Studies On Genome Traceless Homologous Recombination Based On Lac Repressive System And Resistance Screening In Escherichita Coli

Escherichia coli has the advantages of clear genetic background,easy culturing and rapid growth,and is one of the most commonly used host bacteria in industrial biotechnology.It is beneficial to optimizing the metabolic network to establish quick and effective methods which can simultaneously transform multiple sites on the genome.After editing the genome of E.coli by traditional Red homologous recombination technique,the FRT site sequence remains on the genome which will seriously affect the subsequent homologous recombination efficiency,even if the resistance tag is eliminated.However,the two-step traceless recombination based on sacB gene still has the problem of high false positive rate.In this study,we used the lacl gene as a new negative screening marker,and constructed a new two-stp traceless recombination with lower false positive rate.The establishment of this traceless homologous recombination technique is also beneficial to the simultaneous transformation of multiple locis on the genome of E.coli by the combination with other traceless homologous recombination techniques,which is of great significance to the improvement of E.coli strain traits.In this study,the lacI gene,which was activated by the enhanced promoter,was first labeled as a negative screening marker and ligated with the resistance gene to obtain a positive and negative screening gene cassette.The resistance gene in this gene cassette can be selected flexibly,which is possible to cooperate with other traceless homologous recombination technology to realize the simultaneous transformation of E.coli genome at the same time.In this study,we first knocked out the natural lacl gene and its promoter on genome of the initial bacteria EcHW2f and then replaced the natural promoter of kan resistance gene by lac promoter,to complete the construction of the background bacteria.The lacI gene in the positive and negative screening gene cassettes became the only donor of negative screening marker,and inhibited the expression of kan resistance gene before negative screening.On the basis of this,the natural promoter of the talB gene was successfully replaced by artificial promoter M1-93 by using the lacI two-step traceless recombination,and the validity of the technique was verified.Through a series of controlled trials,it was explored to determine 250 mg/1 kanamycin as the optimal screening concentration in negative screening,making the false positive rate reduce to 4%compared to 20%of the cat-sacB system,which greatly improve the screening efficiency.The lycopene unit yield of the strain was increased by 23%after the promoter substitution.In this paper,the lacI gene in the genetically engineered host DH5a was finally replaced by the Plac-lacO-kan gene sequence,and the construction of the bacterium was completed in only one step of recombination,to make this traceless system be used in DH5a genome.It means that other Escherichia coli host bacterias can also be constructed to background bacterias through,simple operation,which can achieve rapid gene genome transformation by using our system.In conclusion,this study is based on the characteristics of lac repressive system,and constructs a new two-step traceless recombination method to meet the needs of traceless and screening.It is helpful to metabolic network optimization through homologous recombination transformation,and is expected to play an important role in the improvement of E.coli strain traits.