Difference between revisions of "Team:OUC-China/Improve"

The result by flow cytometer

The qualitative experiment is not enough to analyze Csy4s. So we test our system by flow cytometer after we cultured them for ten hours in M9 medium. The expression of sfGFP in five groups are showed in Fig.3, and they are Csy4-WT&miniToe-WT, Csy4-Q104A&miniToe-WT, Csy4-Y176F&miniToe-WT, Csy4-F155A&miniToe-WT and Csy4-H29A&miniToe-WT. We find that 5 groups' fluorescence intensities have an obvious order from Csy4-WT to Csy4-H29A, which means the capabilities of cleavage decrease one by one. Their order goes from strong to weak is Csy4-WT, Csy4-Q104A, Csy4-Y176F,Csy4-F155A and Csy4-H29A. As the Fig.3 shown, the relative expression level can be measured by flow cytometer at the same time.

The result by microplate reader

Besides all the works we have done before, we also need to know more information about the Csy4s we design. Even though we have known that our Csy4 mutants have differentiated expression level after ten-hour-culture, the expression of whole cultivation period is also a reference for us to know if our system can work as expectation.

So we tested five Csy4s individually by microplate reader. We have tested them every two hours. The green lines in all the images represent the control group, “miniToe only” group and the green lines keep stable which means the miniToe structure can close the expression of downstream genes. And the test groups show different characteristics. As we can see in Fig.5-A, the Csy4-WT shows the same result with the first system. The switch turn off when the system without IPTG (as the blue line shows). And the expression level is the highest among all the test groups which indicates the highest enzyme activity by Csy4-WT (Fig.5-F). In the Fig.5-B, the tendency of increase of fluorescence intensities by Csy4-Q104A is almost same with Csy4-WT. And the expression level is lower than Csy4-WT. So the Csy4-Y176F is. What is special is Csy4-H29A. We have mentioned Csy4-H29A before. The active site of Csy4 contains an essential histidine residue (H29) that functions as a general base during RNA strand scission. Mutation of H29 to alanine inactivates Csy4 without affecting substrate binding affinity or specificity. So Csy4-H29A is a dead-Csy4 which has high binding affinity but has lowest capabilities of cleavage as we can see in Fig.5-E. In summary, we put all the test groups together in Fig.5-F, the picture shows our prediction by model matchs the result perfectly in Fig.6.

In summary

This year, we used point mutations to redesign four mutants on the basis of Csy4(BBa_K1062004) which are Csy4-Q104A(BBa_K2615004), Csy4-Y176F(BBa_K2615005),Csy4-F155A(BBa_K2615006) and Csy4-H29A(BBa_K2615007). The capabilities of cleavage and recognition are different for each Csy4 mutants, and we name them the Csy4 family. The combination of the Csy4 family members and the miniToe family members constitute a post-transcriptional regulatory toolkit for achieving different expression levels of target genes.

Csy4-WT, the wild type, is a member of the CRISPR family, and also the core member of our project. Csy4-WT can specifically recognize and cleave a 22nt hairpin structure, known as the miniToe-WT. We confirmed that Csy4-WT is the strongest of the Csy4 family through the analysis of the results of our fluorescence microscopy, flow cytometry and microplate reader experiments. And the strength of the remaining members of the Csy4 family shows a staircase pattern.

Csy4-Q104A, which is second only to Csy4-WT in strength in the Csy4 family, coming from point mutation, and we change the CAG(encoding Gln) to GCG(encoding Ala) on the 104th site based on Csy4-WT. It can also recognize and cleave the 22nt miniToe, regulating the expression of downstream genes. When we conducted experiments with the miniToe-WT combination and used sfGFP as the downstream target gene, we could see the experimental results that the sfGFP expression level of Csy4-Q104A was about half that of Csy4-WT.

Csy4-Y176F, the third-strongest in the Csy4 family. It is designed in the same way as Csy4-Q104A, but with the 176th site changed from TAC(encoding Tyr) to TTT(encoding Phe). It can be seen from the experimental results that the expression of downstream genes regulated by Csy4-Y176F is correlated with the stepwise decline of Csy4-WT and Csy4-Q104A.

Csy4-F155A, strength is the fourth in the Csy4 family. At point mutation, we changed its 155th site from TTC(encoding Phe) to GCG(encoding Ara). It has a weaker cleavage and recognition capability..

Csy4-H29A, the most special one of our Csy4 family, whose 29th site is changed from CAC(encoding His ) to GCG(encoding Ara). Csy4-H29A has a high binding affinity but has the lowest capacity of cleavage, so we call it dead-Csy4. There is no doubt that its downstream gene expression is the lowest in the family.