Team:UANL Mty-Mexico/wetlab/mini project




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).

Figure 1. This part (K1140006) changes its behavior in response to temperature variations above and below 37°C. This part is available in pUC57, pSB1C3 and pSB2K3.

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).

Figure 2. This part does not change its behavior in response to temperature. The biobrick part is available in pSB1C3 and pSB2K3.

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.

Figure 3. The names of constructed parts are BBa_K1480003, K1480004 and K1480005. Their respective promoters are J23109, J23106 and J23111. Actually, those parts are just available in pBca.


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:

Table 1. Combinations for clones
Fluorescent protein (in pSB2K3) TetR generator
ptet + mCherry Any
ptet + mCherry J23111 + tetR
ptet + thermometer + mCherry Any
ptet + thermometer + mCherry J23019 + tetR
ptet + thermometer + mCherry J23116 + tetR
ptet + thermometer + mCherry J23106 + tetR
ptet + thermometer + mCherry J23111 + tetR

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).


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.

Table 2. Repression over K1480002.
Without repressor With repressor
3869 2590
4012 2450
4320 2296
4232 2601
3794 2564
Mean 4045.4 2500.2
SD 226.7 128.9

Figure 4. Graph of previous results.

Then, we started to prove our synthetic part (K1140006) with the set generators TetR. The results are shown in the next table.

Table 3. Repression over K1140003.
Without repressor K1480003 K145201 K1480004 K1480005
2770 852 868 731 738
2621 827 766 780 673
3294 866 1212 898 655
3492 946 1212 813 746
3038 725 742 608 450
2710 776 744 827 516
1671 620 536 566 NG*
Mean 2799.9 801.9 868.8 746.6 630.0
SD 590.9 106.3 254.7 120.6 121.1

Figure 5. Mean and SD of every set of results of table 3.

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.

Table 4. Effect of temperature and TetR.
Temperature Without repressor K1480003 K145201 K1480004
25°C 816.2 759.8 1240.7 1478 509.5 510.5 656.8 671.6 565.4 429.9 1271.7 884.3 553.8 306.1 638.3 670.3
37°C 2770.5 2622 3294.9 3492.5 852.4 827.4 866.8 946.2 868 766.6 1212.9 1212.4 731.9 780.9 898.7 813.9

Figure 6. The blue color represent the clones that were cultivated to 25°C and the red is for 37°C. All the clones have ptet and the 37°C RNA thermometer. Below it is shown the repressor used.


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.


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.

[1] Neupert J, Karcher D and Bock R (2008) Design of simple synthetic RNA thermometers for temperature- controlled gene expression in Escherichia coli. Nucleic Acids Res36:e124.

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