HyperCyt on the run

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

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

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

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

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

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

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

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

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

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.

Hot Water? Story of a red pipe

As the work progresses various surprises pop up. As I reported before, we had the water pipes installed all along the lab.

A very prominent pipe is the thick red one. This is the hot water pipe that runs along our lab from the peer on the one end of the lab and into the next one. It is thick because it is covered by insulation sleeves.

In the middle of the room there is a branch point where a branch goes down below the floor and out the other side of the room to connect to where we will have a sink.

Last week there was a site visit by the maintenance people. They pointed out that although the building has a hot water system (i.e., a dedicated set of pipes through all the labs), there is no supply of hot water, and in fact it will probably will never be supplied (I didn't get into the details of why).

So it turns out that this nice pipe is for naught, and we need to install electric water heating system in this room (and the other ones) for local supply of water.