Preliminary Study On The Construction Of High-efficiency Recombinant Engineering Strains Of Escherichia Coli And Its Use In Broadening The Scope Of Gene Editing

Recombineering,also called ?-Red/ET,is a biotechnology through the recombainse-catalyed homologous recombination between DNA molecules.The main recombinases include the Reda?? from ? phage and RecET from Rac prophage.The outstanding advantages of recombineering include that there is no restriction of the DNA locus,size,or restriction enzyme recognition sites.The ssDNA recombineering uses the synthesized oligonucleotide as substrate(instead of the dsDNA obtained via PCR or enzyme digestion),therefore,it is most convenientCRISPR/Cas(Clustered regularly interspaced short palindromic repeats and associated proteins)is the adaptive immune system developed through the long-term evolution by bacteria and archaea.Currently,the CRISPR/Cas9 system exploited in strain Streptococcus pyogenes SF370 is the mostly throughly studied and most widely used gene editing system.Guided by crRNA(CRISPR RNA),Cas9 specifcially recognizes and cleaves target DNA with high efficiencyEscherichia coli primase DnaG is essential for DNA replication,and researchers found that both DnaG Q576A and DnaG K580A increase the homologous recombination efficiency.By coupling the ssDNA recombineering and CRISPR/Cas9 technologies,our group obtained Q576A,K580A,Q576V K580A,and Q576T K580A variants.In the first part of the thesis,a two-step procedure was developed to eliminate the two plasmids in the engineeried strains.The first step involves the transformation of a self-cleavage plasmid pLS3550 which drives out the cas9 expression plasmid pLS3535 via plasmid incompatibility.The second step involves the caleavage and elimination of the two plamids via the heat-inactivated chloroamphnicol(cTc)-induced homing endonuclease I-Scel.The recombineering efficiency of the resultant strains was compared by two methods.One is the dsDNA-mediated chromosomal laZ' deletion while another is the ssDNA-mediated antibiotic resistance gene repair.Both methods showed that the E.coli LS4019 harboring DnaG Q576T K580A has the highest recombineering efficiencyTo futher improve the strain's recombineering efficiency,the recJ,sbcB,xseA and exoX genes were deleted from the E.coli ?mutS strain LS3508.The function of each gene is that recJ gene encodes a 5'?3' single stranded DNA exonuclease,sbcB gene encodes a 3'?5' DNA exonuclease,xseA gene encodes exonuclease III,and exoX gene encodes single stranded DNA exonuclease.The recombineering effienciency of resulting strains were assay via the ampicillin resistance gene repair.It turned out that sbcB knock out strain showed the highest recombineering effienciency(up to 13.8%).Similarily,gene deletions were perfomed on LS4019;however,the recombineering effienciency was decreased.We constructed cas9 and red?-cas9 expression plasmids which will be used for the establishement a plasmid gene editing methodThe third part of the thesis involves the expand of the scope of gene editing Firstly,plasmids pLS4046 and pLS4047 were constructed which harbor the L-arabinose-inducibe toxic gene ccdB with PAM NNG and NG,respectively.Each plasmid harboring strain did not grow or grew weakly under the L-arabinose-induced CcdB expression,suggesting the toxicity of CcdB killed the cells.Then the cleavage function of the ribonecleoprotein complex was tested.The full RNP expressing PAM NGG plasmid pLS4043,pLS4043 derivative pLS4044 in which part of cas9 was replaced by ccdB,PAM NG plasmid pLS4055 and pLS4057.pLS4055 contains Cas9 T1337R R1335V A1322R E1219F G1218R D1335V,while pLS4057 has a L1111R plus.The four plasmids were individually transformed into the cells contraing pLS4046 and pLS4047 under the selection of L-arabinose and chloroamphenicol.It turned out that pLS4043,pLS4055 and pLS4057 all could act on PAM NGG,while pLS4055 and pLS4057 act on NG.On the othe hand,cas9 in pLS4044 was dysfunctional,indicating the feaasibility of the cleavage system.Finally error-prone PCR was applied to onbtain cas9 variants for expanded recoginition scope.In the preliminary experiment,8 clones was sequened and it was found that all the clones were mutated with different sites.The result lays the foundation for further mutagenesis experiments.