Team:UANL Mty-Mexico/wetlab/mini project-esp

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Miniproject

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 proveif 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 (Neupertet al, 2008), 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 generatorswith 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.

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:

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).

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.

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

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	With K1480003	With K145201	With K1480004	With 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.

References
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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