Team:SHSBNU China/Part Collection

• Acknowledgement
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• Judging
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• Parts
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  Inspiration Description Design Results Hardware Demonstrate Prospect

Part Collection

This part collection is the RBS libarary based on the existing BioBrick BBa_K2507008. The 2017 iGEM Team SHSBNU_China stated that this part had a very high leakage level and could result in. Since then, we designed this collection to reduce the level of leakage of BBa_K2507008. This RBS library helped us to obtain more accurate experiment results and provide us with a better signaling platform.

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.

In the end , we selected 5 of them as our collection:

BBa_K3156667
BBa_K3156671
BBa_K3156674
BBa_K3156677
BBa_K3156690