Team:SJTU-BioX-Shanghai/Engineering
Engineering
Part construction and expression verification
Overview
To facilitate the evaluation of the on/off-target level of the dCas9, one part, which is the pBAD/araC upstream of RFP with inhibition unit containing target (BBa_K3365014) is constructed and the RFP is used as one of the reporter proteins.
The RFP gene sequence (BBa_K3365052) is located directly downstream of one target sequence (BBa_K3365006) followed by rrnBT1-T7TE (BBa_B0015). The transcription of the RFP gene is under the control of the inducible pBAD/araC promoter designed to carry PAM and target sequence downstream of pBAD. In our part, the “target” is the target sequence for PDCD1 CRISPR gene editing in one clinical trial, which can be identified and bound by the complex of dCas9 and corresponding sgRNA[1].
This part is used to express RFP protein regulated by the signal of arabinose and dCas9. The pBAD is regulated by the AraC protein, which is both a positive and a negative regulator[2]. The binding of dCas9 to any position within the region between the promotor and RBS might prevent transcription[3][4]. Therefore, the uninduced transcriptional level of RFP is very low. In the presence of arabinose, transcription from the pBAD promoter is turned on and there will be a relatively strong fluorescence expression. In the presence of both arabinose and the complex of dCas9 and sgRNA, the complex might bind to the target sequence and the transcription is partially inhibited because of the block of RNAP. So, a relatively weak fluorescence expression of bacteria indicates a dCas9 with higher on-target rate that effectively inhibits the expression of reporter gene.
Verification of the successful construction of this part
The inducible pBAD/araC promoter (BBa_K3365013) is amplified with the primer F1/R1 to add restriction cutting sites, target sequence and part of the RBS sequence (BBa_K3365002). The RFP with double terminator is amplified with the primer F2/R2 to add restriction cutting sites and part of the RBS sequence (BBa_K3365002). The above PCR products are ligated through overlap PCR with the primer F1/R2 to get the fragment with suitable restriction sites and the product can be ligated into its backbone pUC57 by enzymic digestion and connection.
The electrophoretic profile of the overlap PCR (F1/R2) and colony PCR (M13 fwd/ M13 rev) product and the sequencing result reveal the successful construction of the fragment and the plasmid, named pIn-RTr.
F1: 5’-CGAGCTCTTATGACAACTTGACGGCTACATCATTCAC-3’
R1: 5’-TTCTTAAAGGCAGTTGTGTGACACGGAAGCGGATGGAGAAACAGTAGAGAGTTGCG-3’
F2: 5’-TTCCGTGTCACACAACTGCCTTTAAGAAGGAGATATACATATGGCGAGTAGCGAAGACG-3’
R2: 5’-GGAATTCCCGCCCTAGGTATAAACGCAGAAAG-3’
M13 fwd: 5’-GTAAAACGACGGCCAGT-3’
M13 rev: 5’-GTCATAGCTGTTTCCTG-3’
Lane 1: 5000 bp marker; Lane 2-13: overlap PCR product
Lane 13-15 show positive results.
Verification of the RFP protein expression
To verify the inhibition effect of pdCas9 on pIn-RTr, we set up six culturing system (as shown in the table below) and used microplate spectrophotometer to examine fluorescence intensity. L-arabinose was added to induced the expression of RFP.
| pIn-RTr | pdCas9 | L-arabinose | |
|---|---|---|---|
| MG1655 | + | + | + |
| MG1655 | + | + | - |
| MG1655 | + | - | + |
| MG1655 | + | - | - |
| MG1655 | - | - | + |
| MG1655 | - | - | - |
The results are shown below, from which we could see 64.39 times of fluorescence/OD600 difference between transform group (without the expression of pdCas9) and co-transform group (with the expression of pdCas9), indicating our transcription inhibition system did work.
To know about the inhibition of the expression of RFP by dCas9 in a single cell as well as verify our mutant library, the obtained dCas9 mutant library and the wild-type dCas9 are transformed into MG1655 carrying pIN-RTr. Then, we analyze the red fluorescence by flow cytometry.
The results are shown below. 77.12% cells have positive red fluorescence in the absence of dCas9, while the proportion of positive cells decreases to 0.08% in the presence of dCas9, showing a quite efficient inhibition of wild-type dCas9. And the positive rate rises to 78.37% when the cells are transformed into dCas9 mutant library, which means the lack of function in most of mutant dCas9 and the successful construction of our mutant library.
References
[1] Lu Y, Xue J, Deng T, Zhou X, Yu K, Deng L, Huang M, Yi X, Liang M, Wang Y, Shen H, Tong R, Wang W, Li L, Song J, Li J, Su X, Ding Z, Gong Y, Zhu J, Wang Y, Zou B, Zhang Y, Li Y, Zhou L, Liu Y, Yu M, Wang Y, Zhang X, Yin L, Xia X, Zeng Y, Zhou Q, Ying B, Chen C, Wei Y, Li W, Mok T. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat Med. 2020 May;26(5):732-740.
[2] Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121-30.
[3] Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013 Feb 28;152(5):1173-83.
[4]Vigouroux A, Bikard D. CRISPR Tools To Control Gene Expression in Bacteria. Microbiol Mol Biol Rev. 2020 Apr 1;84(2):e00077-19.
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