Team:ZJUT-China/Experiments
This page provides all standardized protocols used in our experiments, including Plasmid Construction, Transformation, Cas9&gRNA in vitro Experiment, Cell-Free system and Fluorescence-based Analysis.
We constructed a new plasmid that can transcribe CXCL9 mRNA by using two existing plasmids (igRNA-CXCL9 and pTargetF). The primers were designed for PCR, and the PCR products were obtained after template elimination and purification. Then the constructed plasmid was transformed the plasmid into E.coli DH5αcompetent cells to make it self-replicating. Then we did colony PCR using colonies grown on resistant plates. We got the right plasmid after agarose gel electrophoresis and further sequencing.
CGG and AGG were selected as the protospacer adjacent motif (PAM) to alter the original N20 sequence in plasmid pTargetF. Primers gRNA-R and gRNA-F (Figure 1) were designed for PCR, and the PCR products should be dealt with template elimination and purification, and then transferred the fragments into E. coli DH5α to make them self-ligating. On this basis, we obtained five different gRNAs and measured their cleavage activity and electroporated gRNAs into cells with spCas9 and tetR plasmids and obtained highly active gRNAs by resistance selection in E. coli. Meanwhile, we transcribed gRNAs in vitro, cleaved the target segments with Cas9 protein, and obtained highly active gRNAs by agarose gel electrophoresis.
Note
spCas9(kanamycin resistence), gRNA(spectinomycin resistence), tetR(Ampicillin)
These plasmids have different replicons.
Figure 1. The map of gRNA from
SnapGene
| electroporate | gRNA+spCas9+tetR | resistance screening：Ampicillin |
| electroporate | spCas9+tetR | resistance screening：Ampicillin |
On the basis of screening gRNA1 with high cleavage activity, we added the sequence at the 3 'end to make it complementary with the RNA biomarker, and sent the sequence to Tsingke Biotechnology for synthesis. Primers with T7 promoters were designed, and igRNA transcription templates with T7 promoters were obtained through igRNA plasmids. In vitro transcription experiments were conducted according to the kit, followed by purification with chloroform or RNA purification kit, and the integrity of the bands was verified by agarose gel electrophoresis. After confirming the integrity of the strips (no degradation), the next step in vitro verification experiment can be carried out and The igRNA was stored at -80℃.
For details, please refer to Protocol: IVT, RNA purification.
In order to verify whether igRNA and CXCL9 mRNA can have base complementary pairing and bind to Cas9, an electrophoretic mobility shift assay(EMSA) experiment was carried out. First, the RNA in the -80℃ refrigerator was taken out and added into the annealing buffer for annealing, so as to form the correct secondary structure. Then，the experimental group and the control group were set according to the table. After Cas9 protein was mixed on ice for 30 min, samples were loaded into two pieces of 6% non-denatured polyacrylamide gel, and 120 V electrophoresis was performed in 4℃ for 1 hour. Finally, one was stained with GelRed for visualizing nucleic acid and the other was stained with Coomassie blue to check bright blue, and the binding was observed according to the GelRed-gel in the UV light.
For details, please refer to Protocol: EMSA.
| igRNA | CXCL9+igRNA | CXCL9 | Cas9 | |
| Test(CXCL9 mRNA-igRNA-Cas9) | - | + | - | + |
| Control 1(igRNA-Cas9) | + | - | - | + |
| Control 2(igRNA) | + | - | - | - |
| Control 3(CXCL9 mRNA) | - | - | + | - |
| Control 4(Cas9) | - | - | - | + |
To make the spCas9 plasmid better for expression in the Cell-Free system, we used one-step cloning technique to remove unnecessary genes and constructed a simplified version of the spCas9 plasmid.
Considering the difficulty of plasmid ligation due to excessive plasmid assembly and low transformation efficiency, we chose to synthesize a new plasmid P15A ori-CmR. Then we used spCas9 plasmid from laboratory and P15A ori-CmR to construct plasmid spCas9.
We first designed primers ori-F, ori-R, spCas9-F, and pCas-R for the two plasmids to conduct PCR. After clean-up and eliminate templates experiments, we measured the concentration and converted it, using the enzymes cloned in one-step for ligation of the fragments. Finally the plasmid was transformed into E. coli DH5α to culture overnight.
After the colonies grew, we picked out the colonies for colony PCR, and finally we got the right length and sequencing results.
For details, please refer to Protocol: Plasmid construction.
In order to verify whether our components can operate normally, we first perform in-bacteria verification, and mainly rely on electro- transformation for verification. First, we need to prepare BW25113 competent cells with deionized water and store them at -80°C for later use. Then prepare the P70a-σ28-P28-deGFP, P70a-σ28-P28-tetR and P28-tetO-deGFP required for our verification.
Design four sets of experiments, as follows.
| Group | design |
| Control group | BW25113 |
| test group 1 | BW25113+P70a-σ28-P28-deGFP |
| test group 2 | BW25113+P28-tetO-deGFP |
| test group 3 | BW25113+P28-tetO-deGFP+P70a-σ28-P28-tetR |
Conduct the electroporation experiment with an electroporator. After the electroporation is successful, add 970μL of LB liquid medium to the electroporation cup, pipette it evenly, transfer it to the EP tube, and culture with shaking for 1 hour. Centrifuge, discard 900 μL of supernatant, resuspend the cells, spread and culture. For details of the results, please view the Result：Cas9 group.
For details, please refer to Protocol: Electroporation-Transformation E. coli BW25113 cells.
All of the plasmids were validated in the Cell-Free system.
Each Cell-Free reaction system was 12 μL, including 9 μL σ70 reagent and 3 μL plasmid template. All experimental groups were set up as follows:
| P70a-σ28 | P28-tetR | P28-tetR-ssrA | P28-tetO-dGFP | P70a-ClpXP | Purpose | |
| Test 1 | 2nM | - | - | 10nM | - | Verify the effect of σ28 |
| Test 2 | 2nM | 5nM | - | 10nM | - | Verify the effect of tetR |
| Test 3 | 2nM | - | 5nM | 10nM | 1nM | Verify the effect of ClpXP |
| Test 4 | 2nM | - | 5nM | 10nM | - | Control group |
Since the total volume of plasmid template was only 3 μL, the more kinds of plasmids were added, the higher the concentration of plasmids we needed to prepare. Through plasmid extraction, we obtained plasmids with a concentration of about 500 ng/μL, which was concentrated enough for us to carry on.
Then we calculated the concentration, diluted and mixed each plasmid in equal volume, vortex gently, and add 3μL template mixture to the Cell-Free system. After vortex again, we transfer 5μL to the two wells of the 96-well V plate preheated at 29℃ for 30 minutes in advance. After sealing the film, put it into a microplate for fluorescence dynamic detection.
Detailed analysis of the results obtained from the experiment is on the page of Engineering Success.
For details, please refer to Protocol: Cell-Free system.
- LB Media
- Polymerase Chain Reaction（PCR）&Colony PCR
- Elimination template and PCR Clean-up
- DNA Gel Extraction
- Agrose Gel Electrophoresis
- One-Step Cloning
- Preparation of chemo-competent E. coli KL740 cells
- Transformation of chemo-competent E. coli KL740 cells
- Transformation of chemo-competent E. coli DH5α cells
- Plasmid microextraction
- Mass plasmid DNA extraction
- Preparation of chemo-competent E. coli BW25113 cells
- Electroporation-Transformation E. coli BW25113 cells
- In vitro transcription
- RNA purification
- In vitro DNA digestion with Cas9 Nuclease
- EMSA(Gel-shift Assay)