Part:BBa_K3156667

J23105-thsR-PhhsA-RBS lib-sfGFP 1

We designed a RBS library based on 2017 SHSBNU_China part BBa_K2507008 to eliminate the promoter leakage level. The size of our library is 28 and all parts in it have been proved working.

Usage and Biology

For the PROBE system, we adopt our school team of iGEM in 2017’s thsS/R system which can sense the intestine inflammation indicative signal as our signal receiving part. The thsS/R system is a kind of induced promotor which has main component including a membrane-bound sensor kinase: thsS, a DNA-binding response regulator: thsR, and the promotor: PphsA.

The thsR is a repressor of the PphsA，stopping the expression of sfgfp. When thsS senses thiosulfate, it phosphorylate thsR, canceling its repressor activity. Then PphsA along promotes sfgfp expression.

But the actual effectiveness of the repression of thsR isn’t as high as the ideal value, making the thsS/R system leaky. In fact, the thsR has a relatively high leakage rate, referring to a part of the PphsA promotor that can still promote the sfgfp to express.

There is a way to offset part of the leakage of thsR: When alternating the kernel 4 base pairs in RBS, effectiveness of RBS could be changed, which increase or decrease the expression of following sfgfp gene. That means we can build a RBS library to get a large amount of variants to find out the most matched RBS so that the repression to PphsA can be enhanced without changing the regulator protein’s dynamics attribute.

Figure 1.RBS library design.

The RBS library is built in this way: the 4 kernel base pairs are set into random sequence and sent to a company for sequencing and after eliminating the repetitive sequences, including the control group, there are 27 different sequences left.We did a qualitative test at first and find that there are different between variants:

Figure 2.RBS Control group qualitative result

Figure 3.RBS library 1

Figure 4.RBS library 2

Figure 5.RBS library 3.

Since then we did a quantitative test and the relationship between the different RBS sequences and their binding effectiveness is shown by graph:

Figure 6.RBS library 6 hour culture.

As the result shown in the figure above, some of the RBS Library have similar effectiveness as the control group, whereas some of the RBS Library have a dramatical decrease in ribosome binding efficiency, which controls the expression of sfgfp more strictly, reducing the leakage of thsS/R system.

After all, in order to examine whether the variants can successfully activate the sfgfp when there is thiosulfate, we choose part of the variants and add in 1 mM of thiosulfate to induce. The result shows that all of the variants which leakage decrease is accompanying with the decrease of the highest expression quantity when inducer exist, but it doesn’t affect the on/off ratio of signal.

Figure 7.RBS library On/Off ratio.

According to the result, the no.10 RBS library strain is chosen to be a part of our final product.