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Team:UANL Mty-Mexico/wetlab/mini project
From 2014.igem.org
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Introduction It is know that TetR (like others transcriptional repressors) can allow a basal expression. Because of that, it would be a good idea to reduce that basal expression using a molecular tool which does not cause noise to circuits and systems. The last year our team participated with the project “Thermo coli” (Team: UANL_Mty-Mexico). The main biobrick (K1140006) that we used was composed for the fluorescent protein mCherry (E1010) regulated by the ptet promoter (R0040) and a 37°C thermometer. This year we decided to use that biobrick part to develop a mini-project. We want to prove if it is possible to enhance the repression of a gene by a combined use of the repressor protein TetR (transcriptional regulation) and a RNA thermometer (post-transcriptional regulation). Constructions and Parts The fluorescent part was synthetized based on the promoter ptet (R0040), a RBS fused to a RNA thermometer which was obtained on a previous work (1), the mCherry (E1010) and the transcriptional terminator T7 (B0010-B0012). 
Also, it was constructed a part that does not have a thermometer, so that we could see its effect on the fluorescence. This part was constructed using the same promoter (R0040) with the RFP/mCherry protein generator (K081014). 
We constructed TetR generator switch different constitutive promoters. We used the promoters J23109, J23106 and J23111 with the relative force 106, 1185 and 1487, respectively.These parts have a RBS (B0034), the CDS for the TetR repressor (C0040) and the T7 transcriptional terminator (B0010-B0012). Moreover, we used the biobrick part K145201 as a positive control to expression of TetR, because it is a generator with promoter force 396. Also the RBS and transcriptional terminator are both the same. 
Measurements For the measurements, we made constructions in order to have the biobrick parts in compatible plasmids. It means that those which codifies to mCherry are in pSB2K3 and the tetR genes are in pSB1A2. The bacteria E. coli Top10 were transformed to have the next 7 cultures:
The cultures were growth at 25 and 37°C for 15 hours. The conditions were the same as it is indicated in protocols. The wave lengths that we used were 530+/-25 (excitation) and 590+/-35 (Emission). Results First, we had to prove the function of our construction ptet + mCherry (K1480002). So, it was decided to prove its fluorescence with and without repressor. That TetR generator is the one has been proved before that works (K145201). The results are shown in table 2.
Then, we started to prove our synthetic part (K1140006) with the set generators TetR. The results are shown in the next table.
The next experimental section could not be achieved properly because there were issues with the cultures. Even so we here we present some partial results about repression using the combinated effect of a transcriptional factor (TetR) and RNA thermometer under the influece of changes in temperature.
Discussion We can see (table 2) that the construction K1480002 is functional because there was a decrease in the relative fluorescence of the five clones used. So it can be a useful control to prove the fluorescence variation in K1140006 due to temperature changes. The part K1140006 has been proved against the four TetR generators that we have. In the table 3 and figure 5 we are able to see that was effect over the promoter, so we can proceed to the next experimenal section. This last section we can see (table 4 and figure 6) the variation of fluorescence due to temperature and TetR. While the temperature was 37°C, just TetR was repressing. On the oder hand, we have the added effect of temperature dropping where the temperature was lower than in the 37°C experiment. Conclusion Those partial results allow us to know that the combined repression was stronger than just using a transcriptional repressor. We obviously know further experiments are going to be required. References
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