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

The result by flow cytometer

The qualitative experiment is not enough to analyze the Csy4 mutants. So we tested miniToe family system by flow cytometer. The expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A is showed in Fig.3. We find that 5 groups' fluorescence intensities have an obvious order from Csy4-WT to Csy4-H29A, which means the capabilities decrease one by one. Their order goes from strong to weak is Csy4-WT, Csy4-Q104A, Csy4-Y176F,Csy4-F155A and Csy4-H29A.

The result by microplate reader

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

So we tested five test stains individually (strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A) by microplate reader every two hours. The green lines in all the images represents strain-miniToe-only group keep stable. It means the miniToe structure fold well and lock the process of translation without Csy4. And the five test groups show different characteristics. In Fig.5-A, the group strain-Csy4 shows the same result with the first system. The switch turns off without IPTG (as the blue line shows). And the expression level is the highest among all the test groups which indicates the Csy4-WT has strongest capabilities (Fig.5-F). In the Fig.5-B, the tendency of fluorescence intensities by Csy4-Q104A is similar with Csy4-WT. And the expression level is lower than Csy4-WT. The Csy4-Y176F’s capabilities ranks the third. What is special is Csy4-H29A. 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 design Csy4 family including four mutants on the basis of Csy4 which are Csy4-Q104A, Csy4-Y176F,Csy4-F155A and Csy4-H29A. Their capabilities are different.

Csy4-WT, the wild type, is a member of the CRISPR family, and also the key member of our project. Csy4-WT can specifically recognize and cleave a 22nt hairpin structure. We proved that Csy4 has the strongest capabilities of the Csy4 family by analysing the results by fluorescence microscopy, flow cytometry and microplate reader experiments. And the capabilities of the remaining members in Csy4 family shows a staircase pattern.

Csy4-Q104A, whose capabilities rank the second in Csy4 family. By point mutation, and we change the CAG(encoding Gln) to GCG(encoding Ala) on the 104th site. It can recognize and cleave miniToe structure on the RNA level, regulating the expression of downstream genes. By experiment, we find the expression level of strain-Csy4-Q104A was about half that of stain-Csy4-WT's.

Csy4-Y176F, which rank the third in 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). By wet experiments, the expression level of downstream genes present stepwise decline from Csy4-WT to Csy4-Y176F.

Csy4-F155A, No.4 in the Csy4 family. We changed its 155th site from TTC(encoding Phe) to GCG(encoding Ara). It has a weaker capability of cleavage and recognition.

Csy4-H29A, the most special one in Csy4 family, whose 29th site is changed from CAC(encoding His ) to GCG(encoding Ara). Csy4-H29A has a high binding affinity but the lowest ability of cleavage, named dead-Csy4. There is no doubt that the downstream gene expression level by Csy4-H29A is the lowest in the family.

Silver part: Designing a new part

This year, our project can be divided into four systems. Between the different systems, there are different protocols. And we have sorted out these experimentals in detail.

This year, we design a basic part called miniToe, which consists of RBS, Csy4 recognition site and cis-repressive RNA element. As a regulatory element, it can be specifically recognized and cleaved by Csy4 to regulate the expression of its downstream genes from the RNA level. MiniToe is the core of our project design, is RFC10 compatible and works as expected. We have documented its experimental characterization on Part's Main Page on the Registry and submitted the sample to the Registry. Certainly, this part is different from the new part documented for Gold#2. See the page for more details: parts.igem.org/Part:BBa_K2615020

At the same time, the Csy4 (BBa_K2615003) is one of the key role in our system. We also submitted it to registry. We also apply miniToe to polycistron to create miniToe polycistron (BBa_K2615019).

Gold parts: Improving an existing part

Improving an existing Part Standardization and building up on existing parts are the fundaments of iGEM. We have created FOUR new BioBrick Part (BBa_K2615004, BBa_K2615005, BBa_K2615006, BBa_K2615007) that has a functional improvement upon an existing BioBrick Part (BBa_K1062004). The sequences of four new parts and existing part are different, and the new parts are changed by point mutation. We have showed experiments with both parts to demonstrate this improvement.

See the pages below for details:
The existing part
parts.igem.org/Part:BBa_K1062004:Experience

The four improved parts
parts.igem.org/Part:BBa_K2615004
parts.igem.org/Part:BBa_K2615005
parts.igem.org/Part:BBa_K2615006
parts.igem.org/Part:BBa_K2615007