Part:BBa_K2615003

Csy4-WT, the No.1 member of Csy4 family.

Csy4 (Csy6f), a member of CRISPR family.

Csy4 is a 21.4 kDa protein that binds and cleaves at the 3' side of a stable RNA hairpin structure via sequence- and structure-specific contacts. Csy4 binds its substrate RNA with extremely high affinity and functions as a single-turnover enzyme. Tight binding is mediated exclusively by interactions upstream of the scissile phosphate that allow Csy4 to remain bound to its product. Substrate specificity is achieved by RNA major groove contacts that are highly sensitive to helical geometry, as well as a strict preference for guanosine adjacent to the scissile phosphate in the active site. A highly basic a-helix docks into the major groove of the hairpin and contains multiple arginine residues that form a network of hydrogen.

Fig.1 The Csy4/Hairpin complex.

Background of 2018 OUC-China's project

This year, we design a toolkit focused on translational regulation, which is composed of a RNA endoribonuclease (Csy4) and a RNA module (hairpin). In our project, the cleavage function of Csy4 releases a cis-repressive RNA module (crRNA, paired with RBS) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. A Ribosome Binding Site (RBS) is an RNA sequence to which ribosomes can bind and initiate translation.

We want to achieve precise expression of proteins by using different Csy4 mutants. The aim is using one system to realize diverse expression. We focus on the sites which play an important role in binding and cleavage. Gln104 is located in the linker segment connecting the body of Csy4 to the arginine-rich area, which makes sequence-specific hydrogen bonding contacts in the major groove of the RNA stem to nucleotides G20 and A19. His29 is in its deprotonated form and functions as a general base during cleavage by activating the 2′-hydroxyl nucleophile through proton abstraction. The side chain of Tyr176 points into the active site and stacks on top of the His29 imidazole group, which plays a role in orienting His 29. Phe155 is to recognize the ssRNA-dsRNA junctions in RNA hairpin. Based on the molecular simulation and the theory of fluctuations, four mutants are chosen rationally: Q104A, H29A, Y176F, F155A.

Fig.2 Four key sites of wild type Csy4.

Combined RBS and Csy4, it can be more convenient for other iGEMers to use this composite part without putting a RBS sequence on the upstream of Csy4 coding sequence.The length of this part is 588bp, and the electrophoretic result shows the validity of stripe.

Fig.3 The electrophoretic result of wild type Csy4.

Result

Proof of functions about Csy4 family

We have done three kinds of experiments to help us confirm the function of the Csy4 family. Our aim is to get some new Csy4 mutants with different cleavage capacity, so we specifically tested this aspect of them. For testing our system, we use the superfold green fluorescent protein (sfGFP) as our target gene. Our expectation is that the fluorescence intensities of sfGFP can vary upon the rates of Csy4s’ cleavage. That means we have improved four new parts which present various expression of target genes.

Prediction

Before the experiments, we have proved our ideas by model. The predication below shows the possibilities of different expression levels by different Csy4 mutants. So the model help us to get more information for our improvement deeply this year!

Fig.4 The predication: The fluorescence intensities by different Csy4 mutants along with time.

The qualitative experiments by fluorescent microscope

First, we have tested five different Csy4s by Fluorescent Stereo Microscope Leica M165 FC. We have cultured them in the solid medium in plates until the bacterial colonies can be observed by naked eyes. At that time, the sfGFP have been accumulated so we can see the fluorescence by microscope. As we can see in Fig.5, we have cultured the five different strains for same time which both have the same miniToe circuit but have totally different Csy4 mutants. From top to bottom in Fig.5, there are fluorescence images by fluorescent microscope which indicate Csy4-WT, Csy4-Q104A, Csy4-Y176F, Csy4-F155A and Csy4-H29A in sequence. We can observe visible distinctions 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. So the images indicate the Csy4-WT has the strongest capability of cleavage, while 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.5-1 The expression of sfGFP by Csy4-WT&miniToe-WT.

Fig.5-2 The expression of sfGFP by Csy4-Q104A&miniToe-WT.

Fig.5-3 The expression of sfGFP by Csy4-Y176F&miniToe-WT.

Fig.5-4 The expression of sfGFP by Csy4-F155A&miniToe-WT.

Fig.5-5 The expression of sfGFP by Csy4-H29A&miniToe-WT.

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.6, 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.6 shown, the relative expression level can be measured by flow cytometer at the same time.

Fig.6 Fluorescence intensity of sfGFP corresponding to each Csy4. Histograms show distribution of fluorescence in samples with Csy4-WT&miniToe-WT (Black), Csy4-Q104A&miniToe-WT (Orange), Csy4-Y176F&miniToe-WT (Red), Csy4-F155A&miniToe-WT (Blue), Csy4-H29A&miniToe-WT (Green). Crosscolumn number shows fold increase of sfGFP fluorescence.

Fig.7 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 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.8-A, the Csy4-WT shows the same result with the first system. The switch turn off when the system without isopropyl-β-d-thiogalactoside (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.8-F). In the Fig.8-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.8-E. In summary, we put all the test groups together in Fig.8-F, the picture shows our prediction by model matchs the result perfectly in Fig.9.

Fig.8 The fluorescence intensities of sfGFP by microplate reader. A. Csy4-WT&miniToe-WT. B. Csy4-Q104A&miniToe-WT. C. Csy4-Y176F&miniToe-WT. D. Csy4-F155A&miniToe-WT. E. Csy4-H29A&miniToe-WT. 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.

Fig.9 The comparison about model and result by microplate reader.

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 377
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 93