I am going to construct a library where several genes of a pathway are contained and each gene is expressed in different level. The number of variants in this library is about 107. I want to express these genes in the chromosome. I plan to construct these genes into plasmid firstly, then insert them into the chromosome through CRISPR/Cas9 and λ-RED system. But how frequently will homologous recombination (HR) happen in E. coli with λ-RED system and a DSB?

I guess the frequency of homologous recombination (HR) will not be that high to make sure that each variant is integrated into chromosome, so I plan to cultivate E. coli to let it reproduce for several rounds, then induce the expression of cas9 and RED. I presume this can make sure that each variant is integrated into chromosome. Is there any problem if I do so?

Thank you so much if you would like to share any ideas!