Construction And Application Of Size-Reduced Plasmids Based On TMP Resistance Gene DfrB10

Objective1.Construct pET24a-dfrB10 plasmid and transform E.coli to verify that host bacteria carrying dfrB10 gene expression plasmid are resistant to trimethoprim(TMP);2.Replace the ampicillin(AMP)resistance gene Amp R on the pUC19 plasmid with dfrB10 gene(TmpR)to detect the drug sensitivity of E.coli transformed by pUC19(TmpR)plasmid to TMP,which provides a basis for establishing a dfrB10-TMP resistance screening system;3.The miniaturized plasmids of pUC19,pET28 a,pc DNA3.1 and pX330 series with TMP resistance were constructed,and whether dfrB10 as a plasmid resistance screening marker affected the original function of the plasmid by comparing the function with the original plasmid.4.Based on pUC19(TmpR),a miniature plasmid p TMi was constructed to provide a smaller tool plasmid for molecular biology research.MethodsSynthesize the dfrB10 gene sequence,insert it into the polyclonal site of the pET24 a plasmid,transform Escherichia coli,apply kanamycin(KANA)and TMP doubleresistant plates,and observe whether E.coli carrying pET24a-dfrB10 plasmids has the ability to resist TMP drugs.The pUC19(TmpR)plasmid was constructed,the linearized vector pUC19(without Amp R)was amplified by PCR,the vector was homologous recombinant ligated to the dfrB10 fragment,and the drug sensitivity of Escherichia coli TMP carrying the pUC19(TmpR)plasmid was detected by microdilution method.The same foreign gene fragment was inserted into the polyclonal site of the pUC19(Amp R)original plasmid and the pUC19(TmpR)novel plasmid in the same method to compare the cloning efficiency of the two plasmids.The pET28a-eGFP(TmpR)prokaryotic expression plasmid was constructed by homologous recombination method,and the expression strains of the two resistant expression plasmids were transformed to induce e GFP protein expression,and the fluorescence intensity of the two was compared by fluorescence microscopy observation and multifunctional microplate reader.The pc DNA3.1-e GFP(TmpR)eukaryotic expression plasmid was constructed by homologous recombination method,and the two resistant plasmids were transformed into HEK 293 cells,and the cell fluorescence was observed after culture to detect the fluorescence intensity.The pX330(TmpR)plasmid was constructed by homologous recombination method,the AAVS1 gene was selected as the target,two resistant CRISPR plasmids were constructed,HEK 293 cells were transformed respectively,cell DNA was extracted after culture,and the target gene fragment was amplified by PCR,and gene editing was identified by T7 EI digestion and sequencing.Using pUC19(TmpR)as a template,primers were designed,PCR amplification replication start site(Ori),resistance gene fragment dfrB10(including resistance gene promoter),the product fragment was ligated by homologous recombination method and transformed,a single colony was selected,the plasmid was sequenced and extracted,and the miniaturization tool plasmid p TMi was constructed.ResultsAfter the transformation of the pET24a-dfrB10 plasmid into E.coli,colonies grew normally on KANA and TMP double-resistant plates,and control bacteria containing pET24 a empty plasmids grew aseptically on bispecific plates,indicating that recombinant bacteria carrying pET24a-dfrB10 plasmids had the ability to resist TMP after induction expression.The clonal plasmid pUC19(TmpR)constructed by replacing Amp R with the dfrB10 gene(TmpR)reduced its length by 624 bp.Recombinant bacteria can grow normally in the concentration range of 8-128 mg/L TMP,and dfrB10-TMP is most suitable for 64 mg/L when used as a marker for resistance screening.There was no significant difference in cloning efficiency between pUC19(TmpR)and the original plasmid pUC19(Amp R).The newly constructed prokaryotic expression plasmid pET28a-e GFP(TmpR)transformed E.coli into E.coli and induced the bacteria to express green fluorescence,and its intensity was similar to that of the original plasmid pET28a-e GFP(Kana R).The results of SDS-PAGE electrophoresis showed that the e GFP protein induced expression of the two resistant plasmids transformed by E.coli was similar.After the miniaturized eukaryotic expression plasmid pc DNA3.1-e GFP(TmpR)was constructed,HEK 293 cells were transfected,and the results showed that the fluorescence expression and fluorescence intensity of TMP-resistant plasmid were similar to those transformed by the original AMP-resistant plasmid.After the construction of the TMP-resistant CRISPR plasmid pX330(TmpR),it was successfully ligated to AAVS1-specific sg RNA.The two plasmic CRISPR granules pX330(TmpR)and pX330(Amp R)were transfected with 293 cells,and compared with the untransfected 293 cells,the sequence results of AAVS1 gene fragments in both transformed cells showed sleeve peak formation.The newly constructed plasmid p TMi,with only Ori,TMP resistance screening markers and polyclonal sites on the plasmid backbone,was sequenced to be consistent with the design,with a total length of only 988 bp.Conclusion1.E.coli carrying a recombinant plasmid containing dfrB10 gene has the ability to resist drug TMP,and dfrB10-TMP can be used as a resistance screening marker for novel plasmid construction.2.The resistance genes of pUC19,pET28 a,pc DNA3.1 and pX330 plasmids were successfully replaced,and a series of plasmids with TMP resistance were constructed.3.The miniaturized tool plasmid p TMi was successfully constructed,with a total length of only 988 bp,which is the smallest known artificially constructed tool plasmid.4.Using dfrB10 gene as a resistance marker,TMP drug screening can be used to modify various miniaturized plasmids with similar functions to the original plasmid for molecular genetics and other research applications.