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Notebook

Improved Flip Flop

Yolanda Gonzalez and Felix Reyes

• Week 1

  This week we have finished the complete sequence of our bistable improved by proteolysis and asRNA.

  At the beginning we used a temperature-sensitive mutant of the GFP, but finally we decided to use the wild type because we want to be sure that the improvement in the speed of the signal fall is because of the asRNA/proteolysis mechanism.

  In order to choose the restriction sites that will make our construct modular and more versatile (for example going from a 1-2 bistable to a 1-0 bistable), we had to scan our sequence looking for restriction sites already present, so we chose those enzymes that weren’t already present both in the sequence and in the iGEM standard plasmid. In a second optimization step we selected enzymes share buffer for double digestions.

• Week 2

  This week we have designed a plasmid construct that will express our antisense RNA. This is regulated by Prm promoter. By having it on a plasmid we will have an easy control of the whole antisense system by comparing strains containing or not-containing it.

  At first, we decided to use only the first 16 nt of the sequence of the asRNA because it was said to give a greater inhibition in our article (Small RNA Binding to 50 mRNA Coding Region Inhibits Translational Initiation, Bouvier et al.). However, when we where analyzing the kind of terminator they used we discovered that in the complete sequence the terminator was included, and it was a ρ independent one. The fact that the 16 nt asRNA was more effective was proven by chemical synthesis of the asRNA, and it was non-suitable for us as we want to express the asRNA in vivo. So finally we decided to include in the plasmid the complete asRNA sequence shown in the paper.

  For the correct operation of our bistable we need a double ΔrybB-ΔSspb E. coli mutant. We have been sent ΔClpX (control strain) and ΔSspb mutants, and this week we have left the bacteria growing with the pKD46 plasmid in order to delete the rybB gene using the lambda red protocol (Wanner et al.).

  Finally, we have grown strains with the iGEM plasmids pSB4C5, pSB4K5, pSB1T3 and pSB4A5 (expressing RFP) and the pMP01226 Apr lineage expressing GFP.

• Week 3

  This week we have made minipreps of the following plasmids: pKD46, pSB4A5, pSB4K5, pSB1T3, pSB4C5, pKD3, pKD4 and pSB1C3. We have digested the pSB4A5, pSB4K5, pSB1T3 and pSB4C5 iGEM plasmids with EcoRI and PstI in order to get them ready-to-clone our synthetic DNA.

  To obtain the double ΔrybB-ΔSspb E. coli mutant, we transformed ΔClpX (as we will use the double ΔrybB-ΔClpx mutants as a control) and ΔSspb mutants with the lambda red plasmid (pKD46 plasmid with ampicillin resistance) by a heat shock transformation protocol. After transformation, we left the cells growing in ampicillin selective medium and we used as negative control ΔSspb and ΔClpx mutants that hadn’t been transformed. Next day, we observed colonies onto the experiment plates, but not in the negative controls. Two positive colonies of each strain carrying the lambda red plasmid were inoculated to make glycerol stocks.

  We digested the pKD4 and pSB1C3 plasmids with PstI and pKD3 plasmid with SphI in order to have them linear to check purity and concentration by electrophoresis. We carried out the electrophoresis with the pKD3, pKD4 and pSB1C3 plasmid (digested and not digested); digested pSB4A5, pSB4K5, pSB1T3 and pSB4C5 plasmids, and not digested pSB4A5 plasmid.

  Besides we amplify pKD3 and pKD4 markers in order to electropore that fragments according to Wanner et al. protocol.

• Week 4

  Finally our plasmids are already digested and purified, ready to be cloned with the synthetic DNA. The worst part of this process is that during the purifying step, we always lose a great amount of DNA. we obtain, however, enough linearized plasmids.

  This week we tried the transformation. We expected our transformation to be successful but despite our expectations, we obtained no colonies. Besides, something unusual occurred, our ΔSspB strain was not able to continue growing when the culture had got a DO = 0.3 (this kept happening every time we tried to repeat that protocol). At the end of the week, after some analysis, we concluded that our ΔSspB could be transfected with a phage, so we will execute some assays to determinate it.

  Also, we discovered that ΔSspB and ΔClpX have an F’ plasmid. It’s extremely important to remove it since there is a strong LacI gene on it. We think the best way to achieve it would be: first, continue with the deletion protocol; and then we’ll check the loss of F’ plasmid as the plasmid won’t have been selected.

  Furthermore, in the middle of the week, a synthetic device arrived, the asRNA device, (hurrah!) and now we are trying to clone it with the plasmid.

• Week 5

  As we prognosticated before, our ΔSspB is infected with a phage according to our assays (we could see calves!). So we decided to start from the beginning with the ΔSspB strain… Meanwhile we focused on the ΔClpX strain and we carry out the protocol one more time. Thank God (or whoever it is) we acquired some colonies! Now, we have to check it by PCR after holidays (just a couple of weeks to rest).

  The ligation of the iGEM vectors and asRNA device was unsuccessful, so that we will try it again in September.

• Week 6

  After three weeks, we return to work with renewed energy! We start checking the hypothetical ΔClpX ΔRybB strain. We make the PCR very carefully, designing the proper controls and putting our hopes in it. What a relief that at least one of the 20 colonies tested was positive! We got a ΔClpX /ΔRybB strain! The next step is to remove the selective marker used to provoke the delection. We will express a flipase which recombines FRT sequences. These FRT sequences are flanking the marker, so in this way, the strain will be sensitive to this marker.

  We try again the ligation step but we won’t be able to see the results until next week. Once again, we tried the electro-transformation and this failed again. No more words…

• Week 7

  Besides, since we had ran out of fragments to electropore our bacteria, we amplifyed the pKD3 and pKD4 markers. We tried to electroporate the bacteria with the fragments at the end of the week. On Monday we will see the results (hopefully positive, this would be the last time we will try that protocol).

  Also we checked three possible positive colonies which could have lost the marker (cloramfenicol resistance) and the F’plasmid. We have no results yet, next week we will obtain full details about our final strain.

• Week 8

  This week we were working hard trying to arrive well to deadlines.

  On the one hand, finally we got a ΔSspb/ΔRybB!. It was really hard but at the end we got it. After PCR checking, we could say we had our strain. Now, we are removing the kanamicine resistance with the flipase.

  On the other hand, Invitrogen sent us our improved flipflop. We are still cloning it into psB1C3 vector. We’re hurring up!! Just checking the ligations!

  This is a successful week! We hope to have some experimental results for jamboree!

• Weeks 8, 9 and 10

  After Amsterdam Jamboree we arrived with much more energy and ready to start with the work. First of all, we had to make up our strain. We had two problems, (1) the strain had a F plasmid with a LacI inside and (2) we should check our strain and remove the resistance cassettes.

  Firstly, we tried to achieve a strain without F plasmid and to get that we followed two different strategies: on the one hand, we set up a continuous culture eventually checking if any bacteria had lost that plasmid (lost beta-galactosidase activity and proline auxotrophy); on the other hand, in parallel with that, we tried to obtain in another strain with the same deletions as we got weeks ago. Finally we achieved that by continous culture strategy. Secondly, after the strain had lost the F’ plasmid, we remove the resistance cassette and we checked our strain by PCR. Finally, we construct the controls (empty vector and improved flipflop without SspB) and we electroporated our bacteria with all the plasmids. The bacteria were ready to be tested!

  During these weeks, we’ve been intensively proving our improved flip-flop with the fluorometer. We have studied the response of our flip-flop, changing from no induction to IPTG and temperature inductions, proving the basal measures without induction, the results of changing the induction, the duration of the temperature pulse to make the change stable and studied a continuous temperature induction of the previously non-inducted improved bistable. These measurements were carried out in the same strain as the previous measurements of the basic flip-flop, which is not optimal as it expresses the Sspb adaptor protein and the RybB asRNA, being able to interfere in the expression.

  The last week, finally having our new strain, we spent a whole day and night in the lab measuring the improved flip-flip stability, and change speed. It was a long sleepless night, but it was worth it: we got very useful results!