Team:OUC-China/Improve

Team OUC-China: Main

Improve

Overview

This year, we create a brand new family called Csy4 family on the basis of an existing part, Csy4 BBa_K1062004. We redesign four Csy4 mutants by point mutation. The members in Csy4 family have different capabilities of cleavage and recognition. As an important role in project, we tested them by several ways. The Csy4 family works well as expectation. Csy4 family is an improvement based on existing part and is proved work well in our system.

Proof of functions about Csy4 family

We did three kinds of experiments to help us confirm the function of the Csy4 family. The aim is to get some new Csy4 mutants with different capabilities. Superfolder green fluorescent protein (sfGFP) is target gene for test experiments. Our expectation is that the fluorescence intensities of sfGFP change upon various activity of Csy4 mutants. It means we have improved four new parts which present various expression of target genes.

Prediction

Before the experiments, model proved our ideas. The predication shows the possibilities of different expression levels by different Csy4 mutants. It is not difficult to predict that the cleavage rate has an influence in the expression of sfGFP.

Fig.1 The predication: the fluorescence intensities by different Csy4 mutants along with time.

We designed three kinds experiments to test the capabilities of five Csy4 mutants by putting them into miniToe system. So the recombination strains for test both have same pReporter but different Csy4 mutants plasmids in the following. The recombination strains to test the functions of Csy4 are strain-Csy4 (pCsy4&pReporter), strain-Csy4-Q104A (pCsy4-Q104A&pReporter), strain-Csy4-Y176F (pCsy4-Y176F&pReporter), strain-Csy4-F155A (pCsy4-F155A&pReporter), strain-Csy4-H29A (pCsy4-H29A&pReporter). At the same time, we have a control strain named strain-miniToe-only which only has pReporter.

The qualitative experiments by fluorescent microscope

First, we have tested five different groups by Fluorescent Stereo Microscope Leica M165 FC. The sfGFP accumulated during the cultivation period so the fluorescence can be observed by microscope after 8 hours. Because the five Csy4 mutants have different capabilities of cleavage, the distinguishing intensities of fluorescent can be seen by naked eyes. The five test strains have same miniToe part but different Csy4 mutant genes. From top to bottom in Fig.2, there are fluorescence images by fluorescent microscope which indicate strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The visible distinctions have shown in these images. The fluorescence intensities decrease one by one from top to bottom which means the Csy4s' capabilities of cleavage decrease one by one. The Csy4-WT has the strongest capability of cleavage when the Csy4-H29A is a kind of dead-Csy4 (dCsy4) which is hardly to find the fluorescence by microscope. The qualitative experiment is a basis of further experiments.

Fig.2-1. The expression of sfGFP by strain-Csy4.

Fig.2-2. The expression of sfGFP by strain-Csy4-Q104A.

Fig.2-3. The expression of sfGFP by strain-Csy4-Y176F.

Fig.2-4. The expression of sfGFP by strain-Csy4-F155A.

Fig.2-5. The expression of sfGFP by strain-Csy4-H29A.

Fig.2 The fluorescence images by fluorescent microscope. From top to bottom, the images shows the expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The plotting scale is on the right corner of images. The images on the left shows E. coli without fluorescence excitation. The images on the right represent situation when fluorescence excitation.

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.

Fig.3 The fluorescence intensities of sfGFP about Csy4 mutants by flow cytometer. Histograms show distribution of fluorescence in samples with strain-Csy4 (Black), strain-Csy4-Q104A (Orange), strain-Csy4-Y176F (Red), strain-Csy4-F155A (Blue), strain-Csy4-H29A (Green). Crosscolumn number shows fold increase of sfGFP fluorescence.

Fig.4 The Gate Mean of flow cytometer. Histograms show the relative expression of sfGFP. The five test groups present different fluorescence intensities from high to low which prove that they have different capabilities of cleavage.

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.

Fig.5 The fluorescence intensities of sfGFP by microplate reader. A. strain-Csy4. B. strain-Csy4-Q104A. C. strain-Csy4-Y176F. D. strain-Csy4-F155A. E. strain-Csy4-H29A. A-E. The blue line is test group with IPTG. The yellow line is test group without IPTG. The green line is a control group which only has miniToe structure without Csy4s. F. The summary of different test groups which indicates the capabilities of Csy4 mutants. The results are listed in the order: Csy4-WT>Csy4-Q104A>Csy4-Y176F>Csy4-F155A>Csy4-H29A.

Fig.6 The comparison about model and result by microplate reader.

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.

Achievements

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 experimental result in both parts to demonstrate the improvement. We documented its experimental characterization on Part's Main Page on the Registry and submitted the sample to the Registry.

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

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