Construction of the hamS Mutant Library by Error-prone PCR
1. The catalytic module consisted of hmaS gene. The HmaS coding sequence (hmaS), which can express HmaS protein, having key function in the conversion of 4-hydroxyphenylpyruvate (HPP) to 4-hydroxymandelate (HMA). And we constructed a hmaS mutant library by error-prone PCR.
2. The sensing module consisted of mCherry gene, which could transcribes red fluorescent protein. The fluorescence intensity is coupled to the enzyme activity.
3. The cytosine deaminase codA gene that converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), which kills bacteria. And the catalytic activity is coupled with the strength of the reporter.
Construction of the gY9s-dual T7-Trrnb-HmaS(Scpa1)-Bio177 Plasmid
1. The catalytic module contains 2 parts, dual T7 promoter and hmas gene. The high specificity of T7 RNA polymerase and T7 promoters enabled the specific mutations during transcription. The HmaS coding sequence (hmaS), which can express HmaS protein, having key function in the conversion of 4-hydroxyphenylpyruvate (HPP) to 4-hydroxymandelate (HMA).
2. The sensing module contains of PobR promoter and mCherry gene. The PobR gene encoding the allosteric transcription factor PobR protein that specifically responds to 4-hydroxyphenylpyruvate (HPP). And mCherry gene, which could transcribe red fluorescent protein. The fluorescence intensity is coupled to the enzyme activity. The cytosine deaminase codA gene that converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), which kills bacteria.
Construction of Mismatch sgRNAs Mutant Library
Construction of mismatch sgRNA Target eGFP
1. dCas9: the codon GAT for Asp10 of the cas9 gene was replaced by Ala and the codon CAC for His840 of the cas9 gene was replaced by Ala.
2. sgRNA targeted the coding sequence (+40bp) of eGFP
Construction of Polygenic Mismatch sgRNA
1. Replace the ori to obtain R6K-dCas9-eGFP as the base plasmid for subsequent characterization. Replace sgRNA spacers to individually target pykF, tyrB, tyrR and pheA in R6K-dCas9-eGFP, and thus generated R6K-dCas-pykF, R6K-dCas-tyrB, R6K-dCas-tyrR, R6K-dCas-pheA.
2.These four sgRNA fragments were linked together by the Golden Gate method to generate the R6K-dCas9-pykF-tyrB-tyrR-pheA plasmid. Using degenerate primers in the amplifications of these sgRNAs to obtain their mutated fragments, which were subsequently subcloned into vectors to construct sgRNA-mutated plasmids leading to the construction of the mutant library.
Characterization of CRISPRi Platform
1. To assess the capacity of the CRISPRi system in repressing eGFP gene expression, the R6K-dCas9-eGFP plasmid was co-transformed with the reporter plasmid pYB1a-eGFP into E. coli BW25113 to induce expression. Then measure Cell density (OD600) and green fluorescence intensity by 96-well plate reader.
2. To test the effect of gene inhibition based on CRISPRi in HPP production process, pATP and R6K-dCas9-sgRNA were co-transformed into E. coli BWΔCD. The bacteria were collected by and further cultivated at 30℃ for 24 h, HPP levels were detected by HPLC.
