Construction Of Promoters With Strong Regulation On Chromosome Of Escherichia Coli

Bacterial promoter is an important regulatory element of gene expression in bacteria,which decides the strength and opportunity of bacterial gene expression.Promoter constructed on the chromosome would solve the problems such as the burden on the host and plasmid stability when constructing the promoters on plasmid.With the development of synthetic biology and metabolic engineering,the demand of constructing promoter on chromosome is more stronger and stronger.Therefore,this study will construct promoters on chromosome of Escherichia coli,and select promoters with strong strength,including constitutive promoters and induced promoters with tightly control.Artificial promoter M1-37,M1-46 and M1-93 from our lab were constitutive promoters;PrrnD from Escherichia coli as constitutive is often used in literature,which is a ribosomal rRNA promoter.Using GFP as reporter gene,these four promoters sequences(M1-37.M1-46,M1-93 and PrrnD)were assessed after inserted on chromosome.96 microtitor plates(bottom transparent blackboard)fluorescence analysis showed that the expression strength of M1-37,M1-46,M1-93 and PrrnD were 1.5,1,2.67 and 5,respectively,when comparing with the Plac promoter as control.Obviously,PrrnD was the strongest expression as constitutive promoter.In order to obtain constitutive promoter with higher strength,we designed a novel strategy to select strong promoter based on PrrnD promoter using RBS library,of which the resistance kanamycin gene is connected after gfp gene under the same promoter control like operon,and the colonies grown on higher kanamycin medium would be the promoters with higher expression.Using this strategy,some stronger promoters such as P10 and P36 were screened from RBS library of Prrn.Comparing with native promoter PlacZ,the expression levels of P10 and P36 were 5-and 10-fold.As a result,the promoter P36 was the constitutive promoter with highest strength.Four elements including tetO,lacO,araC and rhaR which regulated by TetR,LacI,AraC and RhaR respectively,and two sources of PL and Plac promoter sequence,were selected for designing six promoters including Ptet02,PtetO3,PlacO2,PlacO3,PlacO+ara and PlacO+rha in this work.CRISPR/Cas9 system was applied to integrate these promoters into chromosome of Escherichia coli ATCC 8739.Promoter strength and relative expression with or without inducing agents were determined by the fluorescent quantities of GFP protein.The results showed that the expression strength of Ptet02,Ptet03,Plac02,Plac03,PlacO+ara and PlacO+rha were 0.02,0.02,0.84,0.77,0.6 and 0.02,respectively,without adding agents,and were 4.5,3.7,0.97,0.82,6 and 12,respectively,with adding agent.Among of them,hybrid promoter PlacO+rha was the best one with tight regulation.The expression strength of Plac+rha was 0.02 in absence of the inducer,and the maximum expression intensity was 12-fold higher than that of the induced lacZ promoter,and the range of expression intensity was 600-fold.This study obtained strong promoter P36 with constitutive expression and Plac+rha with tight regulation would provide a good foundation for the application of precise regulation of gene metabolic engineering and synthetic biology in gene expression.