| Conventional DNA cloning is the process of combining exogenous genetic material from various sources with vector DNA,transforming host cells,selecting transformants containing the target gene,and finally forming a DNA molecule with self-replication ability.DNA cloning is a common technique used in biological research and is a major cornerstone for molecular biology.Escherichia coli has always been one of the core hosts of DNA cloning because of its earlier application,rapid growth,and simple preparation of competent cells.DNA cloning consists of two processes:DNA fragments joining and DNA transforming into host cells.In order to construct a highly efficient and simple DNA cloning system,we tried to explore the most efficient methods of DNA joining and DNA transformation.The traditional method of DNA joining relying on DNA digestion by restriction enzymes and re-ligation by T4 DNA ligase has been written into textbooks.In the past 10 years,Sequence based-homology DNA assembly methods have gained adaptation to meet the requirements of higher efficiency,fidelity,and simplicity.The DNA is assembled either in vivo or in vitro.For in vitro methods,DNA fragments are treated with enzymes to join DNA fragments and now is widely used.The in vivo method is simpler,as the DNA fragments are joined relying on the intracellular homologydependent DNA assembly(InHDA)ability of the hosts,such as Saccharomyces cerevisiae or Escherichia coli.S.cerevisiae is a commonly used host for in vivo DNA joining because of its strong recombination ability,but the DNA transformation process of yeast is cumbersome and its growth is slow,which is the main reason that has not replaced the E.coli for daily cloning.E.coli has lower recombination ability,and the mechanism of short homologous recombination is unclarified which limits the application in DNA cloning.The investigation of InHDA process in E.coli may aid the development of more efficient DNA cloning methods with E.coli as the host.Chemical transformation and electroporation are two main methods to transform DNA into E.coli.High transformation efficiency(TE)was commonly more favored,which in turn increases the cloning efficiency,especially for the simultaneous cloning of multiple fragments and DNA library constructions.Though electroporation can reach up to 1010 CFU/μg DNA with supercoiled plasmids,it is not frequently used with the in vitro methods,as the assembled DNA must be purified before electroporation.DNA loss during purification and cell damage from electroporation may lead to low cloning efficiency.Thus,chemically prepared competent cells are usually used with the in vitro methods.A variety of chemically competent preparation methods have been reported,such as Hanahan,Inoue,TSS,etc.However,under standard laboratory conditions,the TE can only reach 108 CFU/μg DNA,which is much lower than commercial competent cells.The development of high competent cells will be benefit to the efficient DNA cloning.In order to solve the above-mentioned problems,my research was as follows.1.The mechanism of InHDA pathway in E.coli was investigated.The RecA-strain XL1-Blue MRF’ was selected for the further study,and a method to measure the InHDA activity was developed to further investigate the InHDA pathway.Firstly,the mutants involved in three known recombination pathways were constructed and they did not affect InHDA in E.coli.The results suggested that InHDA was based on a new mechanism different from the known homologous recombination pathways.Accordingly,we proposed a new hypothesis:The process of InHDA requires the involvement of exonuclease to create a single strand ends.The genes encoding these 3’-5’single strand,3’-5’ double strand,5’-3’ single strand,5’-3’ double strand exonucleases were sequentially knocked out to generate multiple gene deletion mutants.The InHDA assay showed that 3’-5’ dsDNA ExoⅢ and ExoⅩ contributed to the exonuclease activities.The conclusion was verified by complementation and in vitro assays.The DNA polymerase and DNA ligase that may be involved in the InHDA pathway were then studied.The results showed that Pol I was the key polymerase in InHDA.Complementation experiments with site-directed mutations confirmed Pol I’s three activities 5’-3’ DNA polymerase activity,5’-3’ exonuclease activity and strand displacement activity all participated in the InHDA pathway.Finally,the DNA ligase was studied by gene knockout,and the results indicated that LigA participated in the InHDA process.Based on the above results,the short homologous recombination pathway in E.coli was finally determined.2.A strain of E.coli BW3KD with high TE was constructedE.coli BW25113 grew rapidly and had high TE than commonly used cloning strains.We then did a systematic study for constructing a better cloning strain from E.coli BW251 13.First.three different preparation methods were used to compare the TE of E.coli BW251 13 with commonly used E.coli cloning strains.The TE of BW25113 was 100-fold higher than those of others.Second,the known genes that may affect cloning efficienty were inactivated in E.coli BW25113.RecA was important for maintaining the high TE in BW25113.Further investigation showed that the RecAstrains had less live cells,which might contribute to the reduced TE.Since RecA was thought to cause plasmid instability,the DNA assembly efficiency and plasmid stability of BW25113 were tested with four kinds of plasmids:plasmids with direct repeat,plasmids with tandem repeat,plasmids contain repeat sequences with the genome and plasmid with different replicons.These plasmids were successfully maintained in the RecA+BW25113.When endA,fuhA,deoR were deleted,the strain B W3KD and its parent B W25113 had similar TE.Since the deletions might facilitate the cloning with large plasmids,the RecA+strain BW3KD was further developed and used for DNA cloning.3.A high TE method TSS-HI was developed with BW3KD.The TSS-HI method combined the advantages from the TSS,Hanahan,and Inoue methods.The most improvement of the TSS-HI method is the addition of Mn2+ to the TSS buffer and the heat shock in transformation.The TE of competent BW3KD cells prepared with the optimized new method TSS-HI was at 7.21×109±1.85×109 CFU/μg DNA,which was a recored for homemade competent cells.The transformation and assembly ability of multi-fragments and large plasmids were further tested.The method can be used for 7-fragments DNA assembly and transformation of 80-kb large plasmids.Thus,E.coli B W3KD and the TSS-HI method may be applied for diverse DNA cloning jobs.In this study,we determined the mechanism of intracellular short homologous recombination in E.coli,which was involved in in vivo DNA joining.We also constructed a high TE strain BW3KD that was suitable for efficient cloing even with large plasmids and for the assemble of multiple fragements.Further,we developed a new method TSS-HI to prepare competent cells with unmatched TE.The findings contributed to a better understanding of the mechanism of in vivo DNA assembly and developed improved methods for DNA assembly. |
