ariel and David from Merkel Technologies installed these upgrades and today we had a training session to go over the capabilities of these devices.

As part of the installation, Ariel removed the old (big and heat generating) laser, and put in a stack of three lasers instead. These required a panel of one-way mirrors to focus the beams onto the light path of the flowcytometer.

You can see the lasers here (with Ariel explaining) on the left side as small boxes. They are really tiny, but sit on aluminum blocks to disperse heat they generate.

The new light path involves mirrors that take the beams from the lasers

More schematically, the new light path configuration is shown below, where each line segment is either a bandpass filter (that allows only small "window" of wavelengths to pass through) or a half-mirror (that reflect certain wavelengths and passes others). As a result each sensor (FL) measures different area of the spectrum.

The consequence of this design is that we now can measure either GFP and mCherry (a type of RFP) together, or YFP and mCherry. We cannot operate measure together both GFP and YFP since the YFP excitation is very close to the GFP emission.

As before, when the FACS is serviced all the covers come off, and it is a great chance to take pictures of the light beams going through the prisms and mirrors

and finally hitting the target area in the flow cell

Exploring transcription regulation through cell-to-cell variability

2011-03-30T12:38:00.001+02:00

Yay! A paper that was in the works for two years, including more than six months in review, finally appeared in Proceedings of National Academy of Sciences, USA.

This describes Ruty's MSC dissertation, which was done with help from Ariel and me. In this work she took a very large dataset collected by Martin Jonikas and Maya Schuldiner at Jonathan Weissman's lab, and reanalyzed it to uncover new findings.

The key point of the paper is the use of "noise" or variability in expression levels of the same protein between genetically identical cells as a phenotype for genetic screen. Ruty shows that mutations that lead to abnormally high or abnormally low variability are involved in key processes in regulating transcription. Moreover, by using double knockouts she can pinpoint some mechanisms on these defects.

This work is cool as it shows that promise for the general theme that we want to pursue in the lab, of using genetic perturbation and examining effect on protein expression in single cells.

Funny/Sad Moments

2011-03-22T19:37:00.000+02:00

Part of learning to program a complex robot involves learning to debug programs that actually manipulate the real world. As a consequence "bugs" often have more impact than a simple error message on the screen.

Most these bugs are our fault, and so really have no one to blame. Once in a while there is one that seems to come out of the blue, and it took a while to find out what happened. Today, Ayelet tried a simple protocol that worked fine before, and it had dramatic results. I came in to check if this error was reproducible and so got it onto a movie (although a bit out of focus).

After some soul searching, finger pointing, and checks. We realized that the incubator was slighly moved when the FACS was being serviced. As a result the programmed position for lifting the plate was off, and thus the dramatic consequences.

Once we figured this out it was easy to recalibrate the position (we are getting good at that). Moreover, we had the incubator fixed into place the next morning. (We have had the request to do so out for a while, but now we made it clear this was urgent.)

Sooner than expected

2011-03-20T10:00:00.001+02:00

Any installation that involves water flowing in pipes is prone for leaks. We got the first one in our robotic room. Much sooner than we would have hoped.

In the middle of a discussion with Assaf I noticed that the ceiling outside my window was "weeping" water .

A quick examination showed that the whole shelve above the window was flooded with water. We tracked this to a leaking pipe in the water-based A/C unit in the robotic room.

We called the emergency maintenance hotline, and soon someone showed up. He called the person responsible for A/C maintenance who showed up after half an hour. Together they blocked the in & out lines for this unit (and neighboring room) and drained the pipes to avoid additional leakage.

Update from this morning. The contractor who connected the A/C showed up. The fault was a fracture in valve that connected the old (pre-existing) pipes to the new installation. He replaced the valve, and now we hope that the ordeal is behind us.

For leakage, the damage was quite minimal. We might need paint renewal in few spots, but overall all equipment are intact.

Dry Ice Shipping Revisited

2011-03-18T15:20:00.001+02:00

After the catastrophic results of our last attempt to send samples on dry ice, we went back and did research on packaging materials for ice. We learned that we need to use foam and strings as plastic tend to be brittle. We also checked various tubes to see how they hold up in cold temperature.

We finally selected tubes that were less brittle and better packaged in their box. Only the larger version was available, and so we ended using tubes that were much larger than needed, but we decided that this would not harm the content.

Assaf re-run the whole experiment again. This time it went smoothly and he got samples without any probelms (so we do learn from experience).

This time each box was padded both inside and outside, and securely wrapped in foam.

We then packaged them in the box, and wedged them into place with pieces of styrofoam. We broke the big blocks of dry ice to small piece to ensure that there is no big hard mass bumping around during shipping. Since we didn't have a large box, we sent the samples in two separate boxes.

To our relief, the two boxes arrived safe and sound.

Site visit to new Engineering Building

2011-03-06T20:13:00.028+02:00

As you know, I am a member of the School of Engineering at the university. The school was established more than ten years ago, and it was immediately clear that it needs a physical venue. I was part of a committee that helped define the functional specifications for the building with the architects (a collaboration of a Canadian and an Israeli firm, AJ Diamond, and Kolker, Kolker, and Epstein). The result was plans for a very nice building with multiple functions (offices, research, teaching, student lounges, etc.) You can learn more about it here.

Due to complex circumstances, the construction of the building was delayed quite a bit, but in the last two years it has been going on in full steam, and hopefully it will be ready for opening in about a year. Today I was invited for a tour of the construction site to see how the interior will look like. I took the opportunity to take some photos of the place. It was raining last week, and so there puddles still on the roof and in some of the large empty spaces (which will become lounges).

It is a cold harsh world: Lessons in shipping with dry ice

2011-03-03T14:50:00.002+02:00

Two week ago Assaf completed a large scale experiment with the robot. The next step was sending all the samples to Ollie's lab for running the NanoString assays. This involved sending cellular extracts with RNA from ~900 samples. Since the samples are sensitive, they need to be kept cold, and so should be shipped expediently packaged with dry ice to keep the temperature.

It turned out that this is a non-trivial exercise. In our previous iteration, we learned where to order dry ice. Now, however, we needed a much larger box as we were sending hundreds of tubes.

Assaf used the robot to move the samples into 12-strips of small tubes, organized in 96-well format by using tip boxes.

Ayelet and Assaf then sat down and labeled the strips and packed them in boxes. They sealed the boxes with parafilm to keep everything in place.

Assaf and I packed the insulating polystyrene box with blocks of dry ice, and arranged the boxes on top of them. Finally we sealed the box tightly and shipped it to UMass.

After three days, we get an email titled "Disaster!". When the UMass people opened the box they found small rocks of dry ice and many fragments of plastic scattered throughout the box (although few boxes remained intact).

(pictures courtesy of Ollie's iPhone)

The lessons we learned from the exercise is a harsh one. Turns out that the plastic strips we use are extremely brittle at cold temperature. Moreover, parafilm no longer binds when cold. Moreover, as the ice blocks evaporate, there is room for play inside the box, and things start to jump around, leading to havoc.

MODEL-IN semi-annual meeting

2011-02-23T21:56:00.017+02:00

Our group is part of a consortium that is funded by the European Union for a joint project on transcriptional regulation in inflammatory response. The title of the project is MODEL-IN, and you can learn more about it in its website.

As part of the project, we hold meetings roughly every six months. This is a great way to catch up on the progress in each group and also to get better working relationships. Most of the meetings were in the UK, and one was in Rome. This time the meeting was organized in Israel, which was a great opportunity to get project members to see Jerusalem.

We spent two intensive days in meetings going over scientific results.

On the afternoon of the second day we went for a tour around Jerusalem. This was a beautiful spring day (spring started early this year) and we got nice views of the old city and its interior.

Robotic NanoString Experiment

2011-02-21T20:37:00.010+02:00

Today was finished our first large scale experiment using our robotic platform.

Assaf programmed the robot to grow 90+ strains of yeast over two days to reach optimal conditions (as I previously described), then the robot stimulated them and harvested cells at 9 time points. Each harvesting step involved taking small amount of cells from the plate they were growing them and plunging them into a plate of cold liquid methanol. This immediately freezes the cells and "stops" them cold (literally).

Next, Assaf used the protocol he and Ayelet devised for extracting RNA from yeast cells, to process these 9 x 96well plates. These will be shipped to Ollie's lab for running the NanoString assay to measure exact RNA quantities.

This was the first time we had an actual experiment running from start to finish. Although we "debugged" various parts before, putting all of them together uncovered all types of problems. Assaf had to restart the growth several time before we had the whole thing running. But now we are smarter and know how to get things right the next time.

After few days of heavy robot use, Assaf collected all the plasticware he used. Here is a picture of triumphant Assaf at the end of the process.

Robotic Facility Kickoff

2011-02-02T22:02:00.024+02:00

You may recall that we had an event to celebrate the opening of the first half of our lab. Now that the renovation is almost over (only missing few furnitures), we decided to have a more official kickoff. The idea was to say "Thank You" to all the people who helped us build this lab out of nothing.

The last week, Ayelet, Assaf and Avital (also known as "AAA Robotics") worked hard on setting up a demo of the capabilities of the robotic facility. The last two days, I recruited Tommy (who is still waiting for his J1 visa renewal) to help make a movie of this demo. Tommy turned out to to be a capable photographer and has the option of alternative career in the movie business.

Yesterday afternoon, Naomi and I went to buy cheese, cherry tomatos and strawberries for the event. As usual, the selection of cheeses at our favorite fromagerie was tough, but we managed to negotiate it. This morning, Ayelet bought fresh bread from our favorite baker. We set up tables in the corridor leading to our lab, with sparkling wine, bread, cheese, and red tomatos and strawberries.

Since all the rooms are already in use, we had the event in the small meeting room/kitchen next to our lab. This meant very select company (we had space for 24 chairs). It was very emotional as it was a chance to thank so many people at once.

Moments before the 9:00am, we did a quick test of the demo, and Ayelet decided we needed another plate with YPD (media), so I rushed to do the job.

During the event I gave a short talk about the science we do, and then had a movie about the robot.

We then moved to the corridor and had a toast. Sadly, the University President had to rush out before the toast.

We then had real-life tours of the facility while other used the opportunity to chit-chat and catch up. Sometime even serious scientific discussions.

Photography at the event was courtesy of Roy.

Into the cold!

2011-01-23T20:35:00.000+02:00

I am now in New England. I am visiting Ollie to write a grant and work on results. It snowed on the first day here. And now the clouds have cleared away, and it became colder. Today its about -10C and tomorrow it is supposed to get even colder.

Regulation of Blood Cell Differentiation

2011-01-21T19:17:00.002+02:00

Another cause for celebration. A project that was active for several years has made it to publication. Today a paper with Noa as a first author appeared in Cell. This project was mostly done when Noa was at Aviv Regev's lab at the Broad Institute. She analyzed the expression profiles of genes through the differentiation of hematopoitic cell lineages --- how a bone marrow stem cell develops in to different types of blood cells.

(Source: Cell)

The interesting part is that this analysis uncovered multiple "modules" --- groups of genes --- that are reused in different lineages. This shows that differentiation is much more complex than previously assumed about this system.

Congratulations to Noa!

NanoString Tryout

2011-01-20T19:34:00.003+02:00

You might remember that previously I reported on our efforts on RNA extraction from yeast cells. Since then Assaf and Ayelet quantified the amounts and quality of RNA we get by doing RNA purification from the cell supernatant in Assaf's assay. They showed that the amount of RNA we get matches what literature reporters (about 1.2 pico-gram/cell), and that the RNA does not show any degradation.

Last week Assaf performed a time course experiment where he grew yeast, and then subjected them to salt stress, and collected cell at different time points following the stimulation. He froze samples of cells by droping them into very cold (< -40C) methanol, and then extracted supernatant using the technique I discussed in the previous entry.

He packed the supernatant in a box with dry ice and we sent it to Ollie's lab at U. Mass. Medical School by carrier service. We were not sure if the samples will get there in good shape, and it was great to hear that they did, still frozen. Hsiuyi from Ollie's lab run the samples on the Nanostring nCounter machine they have there and sent us back the results.

Few words about the NanoString machine (you can read a nice overview on their website). The basic idea is that we order a set of probes that match genes we are interested in, and then using these probes can count individual molecules in a small volume sample. By counting how many times we see each type of probes we get a very accurate measure of the number of RNA molecules in the initial sample. The cool thing about this method is that it does not use any enzymatic reaction and thus does not have biases due to PCR preferences and such. Moreover, it is very simple to run and does not require RNA purification, labeling and such.

We were very happy that the results we got from Hsiuyi showed high degree of reproducability. Two repeats of the experiments looked almost identical (if they would have been more similar I would start being suspicious).

In this figure each dot correspond to a gene, and the x/y axis are the log (base 2) of the number of counts we had for that gene in the first or second replicate expriment. We can see that the dynamic range here is between 2 to 16, so we have 2^14 (= 4096) fold difference between the lowest probe and the highest one.

This success shows that Assaf's setup is ready for the big experiment of measuring many different mutants. Hopefully we will have this done in the next few weeks.

Visitors

2011-01-11T16:24:00.005+02:00

This week the winter decided to give us a sneak visit. It has been a mostly sunny winter so far, but for a few days we had actual winter (rain and wind).

In the midst of this (mild) winter weather Tommy, an ex-student of mine who is now a postdoc at the Eisen lab at UC Berkeley, showed up for an interview. It was fun to spend time with Tommy again. He was involved with the early steps of thinking about the grant, and so it was nice to show off how much things have progressed.

We enjoyed a lull in the rain to sit outside among the fallen leafs and enjoy a simple lunch.

As part of his visit Tommy had to renew his visa, and seems that courtesy of the US embassy, we will have him visiting for a bit :-(

Here is a picture of wintery sunrise:

Growing yeasts (Robotically)

2011-01-03T21:23:00.001+02:00

The first task we set to do with the robot is grow yeast cells for experiments. This sounds easy? No?

To understand the issue. Lets review the typical yeast life cycle. Suppose you pick a small number of yeasts from a colony or a saturated culture and put in a fresh "rich" media with glucose (sugar). Moreover, suppose that the media is kept in nice temperature (yeast like 30C) and shaken to make sure yeast cells and nutrients keep mixing.

Initially it will take the yeast cells time to realize that they are not in the nutrient-poor environment they were in. They will revitalize themselves and prepare to grow. This phase is called lag phase. During this phase the number of yeast cells will not change.

Once out of lag phase the yeast will start to grow on glucose. They will work hard to use this rich source of energy to grow as fast as possible. A typical yeast cell will divide every 90-120 min (depending on the exact conditions and temperature). This period is called doubling time as the number of yeast will double every fixed period. During this phase the yeast will experience exponential growth. For this reason this is known either as exponential phase or log phase (since it looks linear in logarithmic scale).

After a while the yeast will start exhausting the sugar. If this was bacteria they will stop growing. But yeast has another trick up their sleeve. They switch from using sugar as fuel to using ethanol. During the fast growth phase the yeast use the glucose in a fast way by fermenting it to ethanol. This is what in human metabolism is known as anaerobic metabolism as it does not require oxygen. In nature this trick allows yeast to outgrow the competition (fast growth) and also kill it (by increasing ethanol concentration). Humans learned long time ago to use this property of yeasts to make alcoholic beverages.

Returning to the yeast growth, the switch from glucose to ethanol is called the diauxic shift --- the yeast will go through it once it can no longer import glucose from the environment. Ethanol can be used for aerobic metabolism (or respiration), but requires more work to extract energy from it. As a result the yeast will grow slower. They still grow exponentially but the doubling time is much longer. This phase is often referred to as saturated or early stationary phase although these description are inaccurate as the yeast still grows.

After a while (and this can take much longer), the ethanol reserves are consumed, and the yeast stops growing and enters in to stationary phase. The cells prepare for nutritional hardship and reduce their activity.

When plot the number of yeasts in the tube during this phases we ideally see this type of curve:

For our experiment we want to take yeasts in the middle of the fast growing exponential phase. Moreover, to make sure that the yeast forgot its history, we want to make sure that there were several (>3) cell divisions since the lag phase. This means that we need to yeast to multiply itself by at least 8-fold from the initial amount.

Moreover, we want to make sure the yeast do not come close to diauxic shift, as this stage results in major changes in the yeast metabolism. This means avoiding over-crowded situation. Finally, we also want to ensure that we have sufficient number of cells to work with, so we do not want under-crowded cells either.

Sounds easy. If the lag phase is 60min and doubling time is say 90min, then we need 330min (5 1/2 hours) to grow the yeast. Calculate the desired amount at the end and seed the culture with 1/8th of that.

The problem is that we want to work with many strains of yeast. In fact, we want to grow 96 strains in one plate. Each strain has different lag time and doubling time. This means that while one strain has 90min doubling time, another might have 150min doubling time. This means that for the latter strain we need 450min to get 3 doubling (8-fold increase), but by then the fast strain has two more doublings and has grown by 32-fold from the original number of cells. Due to the exponential growth, small difference in growth rate can lead to dramatic differences in cell concentration.

So, how do we deal with the problem? Ideally, we can measure the relevant times for each strain and then plan the initial seeding to get things right. In fact, this is what we do, but using a robot.

During the last two weeks, Assaf and Avital developed a robotic protocol that grows the plate of yeast for 20 hours. Every half hour the robot took the plate out of the incubator, and put into a plate reader (spectrophotometer for plates) that measures the optical density (roughly equivalent to number of cells). After this incubation time most wells were past the diauxic shift. The program then used the plate to seed a new plate and again monitored growth for several hours. At this point Assaf and Avital's program computed what dillution it need to perform to each well to ensure that at the planned target time the cells will rich a desired density. The robot then applied a customized dilution step for each well.

At the end of this procedure we had a plate with 96 strains (with very different growth characteristics) all in roughly the same density. To our surprise/relief/joy the robot did all of this without a fault.

The end result can be seen like this. In this graph OD corresponds to yeast density, and each curve describe the density in one well on the plate. You can see the yeasts growing fast and then slowing down. You can also see the two dilution steps (the first dramatic one and then the "correction" step). Most importantly you can appreciate that in the end all the wells are fairly close to each other in density.

The nice growth can be more easily seen in a log-scale plot:

For some reason the empty wells (that do not grow :-) misbehave after the dilution step. Do not that before the final dilution there is a large variability in the density and that it mostly removed by the program.

And so now we can start doing experiment with tightly controlled yeast growth. Yey!

One Year Celebration

2010-12-28T15:20:00.002+02:00

Today this blog celebrates its first year. With 102 posts during the year and many exciting events.

Just to remind everyone, a year ago we had two benches, one freshly installed FACS and two devices still packed in their boxes. Today we are past the renovations. The molecular lab has 6 working benches, and two more for equipment. We have a preparation room for making media and agar plates. And, most importantly, our robotics room is setup with all the devices up and running and integrated.

Now our efforts move from the renovation updates you have been hearing about to more scientific issues, such as setting up the experimental system and learning new exciting things.

Cameras and labels

2010-12-27T17:10:00.001+02:00

We are slowly starting to check off the various little details that make a lab functional. Today we had two new improvements in the lab.

First, after long delays, we finally got proper printable labels, and Ayelet set off on the task of putting labels on drawers and cabinet doors. The long searches for where someone has placed a particular item will hopefully be easier now.

In the robot room we installed two IP webcams. These monitor the main Tecan stage and the peripheral area of the microscope and HyperCyt. We now need to hook these to a recording software so we can also browse back in time.

Here we see one of the cameras (the one watching the Tecan) and a view on the screen from the second one that watches the KiNEDx and peripheral devices.

This type of monitoring is important as we are starting to run the robot overnight and want to be able to analyze what went wrong if something does go wrong. Moreover, it will allow remote examination of the system when one of us is not around.

You are what your father ate?!?

2010-12-25T19:32:00.003+02:00

I have been lax at reporting papers coming out, but decided we need to change that. So here is a new type of post - talking about new publication.

Today, Cell published a paper "Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals" by Carone BR*, Fauquier L*, Habib N*, Shea JM*, Hart CE, Li R, Bock C, Li C, Gu H, Zamore PD, Meissner A, Weng Z, Hofmann HA, Friedman N, Rando OJ.

This is culmination of a wild roller coaster ride over the last few years and months. The project has been one of Ollie's (Rando, OJ, that is) for quite few years and we were brought on to help.

For some reason Cell put a press release with the title "You are what your father ate" which seems a bit of an over statement. But it definitely managed to get the press interested. So far there is press coverage by the Time, and various other interesting venues, such as Pravda.

The basic story, is that Ollie and later his students have been feeding mice either control diet or one that is low on protein and high on suger. These mice where then mated with females and then removed from their cage. Ollie then examined gene expression in the offspring livers and found that they were different depending on the father. In particular, lipid metabolism in these mice was different.

(source: Cell)

Our involvement was in trying to help Ollie make sense of the data. As usual he found the cool stuff by himself but needed reaffirmation from computational people :-) Naomi (Habib, N, that is) analyzed the expression data to make sure that the results made sense. She also compared the list of genes that changed in the offsprings to many relevant datasets in the literature and found connections that helped understand the nature of these changes. She also analyzed other datasets, including small RNAs in these livers, and gene expression in sperms from fathers of the two different groups.

All by all, this project was one of the main things Naomi did over the last few years. It is great to have it come out, and in particular in such a prestigious venue.

Ariel sendoff

2010-12-17T12:07:00.001+02:00

In quick succession of event, we moved from a lab retreat to a goodbye event to Ariel, who is going to Stanford to start his postdoctoral fellowship.

Ariel was a joint student of Hanah Margalit and myself, it was a great excuse to have the two groups meet together for a social event. We met in a nice pine grove in Jerusalem.

After a while we started a small BBQ to prepare hamburgers. Assaf, Jenia and Tal found themselves rotating as chief cooks.

In the meantime we all had a pleasant time.

Toward the end of the event we managed to embarrass the guest of honor by ceremonial goodbye speeches.

Clearly, Ariel was a leader in the group and one of the steadfast members who brought knowledge, critique, and useful suggestion to every interaction. We are sure he will have great success abroad, and already waiting for him to visit us.

Lab Retreat

2010-12-01T23:47:00.041+02:00

The last two days we spent on our annual lab retreat. The idea is to go out of the university and spend two days in which we get a chance to hear what everyone has been up to and plans to do next. It is also a chance to bond socially.

This year we met in Netiv HaLamed-Heh, a kibutz south-west of Jerusalem.

In what used to be an old barn there is a site that now hosts weddings and seminars. We had a nice and specious room for the talks.

The talks covered a lot of materials, and so we needed some breaks to enjoy the sun.

In the late afternoon we had a special treat, a talk about therapy using puppets.

We then prepared food for the night. Moran found a yeast-shaped potato, which was clearly an important omen.

The potato went into the pot with all the other ingredients and onto the fire. After an hour or so, we had a tasty meal.

The next day we continued with the talks.

We then got into the cars and drove up to a nice hill, and had a lunch picnic, and a short trip to nearyby ruins.

Lab news - fixes and PCRs

2010-11-29T23:44:00.000+02:00

Today we have quitely marked an important milestone. Itsik and Ami from the "fine mechanics shop" came to fix the Microscope, HyperCyt and KiNEDx rail to the tables. This means that from now we can assume that their relative positions with respect to each other are fixed (and thus the KiNEDx arm can return to the same position reliably). This is also an indication that we are happy with the temporary outline of devices we put down.

We were warned about the difficulties in drilling in the Terspa benchtop we have. It turned out that with a simple drill it was relatively easy to have holes in the benchtop.

Ami threaded the holes, and then screwed in the equipment.

The HyperCyt is held in place by three machined blocks (two in front and one in the back). The Microscope had its holders that allow for screwing into the table and into the microscope body.

Finally the KiNEDx rail has holes in the rail itself, and it was screwed directly into the benchtop without external holders.

While Itsik and Ami worked in the Robotic room, we received a package.

Our new PCR thermocyclers. We got a deal on a pair of devices from Bio-Rad that combine 96-well head with two 48-well heads under a single controller. This was one of the missing large equipment on our "to get" list.

The army marches on its stomach...

2010-11-17T22:00:00.000+02:00

... and science progresses on caffeine. Our newest addition to the lab is the new esspresso machine.

This was gift from Neotec to accompany the integration of the robotic facility.

This definitely improved our quality of life and allowed us to discover new talents of our members. Without much practice, Ayelet created this nicely layered construction.

Blasting yeasts

2010-11-17T16:54:00.000+02:00

In one of our projects we want to extract RNA from yeast for measurements using the nifty NanoString nCounter technology. One of the nice things about this assay is that it uses very small amounts of actual material to measure RNA quantities. Moreover, unlike other assays, we do not need to purify RNA before the assay, which saves a lot of headaches.

The challenge we posed to Assaf, who is about to do the experiment, is how to extract cellular material from a very small yeast sample. We actually need as few as 15,000 cells (just for reference, 1 microliter of happy growing yeast has about 1-2 x 10^7 cells). The problem is that yeast, like many other microorganisms, has a strong cell wall made from proteins.

The classical way to break it is using mechanical action -- beating the yeast using small glass beads. This technique, however, does not scale down for small samples. And so, we decided to go with Plan B. Here we use an enzyme, Zymolase, that digests the cell wall. This creates spheroplasts -- cells without cell walls that are encapsulated by a thin membrane. We can then pop these open by diluting the sample with water with small amount of detergent. This solution also includes protein denaturing agents that block any RNAses from breaking down the fragile RNA, and thus preserves our sample.

Assaf and Ayelet tried this technique, and it seems successful. The zymolase readily digested the cell wall and left with nice spheroplasts. Then when he dilluted these quickly disappeared. Assaf made a nice movie of this process under the microscope. What you see is a small drop with yeast spheroplasts, and midway through the movie this drop is dilluted.

Robotic Arm Integration (and other news)

2010-11-15T22:42:00.002+02:00

The last week I was away on travel. During the week Shai (from Neotec) together with Avital & Assaf worked on robotic programing and hopefully we will have updates from that front.

In addtion, Udi and Yoram from Neotec manufactured a nice station for handoff between the Tecan Liquid Handling Robot and the KiNEDx robotic arm. Today Shai together with Amir and Moshe (also from neotec) worked on fine tuning the integration of the robotic arm into the Tecan control software.

At the end of the day we managed to film a demonstration in which using EvoWare (the Tecan control software) we can take a plate from the deck of the Tecan to the microscope/hypercyt and back. This means that we made a significant step toward integrating the system.

It is impressive to see the KiNEDx arm in action. Its movements are fast and fluid.

From the movie you will also notice that we hang the plastic curtains that surround the Tecan and keep it clean and sterile. We still need to tailor a hole for the KiNEDx to move through, so for now the enclosure is not complete.

Just to get a sense of the flurry of activity that led to these results, here is a time-lapse of part of that day.

HyperCyt and Python Scripts

2010-11-04T22:13:00.000+02:00

Today we had a long day in the robotic room. Yesterday Arik from Neotec came to calibrate the find details of the liquid handling arms on the Tecan which solves some issues we encountered on Tuesday.

Today Shai, who is responsible for "smart" robotic application came. We had a general discussion about different ways of integrating software to the robot controlling software. Afterward he, Avital and Assaf sat down and implement some pythons scripts that call the robot and others that the robot can call.

It seemed that this was very fast pace study as they reported success, and managed to fancy pipetting procedures on the fly.

In the meantime, Ariel and Jenia tried to get the HyperCyt to work in the new location. We decided to move the peristaltic pump onto the hypercyt deck. This however resulted in the arm pushing the pump off when the device was initialized. This was annoying, especially since the pictures of the device showed the pump sitting exactly where we put it.

We searched the manual high and low, and finally realized that one of the figures mentions an L-shaped piece that serves as a stopper in such a configuration. We used this as evidence that this is the right solution and installed the stopper, which indeed solved the problem.

However, now the coordinates of the arm were totally off. And so we learned how to "teach" it where the different locations are. This involved moving the tip of the needle very slowly with mouse controls until it was in the right location.

In the end the system was working, and we even managed to film it. As you can see the sampling needle goes into each well in succession. This means that it creates alternating bubbles of media (+ cells) and air in the tube. If you look carefully in the movie you can see these tubes.

After lunch break and group meeting Jenia run his nifty analysis software to break the long FACs stream of events to specific wells. It worked like a charm and immediately gave him detailed summary of each well. The investment in this software was definitely worth while.

First steps with the robot

2010-11-01T22:54:00.002+02:00

Today we had our first training session with the Tecan robot. We learned how to create a script for doing various steps (e.g., pipetting, moving plate from one device to another and such). The concepts were relatively simple and intuitive, and by lunch we had our first working protocol. It takes a plate, seed it from another plate. Move the plate to the shaker for a minute and then measures optical density.

After lunch we moved to more complex protocols that used multiple plates (e.g., when you want to run something on a large number of plates). At the end of the day we felt that we can start making use of this device.

We also had a our down moments, including a crash when the robot arm that moves plates decided to go to the first available position, which was not available....

Busy busy day (Robots, FACS, cabinets and sinks)

2010-10-26T21:07:00.003+02:00

So another one of these crazy days. So what did we have today.

* David and Ariel from Merkel Technologies came to move the FACS and HyperCyt into the new robotic room (see movie).

* Udi and Shy from NeoTec continued installing the KiNEDx robotic arm. They managed to move a plate from the Tecan to the microscope and back (see movie again).

* We had our first wet-lab group meeting in the room next to robotic room. The furnishing need some work, but the room is usable.

* Yoel, the carpenter from "Wooden Horse" (סוס עץ) brought the new storage cabinets/lockers for the corridor. He also brough cabinets for the robotic room, which made it interesting to work there for a while. The workmanship of the new cabinet was impressive.

* The plumbers came to install a water heater in small yeast preparation room. They also finished the sink in the robotic room.

* We had a regular group meeting in the CS building as well :-)

* And most importantly, Yael came to visit the lab to approve it.

And movie of some of the installation around the robot.... Note the sequence at the end where the arm moves the plate from Tecan to Microscope.

Robot is (finally) installed!

2010-10-25T21:09:00.004+02:00

After two delays, today was finally the day. Early in the morning Udi and Arik from Neotec showed up for installation. After re-checking that the tables are flat, we set out to install the robot.

The first step was to unbox the robot from the huge shiping box.

All empty space inside the device was full of a box with various accessories. It took a while to realize that we don't have any hope of pulling it out as one box, and we resorted to removing the contents one-by-one.

Now we could start seeing the shape of the device, Tecan Evo 200, and admire it.

Next, Udi and Arik connect special handles to the robot frame.

We recruited Alon from the lab and Ayelet and set out to lift the Tecan (220kg according to Udi) onto the smallish cart that the Neotek people brought with them.

Somewhat surprisingly, the cart held the weight, and we slowly moved the procession into the room and next to the table.

A bit of a coordinated heave, and the Tecan was on the table. Few more adjustments and it was located into place.

The curious people could now examine the details of the labels.

We then had a small unwrapping ceremony where we got a chance to remove all plastic protectors from the robot outer shell.

Shy, who is the main software integrator showed up and joined the party. Arik set out to installing the device. This involved removing safety brackets and unlocking the arms. It didn't take long for him to get to a stage where he set the device on a "Random Walk" mode. In this mode the robot tries moving the arms to different X/Y/Z locations to see that there are no obsticles or mechanical problems. It was fun to watch and gave a good impression of what the machine can do.

While the main Tecan was playing at random walk, Udi and Arik started assembling the movable arm. Its called Peak Robotic KiNEDx. It has a flexible gripper that can hold plates and move them from the Tecan to the microscope.

Once Udi managed to get the arm to move, we tested how far it can reach. We had to move the microscope a bit, but now the stage is in reach of the arm.

During this whole procession, Shy started working on interfacing with the external device. He had a quick success in interfacing with the microscope and in no time managed to show that he can control it from the driver he wrote. So, we are hopeful that the integration would be smooth.

And finally, the obligatory stop-motion film of the day's highlights. Given requests from the audience, I edited the sequence to be short and include the main interesting points. Enjoy!

Flat Out Tables

2010-10-24T16:47:00.001+02:00

And so the Saga continues. We still don't have a robot installed. It was a relief to find it waiting in the corridor in the morning. Or as Moran said, the good news it is still there (no one took it) and the bad news is that it is still there (no one installed it).

The crew from PVPlast who were supposed to fix the tables got stuck on the climb to Jerusalem. They managed to get here by re-filling the car heating system with water. When they got here it took a while to convince them that there was an issue, and then how to solve it.

In the end, we to go through several rounds of examining the tables with a long metal ruler until I approved their work. This took three hours and by then it was clear that the robot installation is not going to go as planned.

I give you the "dance of the flat tables"

On other news, we had Ariel, a dedicated reader of the Blog, visit in person. To make sure this is not lost on the crowd here is the relevant images from the movie.

Microscope, Robotics, Tables, and a Window

2010-10-21T21:35:00.000+02:00

So the big day has arrived. Today the robot is finally moving into our hands. Things did not turn out exactly as planned.

The day started early, with Dimitry from the Eisenberg Brothers who came in to move the microscope from the molecular lab to its new station in the robotic room.

While Dimitry was moving things, a nice guy from the communication department showed up to talk about connecting the ethernet ports in the new room. I then got a long phone call from a collaborator.

A bit later, Udi Oz from Neotec and his crew showed up. They brought the Peak Robotics arm. As they started working, Danny from the computer system showed up to talk about communications and potential place to put wireless.

Then Michel the carpenter suddenly came in to ask which doors need safety stops. As he was talking to me the Neotec people realized there is a problem with the new tables for the robot. They were not flat, and the rail for the robot arm was not sitting properly because of that.

We started consultation as to what to do about that when the delivery truck with the robot showed. The huge robot box almost blocked the corridor.

But since the tables are not flat, we had to move to a side area so traffic will not be jammed.

As we were doing this the aluminum profile person showed up and took the temporary window in the robotic room. Michel came in and tried to see if he can help us with the tables. In the end we decided to wait until the PVPlast team shows up and fix the issues.

Just to get a sense of the problematic aspect here, we took pictures of a straight piece of wood as one end was touching the table, the other is seen to be far from it.

These events were squeezed into five intense hours. Just to get a sense, here is a time-lapse movie of this part of the day.

This left me a bit of time to talk some issues with Udi, grab a sandwich and go to group meeting. On returning we found out that the new window is installed.

It has integrated shades inside, between two glass panels, and so allows to close or open the window easily.

Avital is Back! (and the sink is here)

2010-10-19T16:31:00.000+02:00

Latest news from the lab. Avital, who has been studying the last month for here MSc exam, has passed the exam with high marks and is back in the lab. We all have missed her during the last few weeks.

While we are waiting for the robot to arrive, another small unresolved issue in the lab was solved. Today the sink in the robotic room was installed.

It arrived as a nice shiny object.

The installers cut a hole in the cabinet. I was happy that this part takes place before we have sensitive equipment in the room.

The final installation is nice.

Now we are waiting for the faucet installation and wall ceramics.

New Office

2010-10-18T22:42:00.000+02:00

We are still waiting for the robot. It seems that the air-delivery company had problems getting it on the planned flight, and so it is delayed. As far as I understand it landed in Israel today or will land tomorrow. We hope to see it by Thursday.

In the mean time, we finally got locks on the doors of the new part of the lab. This means that we can start using the room. One of them is a small office for myself or Ayelet. For the next few months I will be using it most of the the time.

The office is very small 2.5m x 2.5m. But it has two big external windows with light, and a large window into the robot room. For now the furniture in the office is really minimal. I already used for a conference call to the States and for few discussions (one even involving three of us) and it was fine.

From the robotics room the office looks like that.

The sharp of eye will note that the window between the rooms does not look finish. This is a temporary window that will be replaced with a double glazed window that can be shaded for privacy or when we want to limit the light in the robotic room.

The stage is almost set, waiting for the actors

2010-10-12T23:28:00.000+02:00

After long period of very slow developments, the last two days saw frenetic activity. I am away on a trip to Edinburgh (to attend the International Conference on Systems Biology), and so had to hear about the developments by remote control. All the images here were taken by Assaf, serving as my remote eyes.

Yesterday bright and early (before anyone showed up to work) the nice people from PVPlast came in and start installing the work-benches for the robotic room. The design was non-trivial, but they delivered it perfectly. By 2pm they finished and left.

Today, we had the electricity contractors come in to finish the electricity installation, as there is a non-trivial amount of electric/communication outlets that are mounted on the workbenches.

Few pictures of the work as it progressed today.

The table with the hole is going to hold the robotic-arm-on-a-rail. (The hole is for throwing out used plates.) The table next to it is the "heavy" table for the microscope. This table is not attached to any of the others to avoid vibrations. The bench next to wall is going to hold the microscope monitors and control boxes.

Here we see the tables where the liquid handling robot will sit and the FACS. The table that run in angle to them is the robotic arm table, and in the foreground we see the corner of the microscope table. The clean hood is sitting over the liquid handling table and the end of the robotic arm table, and will enclose this area with super-clean air. The long hole between the two tables will serve for cables. In the background you can see the workbench that will serve for sample preparation, and might also hold some of the equipment monitors. Here is another view of this bench and the clean hood.

Since there many devices will sit on these tables we needed to supply them with electricity and communications. Thus, there is an electricity panel below the robotic table (all along its length).

These cable come from connection boxes along the wall. Thus, there is another panel between the long arm table and the robotic table.

From there the cables go down below the microscope table and all the way to the wall. From the side this look like this.

At the other end of the microscope table the cables come from the rack into another panel. The important part of this construction is that the rack itself is "floating" below the microscope table without actually touching it (so not pass any vibrations).

On the wall the cables are distributed to the various connection boxes (each one housing cables fed from the main electricity and communication boards.

While they were here, the electricians also installed the benchtop lights that we have been waiting for for a long time. Let there be light!

Clean Tent

2010-10-05T21:39:00.000+02:00

One of the main worries regarding the robotic setup is contamination from the environment. Since the robot works with media and open plates, such contamination can ruin many experiments. We started by thinking of housing the robot in a clean room. This however was very expensive and would require harsh operating conditions. The solution we came up with is to have the robotic room over-pressurized, so that dust and contaminants are pushed out. In addition, the robot parts that involve sterile work will be housed inside a "clean tent".

Today the tent arrived, in pieces.

The technicians quickly assembled the heavy aluminum framework (which was interesting to watch).

Then they loaded on top of it the filter units.

Each unit filters air through a series of filters and pushes it downwards. As a result the insides of the tent are continually "washed" by very clean air and kept clean. To close off the relevant volume, the tent has plastic sheet flaps that allow access inside but keep the bulk of the clean air going down in a laminar flow.

The next week the tables and benches for the robot will be installed, and then the stage will be set for the main player.

Lab Party!

2010-10-04T21:25:00.002+02:00

The holidays are over, and everyone gathered back from the four corners of the earth they dispersed to. To celebrate the start of a new year we had a party. The theme was Italian Food.

Ariel made fresh fettuccine in three colors.

Moran, who just returned from Italy, made pizza dough and baked a series of fresh pizza on the premises. One problem is that by the time the pizza tray left the kitchen it was mostly empty, as the smell of the pizza coming out of the oven lured people to come and "taste" the creation.

We set up a WII station in the yard, and although the screen was far from perfect (a tablecloth) we did see some heated action.

The less happy part of the event was to say goodbye to few people. Ruty and Aharon who are finishing their M.Sc. and continuing to other things, and Ohad and Julia who finished their B.Sc. project with us.

The dessert table was exciting and managed to cheer everyone up.

We had a surprise appearance by Matan, a lab alumni, who showed his powers and cradled babies.

And we ended the evening with an optimistic smile.

Back from Hiatus

2010-10-01T20:51:00.001+02:00

After a long break this blog is back in action. In the last posts I covered the celebration of the new molecular laboratory and the start of the works on the second part. Since then there was a long summer. I traveled to the UK (Oxford), US (Boston), Japan (Tokyo, Kyoto, and many other places). I was back to Israel for few days and then off to Austria, and then back again and off to Egypt, and then the Jewish High Holidays arrived.

While I was traveling many other group members were also traveling (some for longer and some for shorter periods), and so action at the lab was a stop-and-go process. Nonetheless, a lot has happened during the last three months, and I will try to summarize by quick snapshots, most of them taken by Assaf and Avital.

The renovation started with a fierce burst of rebuilding walls.

At the lab about that time we finally got proper chairs.

Following the drywall people, the systems people moved in to install electricity, pipes, and A/C.

In the working lab things kept improving. A new cabinet arrived. This required moving out the temporary ones, and covering all equipment again dust.

Some people tried to continue working in spite of the harsh environment.

At this point we (i.e., Ayelet) realized that the office next to the robotic room will be very small, and to make it less claustrophobic we asked the builders to open a window into the robotic room. This changed the perspective of the room quit a bit.

All the while the lab was teeming with activity, with many new faces such as Jenia, Eli, and Alon who came for the summer.

The main work was done and now we got into finishing the systems. The electricity board for the new section arrived, followed by the control board for the A/C. Each was tended to by a team of technician for more than a day.

The main issue at this point with the lab was the set up of the robotic room.

We worried allot about different options, and at some point decided to setup a mock-up to check whether the diagrams match reallity. We took a tape and marked the different parts on the floor.

This led to arguments, rethinking, and eventually we settled on a plan, and ordered the furniture.

Few days later we learned that the UPS has finally arrived. We found several large cabinets, and a stack of batteries that was almost as tall as Naomi.

Construction of the Second Half has Begun

2010-07-01T14:46:00.008+03:00

This week the construction of the second half of the lab has begun.
Because there is a lot of noise and dust, Nir has escaped and left us alone in the lab (and in the dark). Surprisingly, though he is not here, the construction has begun successfully.

This new part will contain three rooms: the office, the robot room and the small kitchen. For now, the construction team has torn down the walls and laid infrastructure for constructing two drywalls that will separate between the new rooms. In addition, they made two halls for new doors that will enable entering and exiting each room without the need to go through the other rooms.

Here are some pictures so that the loyal readers of the blog (including one specific reader, that usually writes it) will know how it looks

The office

The robot room + the kitchen

The door to the robot room

The door to the kitchen

Renovation Halftime Celebrations

2010-06-21T22:55:00.001+03:00

Today we celebrated the opening of the first half of the renovated lab.

The day started by a trip to the Market to buy cheeses, bread, fruits and vegetables. We started by buying breads at Teler Bakery, which is considered the best in Jerusalem, and one of the best I know.

We then moved to Basher's Fromagerie. This is best Fromagerie I know outside France, maybe competing with The Cheeseboard in Berkeley. Eli Basher, the owner, is a very welcoming man, who puts on a show for recommending cheeses, cutting samples with the big knife and giving customers a taste.

We asked Eli for his advice, and he sent one of his workers to check prices for wood-boards to serve the cheese on. We then went through a long (and tasty!) project of tasting and choosing cheeses. Eli recommended serving them with Kava and fruits. So, we bought some Kava bottles from him, and then he packed the cheese.

My respect for Eli's professionalism, which was already high, reached new heights today. His estimate of what is needed was right on target, the cheese and the wine was sufficient with a bit left over, but not excessively so.

After buying fruits and serving dishes we returned to the lab. With the help of Drorit we arranged the place for a reception.

At one o'clock the place was empty, and I was getting worried that we will have to eat all the cheese ourselves. But in few minutes the room was packed. We raised a toast, and then cut the improvised ribbon that Avital and Ayelet prepared (picture credit to Zohar Markovich).

The room was packed with guests and seemed like everyone had a good time.

I gave a tour of the lab to the visitors from Computer Science (I seem to be a tour guide quite often lately).

After the party, we stayed to enjoy the aftereffect and the remains of the cheese and wine. Some of us were in elated mood. Avital claimed a desk and made sure it was her's.

Not to waste any time, we started the move. The first item to move was the Singer RoToR. Avital and I secured it, as per instructions, and then with the help of few neighbors we moved it to its new temporary home.

An hour later we called on the same neighbors to help move the cabinets. To get a sense of the action, see the follow stop-animation taken during the move:

By the end of the afternoon we were very tired, but managed to move quite a bit into the lab, and now it starting to look like a real lab.

New (old) person in the lab

2010-06-21T00:32:00.000+03:00

Congratulations to Assaf! He submitted his M.Sc., got a final grade, and his latest paper with Ollie was sent to the journal.

Now Assaf embarks on a new career and set out to tackle some experimental stuff in the lab. Today he got his official induction into the lab stuff. He donned gloves and cherry-picked ~380 strains from the knockout library for his first experiment. This means picking colonies from the original library plates to a fresh plate with media. The challenge is to maintain concentration as each colony must be identified by row/column address and then copied to the right target position.

Avital kept a close eye on him to help ensure that no mistakes creep in during this crucial step.

Acceptance Tests

2010-06-17T00:20:00.000+03:00

Today we had a semi-celebratory event. We performed acceptance tests for the lab. This involved all the contractors, planners, and various university representatives that showed up to examine the work and make a list of accept/reject.

Early in the morning various crews showed up for last minute touches. The electricity folks were in a good mood while testing each circuit breaker.

In the meanwhile the construction workers continued to work on the small Yeast Mating room.

At 10am people started to show up at the lab.

Some of the checks were very thorough and involved detailed examination of every pipe and faucet.

We tested the A/C heating function and nearly suffocated from the heat.

At times the place seemed like a cocktail party.

One of the tests involved the emergency circuit breaker.

There were arguments as to the language on the breaker. And, as expected by some, it did not work the first time around.

Some of the rejects we had

* We didn't have a phone line
* The hot water boiler was leaking and not tightly connected to the beam (!)
* The fire alarm detector system was not properly connected and so couldn't be tested
* The electricity ports below the computer benches are too bulky and could cause knee damage

Most of these will be fixed by Sunday. And so we are about to move into the lab next week!

Clean up

2010-06-15T23:21:00.000+03:00

At the finish line of work to the "release" of the molecular lab, the cleaning crew came in today. They removed all the wraps over the tables and other accessories (thereby taking some of the fun of "new" furniture). Then they cleaned everything to a polish.

The place looks brand new :-)

In other renovation news, the work on the small Singer room is progressing at a fast rate.

We now have a closet for the electricity board of the room.

The plaster wall is standing and mostly finished.

New windows are installed

And later in the afternoon the working crew started with the floor tiles. This meant a lot of tile cutting in the corridor.

Catching them midway of the work shows the room taking shape.

Touch ups

2010-06-14T22:53:00.000+03:00

We are now into the last touch ups in the new molecular biology room. Now that there is proper light, various small defects are noticeable and the workers are fixing them.

One of the funny fixes is dealing with the ceiling. When the ceiling was installed there were noticeable deformation in the tiles.

To fix these, the workers brought small aluminum pieces that they installed at the end of each tile.

This fixed the problem, and now we have nice and even lines.

Singer RoToR Fixup

2010-06-14T22:10:00.001+03:00

You may remember the Singer RoToR, our colony replicator. This is a great device, but recently it has been erratic in lifting on of the plate lids.

We contact the nice people at Singer who sent us instruction on how to tune the sensor on the arm. This is very similar to an issue we had in the past with device after it was filled with agar. Tuning the sensor solved the problem but it would reappear.

Last week we got an email from Ian at Singer that he believes the problem is with a faulty relay in the electronic board. It turns out that the device (which is very thoughtfully designed) comes with a spare relay. Ian sent us detailed instructions on how to fix this. The only problem was that it required opening the back panel --- meaning that we need to move the device.

We opened the instructions for securing the arm and plates. Fixed everything in place, again the design was such that given the instructions it was a piece of cake. We then moved the whole device onto a table.

Removed the back panel,

and switched the old relay with the spare one.

In 10 minutes we had the device back on the original table and working.

I can't say enough how impressed I am with the design and operation of the RoToR. It looks as though every aspect was thought of in advance, including how to best guide remote operators in dealing with issues and fixing problems. I'd wish some of the other devices we bought were that well thought out.

Ian also noted that they came up with a new board that does not requires these relays which can fail. So sometime soon we will do an upgrade to the new board. Given the ease of maintenance of the device, I am looking forward to that.

Renovations updates - "Yeast Mating Room"

2010-06-14T19:12:00.000+03:00

As we wait for the final touches on the main molecular lab room, the workers started working on a small side room.

This room will serve as the "Yeast Mating" room. It will have a place for the Singer RoToR, the MasterClave and agar preparation station, an incubator, and supplies for all of these. The idea is that the room will be a more or less autonomic workstation for researchers interesting building and manipulating yeast libraries.

Since this is a small room, we hope the work will progress quickly. The workers tore down the existing interior. Then they rebuilt the foundations of the wall with the neighboring office (to support shelves and to allow for insulation).

The following day the electricians came in and put the tubes for placing the electricity wires.

Today, the plaster wall was finished, and tomorrow we should have tiles in place.

Let there be light!

2010-06-08T20:11:00.000+03:00

After long delays, today the new lab was connected to the electricity system. The electricity crew came in at 6am and was supposed to be done by 10am.

At 3pm when Ilan and me came in, we found them still working on the main board.

Turned out that the 30+ year switches failed and had to be repaired. This meant that the internet connection for the whole wing was disconnected throughout the day and that our lab and neighboring lab had limited electricity.

Nonetheless, and by that time against all expectations, the crew managed to fix the board, and we got to see lights going on in the new lab!

Ilan

2010-06-08T20:06:00.000+03:00

Ilan Wapinski, a long time collaborator, is visiting Israel for Naomi's wedding. Today we spent several hours together. First we had technical chat with Naomi.

Then we went for lunch at one of the nicest places in town for food - MachneYuda restaurant. This was a combination of cool discussion and tasty food. We sat on the "chef bar" and so also had the whole kitchen staff as entertainment.

Naomi's Wedding

2010-06-04T11:30:00.000+03:00

Last night we (all the group) attended Naomi's wedding. It took place in a meadow in a wooded parks on the hills south-west of Jerusalem. We arrived there toward sunset and had a chance to see the forest view and the rolling hills, and then the sunset. The ceremony was at twilight time with the first stars coming out.

It was a happy, energetic and and fun event. We mostly concentrated on the dance "floor" which was full of people, young and older, with an eclectic mix of dance music.

Short renovations update

2010-06-03T17:31:00.000+03:00

Nothing much happened on site this week. But still some updates.

* Next week, we hope, the electricity board will be connected and maybe the new molecular lab would be declared "done".

* Next Sunday work on the small equipment room, where the Singer and the MasterClave will reside, starts. Due to the delays in the renovations on the robotic room, the contractor promised to make fast progress here.

* The one piece of missing furniture in the molecular lab - storage cabinets for consumables - has been finally designed and call for proposals is out. Hopefully we will have it done in two-three weeks.

* We are starting advanced stages of the furniture and electricity design for the robotic room.

Our first GFP-RFP strain

2010-06-02T18:01:00.000+03:00

On aspect of the lab that I haven't wrote about much is our Scan^R microscope. The problem is that I planned to explain it with a lot of details, which meant that this post was being postponed everytime.

In the mean time, however, we have been using it quite a lot. For our purposes, we need strains that contain the right fluorescent markers. We relay on the use of proteins, such as the Green Fluorescent Proteins (GFP) that are inherently fluorescent. This means that when the protein is expressed (made by the cells) we can measure it.

To setup experiments we need to make sure the cells express these proteins in response to the conditions we want to test. As our first attempt in that direction we combined strains expressing GFP attached to different native proteins (one per strain) with a strain that Avital built that expresses a protein called mCherry (a variant of Red Fluorescent Protein or RFP) in a constitutive way. This means that all cells are marked in red, which helps focusing on them and also defining the area of the cell.

We had some set backs with getting the mCherry strain to work, but after some detective work by Avital, she overcame the technical issues. Last week Avital managed to get her mCherry strains to mate with the GFP strains. And next week we are supposed to get the final product of haploid cells with the two markers. In the mean time she took images of the diploid cells, and they look good.

In red:

In Green:

And an image made by overlaying these two images:

You can see that (almost) all cells are red (with varying degrees). Some cells have green in the nucleus (sharp points in the middle of the cells), suggesting that this protein is nuclear one and has variable level of expression under the conditions we measured.

Excitment and Disappointments

2010-05-26T23:42:00.000+03:00

This morning we met with Eyal, the renovations supervisor. We learned that tomorrow we the electricity board will be connected, and that following that it will a take a week to test all the systems, repair minor issues, and then declare the lab ready for work. We already started to setup the steps needed to move all the equipment to the new lab, and set a date for end of the move.

Later in the morning, we learned that some of our neighboring labs were upset due to the planned disruption in electricity (to connect our board the next level of electricity boards has to be turned off). Turns out that a two-week long experiment will be ruined if the electricity will be cut. We learned to our surprise that although the move was coordinated it was done with a two day warning and that the affected labs didn't hear about it until one day ahead.

Consequently, the planned connection was delayed. The electricity contractor was booked in advanced, and so the next date that he could do it is in two weeks (June 8th). And so, due to poor coordination between the relevant parties, the project is delayed by a bit more than two weeks.

Electric Board

2010-05-24T23:32:00.008+03:00

The last missing item to finish the first half of the renovations is the electricity board. After we waited for it for two weeks it arrived Sunday at noon.

Before its arrival, the electricity people setup the main cord that will feed the board.

Once it arrived, it was put into place and secured.

Now the hard work of wiring started. For two days they worked to move the wires in to the board and connect them.

The thick bunch of green electricity wires that lead to that corner of the room was slowly organized.

In the end all of them were tacked in and secured inside.

As a minor bonus we got an impressive sunset on Sunday evening.

Hard decisions: Lighting fixtures

2010-05-09T23:07:00.000+03:00

Today the electricity people showed to install the lighting fixtures in the lab. I was urgently called to make a decision where I want them to be installed. The issue is that the original plan ignored the fact that there is going to be a storage cabinet along the inner wall, and so had the lights overlapping with the cabinet.

I showed up to find 5 workers idling waiting for the decision to be made. The new furniture was covered with plastic wraps to protect for the reminder of the work. After some discussions we decided it is best to see how things look. Very quickly two ceiling pieces were cut

and then had the fixtures installed in them.

We looked at the ceiling with the two offset fixtures for a while

Finally, we decided that the one closer to the central beam is better (you have to imagine a cabinet below the ceiling on the left).

The next question was how to position the fixtures on the other side of the beam. Whether to go for a symmetric setting or mirror the offset of the other half.

After another test, I decided to go with the straight-down-the-middle approach for this side. You can see in the picture the red fire safety lights and the fresh air inlets.

Benches!

2010-05-06T23:26:00.000+03:00

I have been writing a lot about the renovations in the lab. In fact, one reader commented that he thought all we do right now is watch the workers go about building the lab. Although this is not precise, and there is a lot of experimental activity, the renovations are something that takes attention and also makes easily observable progress.

Since I last wrote about the renovations, many things have happened.

The electricity people finished laying down the fixtures all along the walls. We had our own hot water system installed.

A carpenter came with his crew and installed covers over the A/C units

and a new door.

In parallel, another worker installed a state of the art fire alarm system.

On Tuesday a cleaning crew came in and removed all the waste and the dust.

It is instructive to compare to an image I took from a similar angle at the beginning of the work

And finally, on Wednesday the lab furniture crew arrived. I got an excited text message from Ayelet that they are here and came over. The the corridors outside the lab were full of hardware and cabinets.

In a very intense effort by a large team they started assembling the benches, the hood, and the sink.

Few hours later the bench tops were being secured in place.

This morning we came over and the shelves were being installed.

We were curious whether the desks were specious enough. So we took a computer and a chair and tested the desk.

Ayelet decreed that this was a success. Later toward mid-day, the workers finished with the furniture and moved to connect the electricity cables.

Now we are almost done with the first phase of renovations. The missing parts are the light fixtures, and the main electricity board.

A Moment of Naches

2010-05-06T22:52:00.000+03:00

"Naches" is Yiddish for parental joy & pride (נחת in Hebrew).

Yoseph Barash, a graduate of my lab who is now a postdoc in Toronto (with Brendan Frey and Ben Benclowe) is the lead author of article in Nature. Moreover, his work made big splash with as the main item on the cover.

(From Nature)

If you want to get the layman's explanation, Nature provides a nice coverage. It seems that this result will get a lot of attention both in the media and in science. For example, see report in the Science Daily.

Cinco de Mayo

2010-05-05T22:11:00.000+03:00

Most people here did not hear about Cinco de Mayo, but still we had a celebration - today was the Faculty Day of the Faculty of Mathematics and Natural Sciences.

As part of the tradition of the event students show off their research in a poster competition. For some reason every year this event is in weather that is slightly too hot. The faculty set up a big tent to shield from the sun. This is nice, but as the tent is blue, everyone inside has an Avatar-like tint. Although I came in a bit late due to a meeting at the School of Medicine, the place we full of people, faculty and students. It was nice to get a chance to see what other research groups are doing.

As is our own private tradition, my group was well represented. In fact, close to half of the CS posers were ours.

(Ruty explains her most recent work to Shai Shalev-Swartz)

We had a fun visitor - Inbar passed by to show off the latest toy and encourage the poster presenter.

For some reason Tal finished his poster rather late into the event. To keep people on the edge, his poster was constructed in stage, with interesting delays to enhance the curiosity.

Another tradition we have is winning one of the poster prizes. Tal's tactics did not win the judges' attention. Instead, using good science and nice explanations, Noa brought in the prize (2nd overall in the all faculty). Horay Noa!

(A potential judge examining Noa's poster)

50 Years' Celebration

2010-05-04T21:27:00.001+03:00

No, I am not fifty, yet. This year is the 50th year jubilee of the Israeli Academy of Sciences and Humanities. As part of the celebration they held this week a conference in "Frontiers in Biomedical Research". The list of distinguished speakers included Ada Yonath, Mary-Claire King, and many others.

I had to rush from one thing to the next this week, but managed to get to hear (the end of) Ada Yonth's talk, and these by Matthias Mann, Leroy Hood, and Eric Lander.

Eric gave another talk on Weizmann on Tuesday and I encouraged all my students (and my mother) to go hear him, as he one of the better promoters of the biological revolution in genetics and genomics. In both venues the hall was too small for the audience. At the Weizmann people were sitting in the stairways, below the stage, and some on the stage. Definitely and impressive turn out.

HyperCyt on the run

2010-04-25T18:33:00.004+03:00

Last week I reported on the Flow Cytometer and its new upgrades. These allow to start testing the combination of the HyperCyt loader with the Flow Cytometer. We finally have some preliminary results, but they are exciting.

Recall that the Flow Cytometer "sucks" cells from liquid sample and pass them through a flow chamber where they hit a laser beam which allow to measure both fluorescent signal and the size of the cell. The HyperCyt loader is a machine that allow to collect samples from a multi-well plate and send them to the FlowCytometer. You can see a movie in the original blog entry. Thus, the FlowCytometer receives a sample that consists of 1.5 seconds of a "sip" into a well, followed from a 1 second sip from air. At the end of a 12-well row, there is another step of sipping water to clean the pipes.

Today Ariel (our Ariel, not to be confused with Ariel from Merkel who did the Flow Cytometer upgrade), performed a test to see the whole thing in action. Now that we have upgraded the Flow Cytometer acquisition hardware/software we can start examining the results. To see how good they are, we plot the time a cell was collected vs. the measured value. In situations where there were no cells we will not see points.

When Ariel ploted the time vs. Forward Scatter (a measure of the extent to which the laser beam was scattered by the cell, which is a proxy of cell size), we see a band for each row of wells.

Zooming in to look at a particular band, we can see that it consists of intervals that correspond to a sample separated by blank intervals (air bubbles).

We can observe several things here.

First although each "sip" is of 1.5 second, some of them are spread out on longer measurement time. This is probably due to the fact that the bubbles are spread out by the transition in the tube, and the first bubble might have more space to spread out.

Second, although air bubbles are easily detectable by the lack of any events (cells), the empty wells Ariel added (marked by Medium and water) do contain some cells. These are probably carry over cells that got temporarily stuck on the tubing and then released by the flow. We can see that by after the wash by one well, the subsequent water well has much fewer events. This will give us a sense of how much carry over we can expect.

If we look at the GFP measurement for these cells we see similar bands. Here Ariel used strains of different GFP intensities, and we can see from the plot that they differ.

For example, the "no-gfp" strain shows very low level of GFP (basically auto-florescence), while some of the other strains (e.g., Sod1, Rpl25A) show very high levels.

This is a very first run out of many we are going to perform. Clearly there are issues to deal with, such as how to automatically parse the output into wells, how to reduce the amount of carryover, how reproducible are the results and so on. However, I am very excited to start seeing all these different things start to work together.

Weekend Diving Diversion

2010-04-25T16:09:00.000+03:00

After what was a very hectic and busy week, I managed to get a break and go for a two day weekend in Eilat. Didn't manage the usual amount of dives, but the ones we did were very nice. Few photos to give a taste.

Ceiling, Pipes, Electricity and -80C

2010-04-23T00:52:00.000+03:00

The work on the renovation is moving forward in a quick pace. The last few days three teams worked in parallel.

The first put in the ceiling

At this stage the ceiling does not have any features. Later on the electricians will cut in holes for lights, and the A/C people will add vents.

The AC people started preparing the room for the fancoils that will provide hot and cold air from the university hot/cold water system. They cut and welded the pipes and then installed.

Finally, the electricity people continued working on setting up the electricity panels along the walls.

Most of the work on the room will be finished this week.

The furniture is scheduled to be installed in two weeks. The only remaining item is setting up the electricity board. It turns out that the configuration of the board needs to be approved, and only after that it will be ordered. The plans were submitted to approval today and they expect the electricity board finished and wired up in about 5 weeks. This means that the industrious work on the renovation will stand still for three-four weeks. This means that right now we expect to move into the first stage lab only in mid June (wow...)

On another front, we received today our -80C freezer.

This huge beast barely fit in the space we designated for it, and we will have to shorten the table next to it, so that we will have enough space to open the door.

The Naked FlowCytometer

2010-04-19T23:31:00.001+03:00

The last two days Ariel from Merkel Technologies Ltd installed an upgrade to the logic boards of our Flow Cytometer (FACS). The new logic boards, made by Cytek, will allow us to collect data using a newer acquisition station, which will solve compatibility issues with the HyperCyt loader. They also are the basis for the next upgrade of the lasers and the sensors (that will come in few weeks, depending when we are ready).

The upgrade involved replacing all types of boards inside the FACS, and for a whole day the lab looked like an electronic workshop rather than a biology lab.

In the process, Ariel also uncovered the lid off the laser part of the unit, which gave us a chance to peak into the working of the machine. The main lightpath

involves a laser on the left. The laser beam passes through two prisms and then onto the flow chamber on the right, and then from there to sensors (The cylinders beyond).

The flow chamber, has a two input the first is a sheath fluid and the other is the sample. The sheath fluid is flowing much faster and thus creates a thin stream of cells form the sample.

(source: http://www.abcam.com )

In our configuration we have the same setup but the flow is from below:

At the end of the day the device was closed up, assembled, and working.

Tonight was the beginning of the independence day celebrations and so we got to see some fireworks.

Window sills, cables, and agar plates

2010-04-13T22:11:00.000+03:00

News from today.

Our alumnium sub-constructor came in yesterday and installed the two missing windows, and also installed a new nice window sill below them.

The electricity sub-contractor came in last week and finished laying down all the electricity cables. We now have thick green cables running on wireframe shelves along the ceiling and then entering the large panel of tubes to make their way down to the level of the furniture.

In addition they left cables hanging from the ceiling, these will lead into the light bulbs.

The same guys also installed airvents to take airs from the building "fresh air" system into the room (and filter it on the way).

The building people closed off all the tubes and already painted most of them. They also were building a drywall panel to form a wall above the windows.

In addition, you might remember Drorit, who is helping us with chores. Today Ayelet start teaching her to prepare agar plate. She proved to be a very quick learner, so soon we will have help on day to day lab chores.

And as a nice bonus, we got to see a colorful sunset today.

From the bench: yeast mating

2010-04-12T20:59:00.002+03:00

One of the key technological steps for our work in the lab is "constructing" yeast strains that carry combination of genetic perturbations. In our first try to perform such steps in a high-throughput manner, Avital had very nice results.

Some background first. An important approach to study organism is to modify some genes and see the effect, and from that deduce something about the function of the gene. One of the reasons yeast is such a great model organism for research is that performing genetic modifications --- replacing one piece of DNA with another --- is relatively easy.

Nonetheless, creating many combinations of such modifications one by one would be tedious. Here people in the yeast biology invented clever tricks to do so easily. The idea is simple and is based on simple Mendelian genetics. In nutshell, suppose we have one yeast strain with modification X and one with modification Y. If we can "marry" them and have them generate offsprings, than 1/4 of them would inherit both the X and the Y modification. If we somehow could select these specific offsprings we are managed to combine the two modifications.

This description is oversimplified in several ways. It assumes that X and Y are "unlinked" in genetics terms, that is, the inheritance of X does not influence the inheritance of Y. In practical terms this means that X and Y are either on separate chromosomes, or sufficiently far to allow for recombination (in which case the fraction of offsprings with both X and Y gets smaller as X and Y are closer).

Secondly, we do not marry yeasts. Instead, we use the fact that the yeast lives in two forms. A haploid yeast has one copy of each chromosome, and a diploid yeast has two copies. (In case you wonder, all of our cells are diploid, except for cells involved in reproduction.) The yeast prefers to live as a diploid, and thus two haploid yeasts with different mating type (the yeast version of gender) will fuse together to form a diploid cell. However, when the times get tough, a diploid yeast will undergo meiosis and divide into four haploid spores, which are very resistant and will survive until things get better.

The yeast geneticists use this behavior to create combinations of perturbation. Basically the idea is to go through the stages shown here

This method uses antibiotic markers (NAT and KAN here) to select only the spores that have inherited both mutations (shown as filled ovals here). The idea, initially pushed by Charlie Boone from Toronto, is that this process can be carried in parallel for many yeast strains. We do so by growing yeast on agar plates, each with the right combination of nutrients and antibiotic to select the cells that we want from the previous stage in the above diagram. All these steps relay on our Singer RoToR robot for copying colonies from one plate to the next.

The weeks before Passover, Avital run our first mating protocol. She created a yeast with a knockout (KO) of the Gal1 gene, and then mated that yeast with plate of 96 strains from the Yeast Knock-Out library that is supposed to contain all viable knockout strains.

The original knockout plate she choose was one that contains Gal1 KO as well as KO of few genes that are adjacent to Gal1 on yeast Chromosome 2. The original plate we got didn't have all the strains, which is often the case as KOs are very sick and do not propogate.

Avital mated these with the Gal1 KO she created, and at the same time copied the original 96-colony format to 384-colonies, so that each mating experiment was propagated four independent times. The diploid plate looks like the original plate, except each colony was replaced by four smaller colonies.

She then copied the cells onto a plate with "sporulation media" - poor media that drives diploid yeast to sporulate. Since the media is poor, the colonies do not grow much from the original amount deposited by the pinning.

Once the colonies are copied to fresh rich media they grow again, but now only ones that carry the original KO (as we selected for it).

Once Avital moved cells to a plate that also selects for the second KO, some colonies disappeared (circled in red).

You can see a bit of the original copied cells that did not manage to grow. The quartet where all colonies are missing (red arrow) are exactly the Gal1 KO --- since both KOs are in the same genomic location we can create a strains that contain both of them. In nearby regions we get few successes (unlikely event) but not consistently. Elsewhere, we get the double KO colonies.

You might notice that we also lost colonies in the corner (e.g., lower left corner) but this is probably due to uneven agar levels --- something we need to fine tune.

Khamsin

2010-04-11T20:50:00.002+03:00

Today we had one of the special weather phenomena that leave you dumbfounded. Originally the name Khamsin comes from Egypt and describes a wind from the Sahara -- hot and full of sand. In our case it is more often a wind from the desert east of us that carry fine sand in the air.

The visibility today was almost as though there was a fog, a fierce wind was blowing and the temperature climbed almost 15C above what it was the previous day.

Driving back home in the afternoon, one could barely see the view few hundred meters ahead.

(Don't worry, I don't take pictures while driving, I had Inbar as a passanger and she gets the photo credits).

Coming home from work, I could feel the fine layer of dust on my laptop keyboard, which was standing in my office today.

Science wise, this type of weather has immediate implications. The dust carries micro-organisms that can pollute samples, it blocks air filters, and gets in the way of fine mechanisms.

Spring trip

2010-04-08T20:18:00.005+03:00

Today we have a field trip to celebrate the spring. Actually the trip was organized by Hanah Margalit's group, but they suggested that group lab will join them, due to the relative closeness of our interest and the fact that there are two students that are jointly supervised by Hanah and me.

It was a nice day, not too hot and not cold, so we got to enjoy the end of the spring in the mountains southwest of Jerusalem. The partly clouded sky gave us interesting views.

We used the opertunity to take a group photo (although two people are missing).

And managed to see flowers, insects, birds. We even got a chance to perform some comparative studies

All in all, a very nice day. At the end of it some drove home for the weekend, and few got back to the office and lab to actually get some work done :-)

A wet surprise

2010-04-07T22:25:00.001+03:00

Seems like our building does not like holidays. If you recall, during Purim there was a big water leakage in the peer next to our lab. This passover as I came to visit the lab the security people asked me to watch out of the puddles. What? turns out there is a overflow in one of the bathrooms and it leaked through several levels of the building to the base level through which I came in.

The bathroom at our floor didn't look that inviting.

This morning the place was cleaned up without any signs of the catastrophe....

Visitor in the Lab

2010-04-06T21:30:00.002+03:00

Today was the annual Israeli Bioinformatics Symposium, held by the Israeli Society for Bioinformatics and Computational Biology of which I happen to be the President. Our keynote speaker this year was Trey Ideker, and I hosted him on Monday for a sightseeing tour.

My student Moran joined us a a tour guide of the Old City of Jerusalem, which she knews quite well, and left me to document the visit. Few pictures to get your appetite for a visit here.

The Holy Sepulchre Church Jerusalem

Tasting some authentic humus at Lina

Searching for gifts

From there Trey and I continued to the city of Caeseria, an old port city that was built (and destroyed and rebuilt) from the Roman time until the 14th century and now is an archaeological park.