The Construction And Application Of T7 Transcription Screener

Transcription system is an important component for protein expression,and its transcriptional strength directly affects the expression of genes downstream of the promoter.The basic method for measuring the strength of the transcription system is introducing a reporter gene downstream of the promoter and determining the strength of the reporter gene expression to determine the strength of the transcription system.However,due to the large number of factors affecting the expression of foreign proteins,and like the T7 transcription system,its transcriptional ability is so strong that sometimes the transcription process will obviously affect the normal growth and metabolism of the host,so mutations will occur,which will reduce or lose its transcriptional ability.Based on the above problems,a transcription intensity screener was constructed according to the phenomenon of transcriptional read-through of the terminator,in which a pressure-selecting gene("RBS-resistance gene" structure)was inserted downstream of the gene terminator,constituting a polycistronic structure with the upstream gene of the terminator.If the transcription intensity screener has strong transcriptional ability and large read-through probability,the resistance gene will has corresponding a large amount of transcription and the host will get strong resistance.The transcription ability is positively related to the resistance of the resistance gene,so the transcription systems with different transcriptional abilities can be judged and naturally screened out.By changing the antibiotic concentration,T7 transcription systems with different transcriptional intensities can be detected and screened,thus an adaptive and stable T7 transcription system can be obtained through process control.Therefore,the experimental results of this paper includes the following four points:(1)Construction of the T7 transcription screeners.This paper requires the rapid screening of stable,different-intensity T7 transcription systems.By modifying the T7 single-plasmid expression system,the original inducible system was transformed into a constitutive system to increase the burden on the host,which resulting in instability of the transcription system.After that,the screener structure was introduced into the expression vector,and a series of T7 transcription screeners were constructed successfully by replacing the promoter and the reporter genes;(2)Explore the factors that affect the ability of transcriptional to read through.The promoter of the transcription system,the length of the target gene,the structure of the target gene,and the structure of the RBS-resistance gene were studied through the cell growth curve,the longer the gene length,the more complex gene structure and the lower read-through rate,the more obvious the growth inhibition;(3)Detect and screen transcription systems with different transcriptional strengths by screeners.Through subculture experiments,the strains BL21(No lacl-1 × GFP-RD17)and BL21(No lacl-1 × GFP-RD7)can be screened by spectinomycin to transcription systems with different transcriptional intensities by the results of average fluorescence intensity and flow cytometry,and an adaptive and stable transcription system was obtained in this paper.(4)The applicability of the screener was studied from the length of the reporter gene and the structure of the gene,the stability problem can be solved by changing the resistance concentration.Furthermore,the constructed transcription screener KT2440-RD1 was verified by flow cytometry to eliminate non-fluorescent cell peaks in the P.putida KT2440 from self-expressing read-through plasmids pATG-ZBD-99-Gm-No CAP.