Two Strategies For Chromosomal Integration Of Multiply Copies Of An Insert In RecA~+ Escherichia Coli Strains

Currently,a great deal of work on metabolic engineering and synthetic biology is still based on a plasmid system.The plasmid system is feasible in the laboratory,but not suitable for large-scale industrial applications.This is mainly because the plasmid system generally requires the maintenance of an external pressure which will increase the cost in a great extent.Additionally,the existence of structural instability and genetic instability makes the plasmid system more unfavorable.The genomic integration method is the only way to solve this problem.In the large amount of reports about genomic integration method,the single copy gene integration is the mainstream.However,the single copy chromosomal integration of external DNA often results in decrease of protein production.So the multi-copies gene integration and its related methods have aroused people's attention.However,there are also shortcomings in the current multi-copies chromosomal integration methods in bacteria.It is mainly because the multi-copy integration methods reported in bacteria can only be carried out in the RecA-strain with homologous recombination defects,but RecA-strains normally have more slow growth rate and more poor robustness,which seriously limits the utilization of these methods in industry field.Our laboratory has developed a method of multi-copies chromosomal integration by using transposome in vitro,multiple copies of the external DNA could be integrated into RecA+E.coli strains which has homologous recombination ability.However,there are still many shortcomings of this method:the number of clones obtained by transposon is few.the results are not stable and some defects are complicated.These faults affect its application.Basing on this work,we proposed two new strategies to complete the integration of external DNA into E.coli chromosomal DNA in multiply copies.1.The integration of external DNA with repeated operations for multiply copies.We constructed a plasmid "pKD46::Ptrc-TnpA" and have TnpA induced by IPTG under the control of the Ptrc promoter.Then,an artificial DNA fragment "ME-FRT-Kan-FRT-Ptrc-eGFP-ME" with both ends have 19 bp 5'-phosphated ME sites that could be recognized by Tn5 transposase for transposon reaction was constructed and amplified.Then,this DNA substrate was electroporated into the E.coli cells with induced Tn5 transposase.This method is successful,but its efficiency is still low.Next,we construct a plasmid "pKD4::2ME-Ptrc-eGFP" which has ME-FRT-Kan-FRT-Ptrc-eGFP-ME and has R6K ori.By using this plasmid as the substrate for Tn5 transposase,the efficiency of transposon increased more than 10 folds.By using this method,we successfully integrated the E.coli BL21(DE3)with 3 copies and one PHB gene was also successfully integrated into the genomes of BL21(DE3)for 1.5%PHB production.However,the attempt to construct "mini-Tn5" plasmid for the integration of Ptrc-tnpA operon with its substrate ME-FRT-Kan-FRT-Ptrc-eGFP-ME was failed,further demonstrating that our method is more reliable than the traditional method.2.The multiply copies integration of external DNA in E.coli cells with single operation in the help of transconjugationIn this part,we successfully developed a new method for multiple copy integration of external DNA in E.coli cells with single operation.With the help of S17-1?pir and"mob" sequence,we constructed a transconjugation system,which could mobilize the plasmid that have ME-FRT-Kan-FRT-Ptrc-eGFP-ME from S17-1?pir into receipt E.coli cells.The efficiency was increased more than 100 folds than that of electroporation We have tried to screen for multiple copy insertion with single transconjugation operation.However,we found a lot of false positive colonies.Later,we find the reason is due to the gene encoding for pir protein was also mobilized into the receipt cells due to the intrinsic reason.MFDpir strain was used instead of S17-1?pir strain and was found to reduce the false positive colonies in a great extent.Three selection markers,Cmr,tricloson and Kan.were tested for its ability to be used for high copy selection with increased concentration.However,only Kan was found to behavior as expected in practice.Besides,we further compared the electroporation and transconjugation method in the multiply insertion,it is found that colonies were only found on plates with elevated Kan concentration.It demonstrated that transconjugation was the only selection for our purpose.By using this method,we successfully integrated 5 copies of Pkat-eGFP DNA into RecA-XL-1 Blue MRF' strain and 8 copies of Pkat-eGFP DNA into RecA+ MG1655 strain.In conclusion,we developed two different methods for multiply copies chromosomal integration by using different transformation/mobilization method.It is the only multiply copies chromosomal integration method that could be used for RecA+E.coli cells.It has more potential to be used in engineering strains that normally have RecA gene on its chromosome.