Team:ZJUT-China/Results

Results

The results page involved almost all of achievements in our experiments. There are many flow diagrams before the detailed procedure. We hope it will improve the feeling of reading and makes the results more visual.

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What did we achieve?

1. We constructed a simplified plasmid of spCas9 and a plasmid that can transcribe CXCL9 mRNA.

2. We designed an igRNA based on a gRNA with high cutting activity, and carried out an in vitro verification experiment for igRNA and RNA biomarker.

3. We constructed P70a-σ28-P28-deGFP and P70a-σ28-P28-tetR plasmids to verify the intracellular function of transcription factor σ28 and inhibitor protein tetR in gene circuits.

4. We constructed plasmids with fluorescent genes that remove ssrA degradation tag to explore the effect of degradation tag ssrA on deGFP.

5. We successfully constructed the plasmid in the gene circuits and completed the function verification in Cell-Free system.

Plasmid spCas9 construction
figure-1 Figure 1. Schematic of the procedure of plasmids construction. After colony Polymerase Chain Reaction (PCR), the monoclonal colonies were cultured and sent to the company for sequencing.

Plasmid spCas9 (Figure 1) was constructed from the original plasmid spCas9 and the P15A Ori-CmR gene fragment of the synthetic plasmid puc57, which for expressing Cas9 protein and complex the corresponding CXCL9 mRNA-igRNA for cleavage of target genes in Cell-Free system. The building process is shown in the figure above.

figure-2 Figure 2. Schematic of the plasmid spCas9 construction.
(a) The map of spCas9 from SnapGene.
(b) Gel electrophoresis analysis of PCR products. The marker is 5k (lane 1), the fragment is spCas9 (lane 3&4), the vector is P15A Ori-CmR (lane 5&6).
(c) Gel electrophoresis analysis of cloning PCR products. The marker is 5k (lane 1) , the colony PCR fragment is a part of the goal plasmid (lane 2&3).
Plasmid CXCL9 mRNA construction
figure-3 Figure 3. Schematic of the procedure of plasmid CXCL9 mRNA construction. After cloning PCR, the monoclonal colonies were cultured and sent to the company for sequencing.

We constructed a new plasmid which can transcribe CXCL9 mRNA by using two existing plasmids (igRNA-CXCL9 and pTargetF, Figure 4a). Then designed primers CXCL9-F and CXCL9-R to conduct PCR, clean-up, and eliminate templates experiments (Figure 4b). Transformed the fragment andvector with one-step cloning enzyme into E. coli DH5α to make it self-replication. Colony PCR using colonies grown on resistant plates. We had fundamental success after agarose gel electrophoresis and a final success after sequencing.

figure-4 Figure 4. Schematic of the plasmid construction.
(a) Maps of plasmid pTargetF-gRNA and CXCL9-igRNA from SnapGene;
(b) On the left is 5k marker, in the middle is CXCL9 segment, on the right is the vector from plasmid pTargetF reverse PCR;
(c) On the left is 5k marker, on the right is colony PCR verification strip.
Construction of plasmid gRNA and screening of cleavage activity
figure-5 Figure 5. Schematic of the design procedure of the gRNA construction and screening. There are five 20nt sequences to change into five different gRNAs.

In the CRISPR/Cas9 system, gRNA can direct Cas9 protein to cut dsDNA at the sequence target site. Simultaneously, a hybridization region of igRNA is adding additional sequences in the 5' of sgRNA. Therefore, designing an active gRNA with high cleavage ability is important.

Based on the target gene (tetR), we chose CGG and AGG as PAM, and designed 5 gRNAs in the plasmid pTargetF (Figure 5).

In vitro cleavage validation
figure-6 Figure 6. Gel electrophoresis analysis of in vitro cleavage products with different gRNA.Target dsDNA was incubated with the following gRNA for 1 h at 37 °C under the condition of in vitro Cas9 cleavage assay: marker (lane 1); gRNA1 (lane 2); gRNA2 (lane 3); gRNA3 (lane 4); gRNA5 (lane 5).

After the plasmid of gRNA was constructed, 5 corresponding RNAs were transcribed by in vitro transcription. The gRNA, Cas9 and the target gene fragment were mixed together for reaction according to the kit handbook. Then carry out the agarose gel electrophoresis (Figure 6).

Subsequently, we analyzed why the cutting ability of different gRNAs varies greatly. According to some research, we explored this question with the professionals from Genscript and found an answer from their official CRISPR handbook: "The 10 to 12 bp base of gRNA near PAM, also known as the seed sequence, determines the specificity of gRNA and target recognition. The results showed that controlling GC content in seed sequence at 40%~60% during gRNA sequence design could significantly improve the specificity of gRNA and target recognition."

CG content of 5 gRNA sequences designed is:

gRNA sequence CG (%)
gRNA1 ttagagctgcttaatgaggt 30-42
gRNA2 attggcatgtaaaaaataag 10-17
gRNA3 caaaagtacatttaggtaca 30-33
gRNA4 tagccattgagatgttagat 30-33

Obviously, the CG content of gRNA1 seed region with obvious cutting ability obtained from in vitro validation experiments is between 40%-60%, but gRNA4 does not meet this requirement. More strangely, gRNA3 and gRNA4 have the same CG content, but gRNA3 does not show obvious cutting ability. Therefore, we speculated that gRNA3 could not guide Cas9 protein to perform the cleavage function because of the sequence of bases or the easy degradation of RNA transcribed in vitro.

After comprehensive consideration, we chose gRNA1 with higher CG content as the basis of igRNA design.

In vitro validation of RNA biomarkers and igRNA
figure-7 Figure 7. Polyacrylamide gel-shift assay analysis of the igRNA hybridizating with CXCL9 mRNA.

We selected the CXCL9 mRNA, a biomarker related to rejection reaction after kidney transplantation, and designed the sequence of igRNA. Please refer to Enginering Success for the specific design scheme. In order to verify that igRNA can complement CXCL9 mRNA, we conducted EMSA experiment in vitro (Figure 7).

In column A, non-igRNA and CXCL9 mRNA could not complement each other, and the bands were separated after binding Cas9, and the migration rate of the complex was slower as the number of Cas9-binding RNA bases increased. The column D was CXCL9 mRNA-igRNA-Cas9 complex, and the migration rate was slower than the column A. The results indicated that igRNA was successfully combined with CXCL9 mRNA in vitro.

How to verify that every part is functional in gene circuits?

In Cell-Free systems, validation of each component is also important to ensure proper use of the whole function. Therefore, the σ28, tetR repressor in the gene circuit was validated in bacteria prior to the validation of the Cell-Free system.

Construction of P70a-σ28-P28-deGFP plasmid
figure-8 Figure 8. Schematic of the procedure of plasmid P70a-σ28-P28-deGFP construction. After colony PCR, the monoclonal colonies were cultured and sent to the company for sequencing.

The plasmid P70a-σ28-P28-deGFP was constructed from the gene fragment of the original plasmid P70a-deGFP and the synthetic fragment UTR1-σ28-P28 (Figure 9a) to verify the normal expression of σ28 and provide the original plasmid for the synthesis of P70a-σ28-P28-tetR. After PCR, the results of agarose gel eletrophoresis were as follows, with correct band sizes (Figure 9b).

figure-9 Figure 9. Schematic of the plasmid construction.
(a) The map of plasmid P70a-σ28-P28-deGFP from SnapGene;
(b) On the left is the 5k marker, in the middle is UTR1-σ28-P28 segment, on the right is the vector from plasmid P70a-deGFP reverse PCR;
(c) On the left is the 5k marker, and on the right is colony PCR verification band.
figure-10 Figure 10. Photos of cultured plates. There are some colonies with plasmid P70a-σ28-P28-deGFP theroratically on the left plate, and the negative control is on the right plate.

After recombination and transformation, overnight culture, bacterial colonies grew on ampicillin plates (Figure 10).

Through the colony PCR verification band, our agarose gel eletrophoresis results were consistent with expectations (Figure 9c), and the plasmid was successfully constructed after sequencing.

Construction of P70a-σ28-P28-tetR plasmid
figure-11 Figure 11. Schematic of the procedure of plasmid P70a-σ28-P28-tetR construction. After colony PCR, the monoclonal colonies were cultured and sent to the company for sequencing.

Plasmids P70a-σ28-P28-tetR were constructed from the gene fragment of original plasmid puc57 and the gene fragment of P70a-σ28-P28-tetR. After PCR, the results of agarose gel electrophoresis were as follows, with correct band sizes (Figure 12b).

figure-12 Figure 12. Schematic of the plasmid P70a-σ28-P28-tetR construction.
(a) The map of plasmid P70a-σ28-P28-tetR from SnapGene;
(b) On the left is 5k marker, in the middle is P70a-σ28-P28-tetR segment, on the right is the vector from plasmid P70a-σ28-P28-tetR;
(c) on the left is 5k marker, on the right is colony PCR verification strip.

After recombination and transformation, overnight culture, bacterial colonies grew on chloramphenicol plates. Through the colony PCR verification band, our gel results were consistent with expectations (Figure 12c), and the plasmid was successfully constructed after sequencing.

Construction of P28-tetO-deGFP plasmid
figure-13 Figure 13. Schematic of the procedure of plasmid P28-tetO-deGFP-ssrA construction. After colony PCR, the monoclonal colonies were cultured and sent to the company for sequencing.

The original plasmid P28-tetO-deGFP-ssrA was used to construct a plasmid with ssrA degradation tag removed (Figure 14a) to explore the effect of ssrA degradation tag on deGFP protein.

figure-14 Figure 14. Schematic of the plasmid construction.
(a) The map of plasmid P28-tetO-deGFP-ssrA from SnapGene;
(b) On the left is 5k marker, in the middle is deGFP fragment, on the right is the vector from plasmid P28-tetO-deGFP;
(c) On the left is 5k marker, on the right is colony PCR verification band.

PCR and Agarose Gel Electrophoresis (AGE) results were shown in Figure 14b, and the band was correct.

After template removal and purification, the plasmid was cloned and transformed into E. coli trans1-T1 competent cells for culture.

Colonies on resistant plates were selected for colony PCR, and the results were shown in Figure 14c with correct bands.

Validation of σ28
figure-15 Figure 15. Schematic of the procedure of Electroporation-Transformation E. coli BW25113 cells.

To enhance the expression intensity of each component, we use σ28 as the promoter factor. σ28 is a special transcriptional factor concerning the FliA, which is an alternate sigma factor for the class 3 flagella operons, and it does not exist in Cell-free system. To verify the functional availability of σ28, we build P70a-σ28-P28-deGFP plasmid. If the expression of σ28 is normal, it shows fluorescence. Conversely, there is no fluorescence.

To verify the normal expression of σ28, we performed electroporation experiments with E. coli BW25113 (Table 1).

Group set
Control Group BW25113
Experiment Group BW25113+P70a-σ28-P28-deGFP
Table 1.
figure-16

No colony in the control group fluorescenced (Figure 16a) , while the single colony in the experimental group fluorescenced (Figure 16b) , indicating that P70a-σ28-P28-deGFP was successfully transferred, all gene elements on the plasmid were functional, and σ28 was normally expressed.

Validation of tetR

Tet Repressor (tetR) is a repressor protein that inhibits fluorescence and indirectly feedback the sensity of the entire gene circuits. TetR repressoracts as a guard on gene circuits. Normally, tetR inhibits the expression of deGFP, but when it is cut by Cas9 protein, it will lose its inhibition effect. And it sends us a message by means of fluorescence.

TetR binds to the tetO site and thus blocks deGFP expression. Therefore, if P70a-σ28-P28-tetR is normally constructed, fluorescence is present in the control group and inhibited in the experimental group.

To verify the normal expression of tetR, we carried out an electroporation experiment with E. coli BW25113 (Table 2).

Group set
Control Group BW25113+P28-tetO-deGFP
Experiment Group BW25113+P28-tetO-deGFP+P70a-σ28-P28-tetR
Table 2.

The P28-tetO-deGFP plasmid was present in the control group (BW25113 + P28-tetO-deGFP), the fluorescence was normal (Figure 16c). The fluorescence in the experimental group (BW25113+ P28-tetO-deGFP +P70a-σ28-P28-tetR) should not exist theoretically. DeGFP is theoretically suppressed. However, in fact, there were three colonies with strong green fluorescence, as shown in Figure 16d, which may be because of the failure of transforming P70a-σ28-P28-tetR plasmid during electroporation, resulting in some colonies with strong fluorescence.

Some single colonies were selected for culture in the above three groups and their fluorescence intensity was measured, as shown in Figure 17 and Table 3.

Figure 17. Average ratio-standard deviation of fluorescence intensity from E. coli BW25113 cells containing the plasmids carrying P70a-σ28-P28-deGFP, P28-tetO-deGFP, P70a-σ28-P28-tetR with P28-tetO-deGFP.
Group 1 2 3 4 5 6 Average
BW25113+P70a-σ28-P28-deGFP  33456 29526 33285 30745 28582 35611 31868
BW25113+P28-tetO-deGFP  13225 13112 14765 15351 14796 14305 14259
BW25113+P28-tetO-deGFP+P70a-σ28-P28-tetR  4125 5736 5031 4098 5421 4579 4632
BW25113 2467 3308 3428 1980 2340 2766 2715
Table 3.

After transforming plasmids P28-tetO-deGFP and P70a-σ28-P28-tetR into E. coli BW25113, the fluorescence of BW25113 was significantly decreased compared with that of BW25113 and P28-tetO-deGFP, indicating that tetR reprssor could bind tetO site and thus reduce the intensity of deGFP.

Gene circuits

We aim to use Cell-Free system to detect RNA with CRISPR/Cas9 system. Based on the cleavage of Cas9 protein, we designed special gene circuits.

Plasmid P70a-σ28 Construction

Vector: P70a-deGFP (Figure 18a) segment: puc57-σ28

The vector and segment have been amplified by PCR. We verified all our PCR fragments, the fragment length of σ28 is about 720~750 bp by agarose gel electrophoresis, the fragment length of vector is about 2500~2550bp by agarose gel electrophoresis as we expected (Figure 18b). Then we conducted plasmid recombination.

We verified the recombination plasmid by colony PCR, the fragment length is about 1000bp by agarose gel electrophoresis as we expected (Figure 18c).

Plasmid p70a-σ28 was proved to be constructed successfully after sequencing.

figure-18 Figure 18.Schematic of the plasmid P70a-σ28 construction.
(a) The map of plasmid P70a-σ28 from SnapGene;
(b) On the left is 5k marker, in the middle is σ28 segment, on the right is the vector from plasmid P70a-deGFP reverse PCR;
(C) On the left is 5k marker, on the right is colony PCR verification strip.
Plasmid P28-tetR Construction

Vector: P28-tetO-deGFP-ssrA (Figure 19a) segment: puc57-tetR

The vector and segment have been amplified by PCR. We verified all our PCR fragments. The fragment length of tetR is about 720~750 bp by agarose gel electrophoresis. The fragment length of vector is about 2500~2550bp by agarose gel electrophoresis as we expected (Figure 19b). Then we conducted plasmid recombination.

We verified the recombination plasmid by colony PCR, the fragment length is about 1000bp by agarose gel electrophoresis as we expected (Figure 19c).

Plasmid P28-tetR was proved to be constructed successfully after sequencing.

figure-19 Figure 19.Schematic of the plasmid P28-tetR construction.
(a) The map of plasmid P28-tetR from SnapGene;
(b) On the left is 5k marker, in the middle is tetR segment, on the right is the vector from plasmid P28-tetO-deGFP-ssrA reverse PCR;
(c) On the left is 5k marker, on the right is colony PCR verification strip.
Plasmid P28-tetR-ssrA Construction

Vector: P28-tetO-deGFP-ssrA (Figure 20a) segment: puc57-tetR

The vector and segment have been amplified by PCR. We verified all our PCR fragments, the fragment length of tetR is about 720~750 bp by agarose gel electrophoresis, the fragment length of the vector is about 2500~2550bp by agarose gel electrophoresis as we expected (Figure 20b). Then we conducted plasmid recombination.

We verified the recombination plasmid by colony PCR, the fragment length is about 1000bp by agarose gel electrophoresis as we expected (Figure 20c).

Plasmid P28-tetR-ssrA was proved to be constructed successfully after sequencing.

figure-20 Figure 20.Schematic of the plasmid P28-tetR-ssrA construction.
(a) The map of plasmid P28-tetR-ssrA from SnapGene;
(b) On the left is 5k marker, in the middle is tetR segment, on the right is the vector from plasmid P28-tetO-deGFP-ssrA reverse PCR;
(c) On the left is 5k marker, on the right is colony PCR verification strip.

For more details about the verification results of "Gene Circuits", please refer to Engineering success.