Part:BBa_K3365017
pBAD upstream of eGFP with inhibition unit containing lure3
To facilitate measurements, we use the eGFP as a reporter protein, which can be exchange to other reporters according to the users’ needs. The eGFP gene sequence (BBa_K1875003) is located directly downstream of BBa_K3365009 (Lure3 sequence downstream of pBAD) followed by BBa_B0015 (rrnBT1-T7TE). The transcription of the eGFP gene is under the control of the inducible pBAD/araC promoter designed to carry PAM and lure3 sequence downstream of pBAD. In our part, the “lure3” is the potential off-target sequence for PDCD1 CRISPR gene editing predicted by our off-target predictor software built in 2019, which might be identified and bound by the complex of dCas9 and sgRNA. According to the off-target predictor software, the relative off-target rate is 73.6673%.
Usage and Biology
This part is used to expression of eGFP protein regulated by the signal of arabinose and the off-target or not of dCas9. The pBAD is regulated by the AraC protein, which is both a positive and a negative regulator. The binding of dCas9 to any position within the region between the promotor and RBS might prevent transcription. Therefore, the uninduced transcriptional level of eGFP 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 lure3 sequence wrongly 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 off-target rate that inhibits the expression of reporter gene.
Results
The promoter of the L-arabinose operon of E. coli (pBAD) is amplified to add restriction cutting sites, lure3 sequence and part of the RBS sequence (BBa_K3365002). The eGFP with double terminator is amplified to add restriction cutting sites and part of the RBS sequence (BBa_K3365002). The above PCR products are ligated through overlap PCR to get the fragment with suitable restriction sites and the product can be ligated into its backbone pUC57 by enzymic digestion and connection. The eletrophoretic profile of the overlap PCR and colony PCR product and the sequencing result reveal the successful construction of the fragment.
This part is characterized in combination with BBa_K3365014. It is ligated to pIn-RTr by enzymic digestion and connection and the the colony PCR product reveal the successful construction of the plasmid, named pIn-RTL3.
To verify the inhibition effect of dCas9 on pIn-RTL3, 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 induce the expression of RFP and eGFP.
The results are shown below, from which we could see 38.5 times of fluorescence/OD600 difference between transform group (without the expression of dCas9) and co-transform group (with the expression of dCas9) in RFP and 3.96 times in eGFP, indicating our transcription inhibition system has better effect on target site than lure sites, which matches our prediction.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1053
Illegal XhoI site found at 1062 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 74
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 56
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