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Notebook

Basic Flip Flop

Amalia Martínez

• Week 1

My first week of work in the lab has just ended up, my first approach to work in Fernan’s lab.  My function in the team is learning how the basic toggle switch works, for later improvement of its performance. We obtained the toggle switch from a biobrick, that need to be introduced in a lacI-deficient E. coli strain to work correctly. Before that task, we need to check that the biobricks that the Headquarters sent us were the right ones.

I made inocula from all the pieces that were sent, and then extract the plasmid from all of them. At the same time, I took out from the lab collection a lacI-deficient E. coli strain, called MC4100 and set an inoculums for this strain too. This way, I’ll have the strain ready to be transformed with the plasmid that contains the toggle switch.

To confirm the identity of the biobricks, I set a digestion reaction with to enzymes: EcoRI and PstI. This will separate the biobrick from the plasmid backbone, so we can check their sizes in an electrophoresis gel. I carried out this experiment twice, most of the pieces were confirmed, being the toggle switch among them.

Finally, I made the transformation reaction to introduce the toggle switch into the strain MC4100. Next week, I will check how this device works by fluorescence assays.

  

• Week 2

The transformed bacteria I made last week, wouldn’t grow on minimal media so I tried different concentrations of casaminoacids and checked the composition of the recipe we have in the lab. To measure fluorescence, it’s better to use minimal media than LB, a media that has a strong fluoresce signal because of the high amount of aromatic aminoacids. After four days of trials, I repeated the transformation protocol because I thought the mistake should be there since the strain I use has no auxotrophies. Finally, my LacI deficient strain with the plasmid containig the basic toggle switch grew in the media, ready to be tested in all the conditions I can!

• Week 3

My bacterium has finally grown in minimal media, so it’s time to try out the toggle switch!

I set an overnight inoculum at 30ºC, to use it as a starting point for the experiment because none of the states is induced in this conditions. Then I made plaques with different dilutions and conditions to induce each of the states (IPTG for GFP, 42ºC for RFP). The next day, I llooked them in the UV lamp... Awesome results! The induced plaques gave the expected results; whereas the one at 30ºC without IPTG, was almost the same as the one with IPTG. To check if the GFP was expressed constitutively or the colonies were mixed, we used a fluorescence microscope and a fluorescence magnifying glass.

The magnifying glass helped us to probe that the colonies really expresed the corresponding fluorescent protein. Then, in the microscope we could see which color had each bacterium, and see the differences in populations in the induction conditions.

The results showed us that the switch to red was weaker than the other one, so I grew a liquid culture at 30ºC until it reached at Optical Density of 0.2, and I put it under the red-inducing temperature: 42ºC. Al every half an hour, I took a sample and looked it under the microscope, seeing how the population changed from green to red.

Also, we needed to check the stability of the red condition so some of the induced colonies we grew them at 30ºC, to see how long the RFP expression lasted. It took it one day to start dissapearing, but I hope that with the measures that the other members of our grup are developing, the performance of this genetic device will improve.

• Week 4

After the trials I made last week, where I tried our the behaviour in a qualitative way, I needed to quantify the changes in the fluorescence in both proteins. First, we set the conditions where the measures didn’t interfere with each other. For that, I used two E.coli carrying expression plasmids with GFP and RFP.  As easy as it may seem, I noticed that the RFP is also excited by the wavelenght we use for GFP, lowering the accuracy of this data. However, the plasmids we use for testing had very strong promoters compared to the ones used in the tested toggle switch, then the interference will be disminished when using the actual construction. Following the same protocol, I measured fluorescence and optical density in a microtiter reader each hour during a maximun of four hours. I divide the absolute fluorescence data between the optical density, then the values would not reflect the amount of bacteria in a particular moment.

• Week 5

Our team is developing a system to integrate BioBricks in the bacterial genome, so I’m going to check this device using the basic toggle switch. I have to purify the fragment that contains the toggle switch, and introduce it in the appropriate vector, and then look for differences in the performance of the new construction. At first, I planned to have everything done by Friday but the restriction cuts didn’t work, so I will try it again next week.

• Week 6

After our arrival from the European Jamboree, we set a meeting to organise the work in the following weeks. My duty is keep on testing the basic toggle switch, repeating the experiments I made before the Jamboree.

• Week (3th-7th October)

This week I tried to do a double switch experiment, by makin two consecutives changes of state. The experiment takes two days, making dilutions to keep the bacteria in the exponential phase of growing. Sadly, due to some problems of organization, only one switch could be made.

Also we took samples for flow cytometry, a technique that allows the differenciation of the different populations of fluorescent bacteria that may appear in our culture. This way, we can asses wheter the induced population are expressing just one protein or not.

• Week (17th-21th)

As the construct of the improved toggle switch is now completed, we decided to make both characterisation experiments at the same time, so now I’m part of the V’s subteam!. We used the same experimental methodology.

• Week (24th-28th)

This week we made a 24-hours experiment with both toggle switches. A long long day, but it was worth it because of the data we obtained. Also we processed all the samples obtained before by flow cytometry.