According to the problem we mentioned in the demonstration, the expression of the NicA2 protein may be toxic to E. coli so that its gene cannot be stably present in E. coli. Similarly, genes that are toxic to E. coli are difficult to achieve genetic manipulation in it, especially using replication-dependent recombination methods such as Red/ET homologous recombination. However, many proteins that are toxic to E. coli are normally present in Pseudomonas, and during the establishment of the Pf-5 promoter library of Pseudomonas fluorescens, we found that most of the promoters of P. pf-5 cannot work in E. coli.
Therefore, in addition to the transformation of Pseudomonas fluorescens pf-5 into a new chassis for heterologous expression, we are also interested in directly implementing genetic manipulation in Pseudomonas. The genetic manipulation of Pseudomonas was carried out earlier. The early genetic manipulation method was to use the homologous recombination of the endogenous RecA recombinase, but the efficiency was very low and it was prone to off target. In 2017, Zheng Wenzhao et al[1]. studied the construction of the Pseudomonas Red/ET recombination system and achieved preliminary results in Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas syringae and Pseudomonas fluorescens.
Regardless of the type of cloning, screening markers are indispensable, and our part BBa_K2569032have improved one of the most widely used parts Baa_J04450 in iGEM. lacI is present on the genome of many host cells, which suggests that he normal expression of RFP requires the induction of IPTG and need 18 hours of waiting. We replaced the lac promoter in front of the RFP gene with the strong pf-5 promoter PampC. And hope that RFP, as a visible marker for the naked eye, can be expressed more pronouncedly and faster in in Pseudomonas fluorescens pf-5.
