Evolution of Transcription Factors Binding paper is accepted!

Yay!

Just got email that a paper by Naomi, Ilan Wapinski, Aviv Regev, Hanah Margalit and me was accepted to Molecular Systems Biology. This paper represents almost two years of work by Naomi and Ilan, and undergone several dramatic ups & down during this time. So it is great to hear that it is officially accepted.

It made just in time for Naomi to print her dissertation listing this paper as "accepted for publication" instead of "in review".

We celebrated by an impromptu toast in the lab.

More details once the paper appears....

This is also a good chance to add a picture of Naomi & Ilan from a trip to CSHL at the time we started talking about this project.

Light box

As you may recall from early posts, we use agar plates for growing and manipulating libraries of yeast strains. We also keep "replicating" copies of these plates to maintain a "live" copy of the libraries.

One of the potential problems is that we mis-copied a plate, or introduced a contamination. One way to check that is to examine the plate pattern. Basically, each library plate has different set of empty spots. This means that we can easily identify plates by these "finger prints". Moreover, a contamination would fill the empty spaces by new colonies. 

Thus, it was clear early on that we need a tool for taking pictures of plates and recording/analyzing the colonies. We could use the standard gel camera we have in the shared room, but that was both unwieldy and exposed our plates (and us) to ethidium bromide (which is used by other labs for dying DNA on gels).

Instead we aimed for a dedicated station for imaging plates. We found some solutions by commercial companies. These however, were very very expensive. And so we decided for a Do It Yourself solution. We consulted with the local electrician, and helped by Meretz, a retired electrician built our own solution.

The box is an old wooden box that was originally for a nikon microscope. The local workshop built us a device to hold the plate in a precise location. Since the plate is asymmetric this ensures that the images are of the correct orientation. We bought a Nikon camera that can have external power supply and remote operation. It is an overkill for the image quality we need, but it does work without battery replacement or manual operation.

As a light source, Meretz suggested using a led strip. He built these leds below the plate holder. This meant that the image colonies showed up as silhouettes.

Image with background light - colonies are dark circle on light background

I wrote a simple program that identifies colonies, their location and their size (which is useful for estimating the fitness of different strains). Eli Peker, who worked as a summer student, wrote a program for uploading images and storing them in a database. This interface allowed different people in the lab to keep track of libraries and compare to previous versions.

Recently, Amit (who deserve his own post soon) suggested we also examine the color and texture of the colonies. This appears to be a known phenotype. However, since the light source was below the colonies we could not use our box. We again contacted the local electricians who installed additional led strips for forground lighting.

"Disco" foreground lights

As you can see, the forground lights turned out to be a bit excessive ("disco" as Amit put it). Now we could make out the color and the texture of the colonies.

Same plate, with foreground lighting

However, now some of these disco lights are reflected in the image and look like small colonies (top part of the image). To deal with this, we added our own DIY screen (paper) to soften the light. 

Foreground lights with diffused screen

Which lead to removal of the annoying reflections on the agar.

Same plate, with diffused foreground lighting

Notice that in this plate there are few white colonies (two small ones) and some that are more yellow. These differences are easier to see if zoom in on a region of the image.

Moreover, some colonies have a different texture, these differences are enhanced when we examine a gray-level version.

For now we use the lightbox to record the plates with different strains. We didn't yet write an automated color classifier (relatively easy) or a texture classifier (a bit harder).

Robot Updates

As part of ongoing attempts to get the system to work to our liking we run into two problem.

First, many of the steps in our yeast growth protocols use individual pipetting actions for each well. For this reason we have an arm with individual pipettes. Initially we configured the arm with four fixed tips and four disposable pipettes. The fixed tips are easier to use and do not consume plasticware. On the other hand to get perfect sterility we need to use plastic tips.

So far we learned that the fixed tips (with a wash between uses) is sufficiently clean for our purposes. And so we use these a lot. The problem is that the time it takes to "process" a plate (e.g., remove individually tailored amounts per well for growth dependent dilutions) was too long. The main time consuming steps is the tip washing. 

After consultation with Neotec people, we decided to switch two of the disposable pipette heads with fixed tips. This will give us more tips and thus less washes (as the washes are done in parallel and take the same amount of time for 4 or for 6 tips).

Six fixed tips in action

The second problem is much more serious - the robotic manipulation arm kept getting out of alignment. This lead to serious of failure in our longer experiments. The blame fell onto the plate shaker, which we use a lot in our protocol. The design of the shaker has to allow a robotic arm to place/remove a plate, but at the same time hold the plate firmly during the shaking. The solution in the shaker we had was a spring that held the plate in place. Every time the arm came down to the shaker it would press on a lever and release the plate from the spring. Apparently these repeated presses moved the arm out of place (not by much but sufficiently to cause problems)

The solution was to replace the shaker head to one with a different locking mechanism based on a higher stand that kept the plate sitting down. This required adaptation of a solution Tecan built for arms that hold the plate from above (and not from the side like ours).

New shaker head

So far it seems that the system is indeed more stable. It worked for two days without issues and we are counting.

Gone Sailing

One of the events that took place during the "blackout" period (when I did not blog) is worth reporting.

Ayelet, the command center of our lab, has collected all her saved vacation days (and borrowed some from the future) to take a 6 month vacation. To be honest, she warned me about that plan when we first met to discuss her position as a lab manager. Ayelet's plan was to take a boat and sail with the family in the Mediterranean from Israel to Italy and back, visiting Cypres, Turkey, Greece, and Croatia on the way. Back then it seemed far away and beyond care.

When Ayelet told me they bought a boat, I realized that the plan might actually be carried out. I even got a chance to sail with Ayelet and Udi  one nice day out from Jaffa port (where they had a mooring point).

But since September the threat become more realistic. As the target date in April was closing in, Ayelet worked hard to make sure there are no loose ends left in the lab. She finished all the experiments she was performing, made sure our supply and daily operations are defined by a robust set of instructions. She left different lab members in charge of different aspects of the lab, and left strict instructions how to behave.

Surprisingly, we managed to avoid a major crisis so far. Things are working relatively smoothly, and we carry on the momentum Ayelet left us. This does not mean she will not have a lot of work when she comes back :-)

So what about the trip? Well, a week after passover, they set off. I was recruited on the last minute to drive some of the family and last of the supplies to the port. And so I got to send them off from the boat's berth. (They actually sailed some few kilometers north to Hertzelia where they could go through official border check to legally leave the country.) I also took the official goodbye picture.

Ayelet has been keeping a blog of the trip. It is worthwhile to take a look, as there are some amazing stories.

The observent readers might point out that Ayelet shows up in the pictures from Moran's sendoff party. This is not a mistake. The party was planed for the week Ayelet came back to Israel for a "mid-vacation" break to see her older kids and meet with her extended family.