Back from vacation

The last week I went on vacation with the extended family to Samoens, France part of the Grand Massif ski area. We enjoyed the slopes,

the wine

and also the fresh snow...

In the meantime the lab was busy. Ayelet, Avital, Ruty and Ohad worked and managed experiments with the Microscope and the FACS (more on these during the week).

We also had some new arrivals, the most impressive one is the MasterClave - a device for preparing and pouring agar media.

Quick update(s)

So there haven't been much new recently, but some updates.

If you remember, last time we discussed agar plates, we did a test with a bunch of plates where we UV exposed half, and used the others straight from the box. It seems that all of them did not have contamination. This good, as we have plates we can use, but leaves the question of contamination wide open. Currently, the theory is that there might be days where the air in the lab is cleaner? or maybe certain batches of plates are better sterilized. In any event, to be on the safe side, we had the A/C people come in and clean the air-conditioner filters (they were supposed to do it before we moved in, but apparently didn't).

Another problem we had was plate drying. We switched to using the 30C hot-room (two floors up), and we do not have any dryness problems. We are trying to check with the incubator supplier if there is something we can fix in the incubator, or we will replace it by a less aggressive one.

On other fronts, we had a microscope training on Tuesday. I will expand on how the microscope is built and how it works in a future entry, as we need more rounds of photography for that. I am going on a one week vacation and so will report on progress when I return.

Double agar troubles

Although most of the recent posts where about receiving new equipment and plans to buy additional ones, we also have to push the science forward. One of the things that worries the last few days is getting reliable agar plates for growing yeast strains.

The troubles come in two different areas. I wrote before that we have contamination in plates. Well, we still have contamination in the plates we pour. Moreover, even when we do not have contamination, the plates start drying when we incubate them. For example, here the agar dried and then cracked.

In another plate, it didn't crack, but we see it drying toward one corner (the agar is thinner).

We know that the square plates are prone to water loss (much larger perimeter than round plates). For this reason we seal the plates either in plastic wrap (aka Saran wrap) or inside plastic bags. The two plates above were in the incubator for two days, one with plastic wrap and the other inside a plastic bag and both show bad signs of dryness.

We believe that part of the problem is the aggressive air circulation in our incubator. We called the company and they claimed that there is no way to turn down the fan speed. We now need to consider how to address this. One possible solution is to humidify the incubator by putting a container with water. If this will help, we might consider a more intelligent way of ensuring that it is humid.

The problem of contamination is a serious one. Since we want to grow libraries that will serve as resource for further examinations, having contaminants grow on the same plates is an issue. Since we first noticed the problem we talked with other yeast people in the building and they report similar problems.  All of them recommended shutting down the A/C circulation before pouring plates. Apparently the air supply introduces yeast and bacteria from outside the building into the lab.

One of the debates in yeast sterile techniques is whether to use flame. On the one hand, working with a flame you sterilize the opening of the bottle before you close it, reducing chances of contamination. On the other hand, an open flame heats air, which in turn goes up, leading to a circulation of air toward the area you are working in.

To test the effectiveness of different techniques, Ayelet tried a test where she compared pouring inside a biological hood, without flame sterilization and with flame sterilization. The results are inconclusive (small numbers) , but it seems that flame sterilization is a bit worse. It also seems that with A/C off the total amount of contamination was down. Surprisingly, even working inside a hood we got contaminants. This experience might be due to the fact that while waiting for the hood the agar cooled a bit (although it was in a sealed autoclaved flask).

To make sure that the source of contamination is the air and not the plates (that are supposed to be sterile), we tried today another round. This time we exposed half of the plates to U/V (using the Singer RoToR as a UV "oven") and the other half without exposure. 

We need to wait another day to see how this batch fared. In the mean time, the number of plates we throw is alarming, and so we hope to solve these issues soon.

Robotic Platform - A choice is made

Our aim in the long run is to establish a platform to "phenotype" (that is measure behavior of) many promoters in different genetic perturbations. The plan is to use the microscope and the FACS to measure the activity of these promoters in living yeast cells using what is called a fluorescent reporter. (I will expand on this point in a future post.)

To establish such a platform we need to be able to continuously feed the microscope with new samples so that we maximize the throughput in terms of the number of measured phenotypes. We are aiming at numbers in the 10,000s, which means that even when we work with 384well plates, we still need many runs.

An important ingredient in achieving this goal is a robotic platform that will allow us to prepare samples for the microscope and then move them onto it. The goal is that the robot will integrate several devices:

• Incubator
• Plate reader for spectrophotometer reading of cell density
• Microscope
• FACS loader

In addition the robot should be able to do basic manipulations such as moving liquid from one plate to another, add media, to a plate, and so on. Doing this requires that the robot also have what is called liquid handling capabilities. In other words, that the robot should be able to perform pipette operations: from one well to another, or from multiple wells in parallel. It also means that we the robot needs places to store pipette tips, plates, and media.

During the last year we spent a lot of effort in trying to define our needs and to understand the options available for devices that integrate all of these capabilities. We had long interactions with three companies, Eisenberg Brothers who represents Perkin Elmer, Neotec who represents Tecan, and Agilent, where we interacted directly with their European office.

The process was interesting as we learned a lot about the capabilities of different devices, different ways of using them, and ways of integrating them. This included site visits to installations in Israel, Germany, and the US. The main issue with the choice of robotic platform how to integrate the multitude of devices in a way that will allow seamless operations.

I will not go through the gory details. Agilent has dropped out of the discussion sometime in September as they felt they cannot meet the target price we aimed at. Neotec/Tecan and Eisenberg Brothers/Perkin Elmer stayed in the running and we had long correspondence with people from both. We got to financial negotiation stages with both and finally, after much delays we decided to go with Neotec/Tecan solution.

In the next few weeks I will provide outline of the robot and how we plan to use it.

Renovations update

A short update on renovations. Or more precisely plans for renovations. I met today with Itsik and Eyal to go over the plans. We discussed the types of floor, windows, ceiling, and other such matters.

It seems that here the plan is more less what we want and there isn't much to change or cut costs. We can cut some cost by leaving the current doors intact.

The main point of the discussion was regarding electricity. Our current plan has space for two electricity boards (one for each room) of about 120cm width. There two issues with this. First, outside our lab mounted in the corridor is an electricity board of a cold room, that no one wants to touch. So we cannot touch that wall. Moreover, due to standards electricity boards we might need wider board. If we want to have the electricity main switch accessible from outside the lab this presents a problem.

We discussed several solutions and the pros/cons of each, and at the bottom line we are waiting for clarification from the electricity planner on the precise size needed and for Itsik to check the feasibility of two alternative. 

The good news is that there are three current proposals from contractors for this part, and so soon we will have a contractor that can start some of the works. In parallel the order for the furniture is going out, and so we should soon have detailed plans of the furniture for our approval prior to their manufacturing.

We did a quick tour of the neighboring lab where the first round of renovations will start.

 It is a bit desolate right now, but will turn into a new lab hopefully in no time.

Scan^R is here....

Today at 7:30 Ayelet got a call from a delivery person that he is waiting for her with a bunch of boxes.
By 9am when I arrived Dimitry from Eisenberg Brothers was busy setting up the Scan^R - our new automated microscope system. Here are the boxes that were still unopened at that stage:

The basis for the system is a regular (advanced) microscope body

with objectives (lenses) like the ones we had when we were kids (but of somewhat better quality)

The main heart of the system is the light source, sets of filter wheels for fluorescence imaging, high quality camera, and automated stage (that can move very precisely). Most of the components are "ours" but some (like the objectives above) are from demos until ours arrive.

When Dimitry started to assumble the light source we realized that we have a problem with the optic fiber cable that connects the light source and the microscope. We decided that instead of having it in front of the storage cabinets we need to rearrange the room

Finally, the system is built and actually works. We still need to get the computer monitors (two), and few other gadgets.

Thursday will be our first training session and then I will give more details about this amazing system.

Handiman at work

After installing the Singer RoToR we realized that we don't have tabletop area around it for putting plates while operating the robot. Since in the future we plan to copy dozens of plates in one go this is a problem.

Luckily the old benches we have in the temporary lab have extension surfaces. These right now are placed in an usable location. So, we decided that we can move a couple next to the RoToR.

Yesterday I bought a toolkit (screwdrivers, pliers, hammer, and the works) and a small rechargeable driver. I disassembled the shleves and wanted to install them in the new location. Unfortunately, the bench where the Singer is has different frame than the other benches and so the pieces I disassembled did not match.

Today I brought a saw from home, and cut the pieces to the correct length. After an hour of work we had two retractable shelves next to the RoToR.

(you also can see the new color printer next to the RoToR robot).

To celebrate the achievement, I took pictures of the final product with a funky camera app on the iPhone.

We are online

One of the handicaps for working in the lab was the limited network access here. After consultations with the Computer Science system experts we decided to set the lab computers as remote extension of the CS environment. This will allow us to store data and analyses  on the CS servers and to access it in the same manner from both wet and dry labs.


During the last few days equipment started to arrive. We now have two computers, a docking station for laptop, and a printer. Here is a picture of "iWet-01":

More important are the behind the scenes developments. These involve a new fiber optic cable from the life sciences building to the computer science building, combined with the appropriate switches these will make us feel as though we we are sitting in the CS building.

Singer followup

Just to demonstrate the results of yesterdays demo, here are two plates that we copied yesterday from the same 96-well liquid plate, once into 386 and once into a denser 1536 colonies format (by four iterations).

As you can see, the "holes", we left in the original plate appear in the same places in the replicated ones, except that in the 384 one it is a 2x2 colony hole and in the 1536 it is a 4x4 colony hole.

Another plate is one we copied from a "lawn" of yeast. That lawn dried up in the incubator (it is much drier there than we expected), and so the agar level was uneven. As a result we got stricking colony pattern.

The impressive part is the very uniform and nice colony patterns we see.

Singer RoToR - official liftoff

You might remember the big crate that we received a while ago. Today Ian from Singer Instruments (UK) showed up to unpack the box and install the Singer RoToR.

At this point we needed to lift the device onto the bench. As an example of good design, it had comfy handles attached to both sides, and so four of us relatively easily lifted the 120kg device up and onto the bench.


Here is a zoom on the handle.

Notice the silver globe. This is one of the important features of the device -- an integrated beer bottle opener. In recent years they added an option also to get instead a corkscrew. We decided to go with the traditional choice :-)

So what exactly is this device all about. The job of the Singer RoToR is to copy multiple colonies between plates. Either agar plates or multi-well liquid plates.

As you can see it has a movable arm (parked on the right here), locations to put plates (color coded, black, blue, red, yellow, and green), and two drawers. On the drawer on the right it can pick up sterile "pads" which are essentially plastic pincushions. It then uses the pins to copy cells from one plate to another (or from one to many, or vice versa). It disposes of the pad in the left into a waste drawer.

While this sounds simple enough, the beauty of the machine is that it is very precise and can copy very dense plates (upto 3074 colonies on a plate) in a precise manner. In addition all the work is done in a sterile environment. When the protective wall is in place, all the operations are done in a space that is U/V sterilized. The device opens the lid, does the copying, and then closes them.

The best way to see this, is to watch a movie. In this movie we asked the robot to copy from 96 wells onto an agar plate in 384 colonies format with four repetitions of each original well.

 

The result of this process is a dense array of colonies on the agar. In fact, we can see them if we look carefully. Here Drora (a local yeast expert from another lab) shows off the resulting plate.

Looking at an electric future

As part of the ongoing efforts to push the renovations, we need to go over the plans and make sure they fit and do not contain unnecessary elements.

Today we met Itzik and Eyal, who are in charge of the renovations, to go over the electricity plans from the electricity consultant.

We spent an hour and half going over detailed plans, marking missing outlets and removing superfluous ones. We managed to reduce the 200+ sockets (!!) to a more reasonable number. Hopefully this will translate into some savings in the cost of the electricity works.

Spectrophotomer correction

An alert reader pointed to am error in a previous entry. Although absorbance is one source of optical density, the situation in our case is different. To quote

A solution absorbing at 600 nm has a blue color, the cells do not absorb light at this wavelength. You actually measure the light scattering caused by the cells. The scattered light is not detected by the spectrometer, this is the reason that you measure OD >0.

Thanks!

An additional note regarding the spectrophotometer is that we compared the readings on ours to three others in the institute. To our surprise we discovered that one of them has the same reading as ours, another is slightly off, and the third is way off (almost half the value we measure). We are not sure what to make of this, but it is a point of concern.

Renovations update

As you may recall we are trying to get renovations for a permanent lab. Here are few updates from Itzik who is in charge of the project.

• We have detailed plans for A/C, water, and for electricity. The A/C + water plan is within the budget outline. The electricity plan turned out very complex (in terms of number electrical boards, panels, circuits and such) and thus expensive. Itsik is working re-examine our specifications to see if we can make some concessions toward simplifying the details.
• The actual renovation (breaking walls, building walls, and such) plans have are waiting for bids from contractors. Tomorrow there is a planned site visit for interested contractors. This means that soon we will see how much they will cost.
• The furniture quote is in, and it is about the expected price.
• We are still trying to finalize the complete budget for the project and are in contact with the university management to sort out differences between the expected cost and the actual budget.

 I am trying to be optimistic that the process is continuing although so far no actual work has been carried out.

Spectrophotometer, OD, and yeast growth

As you recall one of our new toys is a spectrophotometer. How does it work? You can check the entry on Wikipedia, but idea is very simple. A light beam of a known wavelength is passed through a small cuvette (fancy name for a container) that holds a fixed volume of sample. On the other side it hits a detector that measures the amount of light that has passed through the sample.

The amount of light that has been absorbed is informative on the content of the sample. Certain wavelengths for example are absorbed by poly-nucleotide and thus can report on DNA/RNA content. In our case, we use 600nm wavelength which is absorbed by cells. Thus, the amount of "lost" light intensity reflects the amount of cells.

As you might guess once you have sufficiently "dense" liquid, no light will pass through, and you cannot really learn much from the measurement. Thus, it is important to understand at what values the measurement is linear in the number of cells.

Today Avital and Ayelet tested the new spectrophotometer by performing serial dilution of dense culture of yeast cells to see how they vary. If we are in a linear range, than a dilution of 1:1 should reduce the loss of light (reported value) by 1/2. They report that they get a linear range from about 0.65 OD (units of Optical Density) and downwards.

On the cool side, we found out that the spectrophotometer is based on batteries and can be moved to the bench you are working on, which is really bonus.

In addition, they used the new incubator to perform a growth curve experiment. They seeded yeast cells in different media and grew them in the incubator.

Then they measured the OD every hour using the spectrophotometer. During exponential grow we expect that every XX minutes the OD will double. The value of XX is the doubling time of the yeast in that media and tells us how "happy" the yeast is there. Hopefully I will load some results in one of the next entries.

Incu Incu Incubator

In the endless list of equipment that one needs to run a yeast lab, the incubator plays a special role. It is crucial for growing yeast! and what would a yeast lab if you cannot grow yeast. After much hesitation we decided to buy a large one that contains both shaking tray and space for revolving wheel. It arrived last week and today it was installed.

After some work (which turned out to happen in unnecessary hard way) we managed to bring the incubator to its designated place and land it there. The tech, after finding out the hard way that only some of the plugs in the wall carry electricity (!), managed to get the incubator working, and now we can keep 30C samples going.

One a less happy note, Ayelet retrieved the plates that we poured last week from the hotroom. Quite  a few of them had nasty contaminations.

This means we have to go back to our pouring strategy and figure better sterile technique. We might also consider a sterile chamber for pouring as this is going to be a big issue for us.