As a beginner to work with pseudomonas, we made a lot attempts to culture and prepare workable P.F.strain. So we tried culture medium with different ingredient. We discovered that it can grow on LBmedium ,but it will grow better on YPDA. In regular LBmedium, the max od600 will around 0.9 ,and in YPDA the max od600 will rise to around 1.4,which significantly rises. Then we made some attempts to make competent P.F.cells. Both electroporation method and CaCl method were tried and we have success in CaCl method. Those competent P.F. will be used in further experiments. We also tried to make culture curve of pseudomonas. 12 culture tubes were prepared and placed in the incubator with a gap of 1hours. We took them out every 12 hrs to record th od600. We tested our plasmid extraction kit on P.F. and the result turns out that the regular plasmid extraction kit is not suitable for P.F. latter plasmids will be amplified in E.coli. We tested several expression system that we can acquire from our PIs. Mainly pET28a, pGEX6p and pMR* system. The system with pET28a backbone works really well. While pGEX6p will strongly inhibit the growth of P.F. and pMR* system is not working at all. However the T7 promoter will not work properly in PF0-1 strain. So we have to re place the promoter.

In our previous experiments we chose pET28a as our vector. The vector introduces a lactose operating system . the plasmid should contain two features:encodes a protein for lactose intake and the anti fungal peptide we use. The expression of the peptide should be controlled by lactose concentration. So we first build a artificial inducible promoter with constitutive promoter PpltR, which mentioned in team SKLMT 2018, and a Lac operator. The antifungal peptide coding with signal peptide assembled toit were atacched to such artificial promoter. Secondly,we use another gene LacY to ensure lactose intake.

We use a dual vector system to make protein expression in yeast: pGADT7 and pGBKT7. In pGADT7 we insert B4GalT1 gene with CYC1 promoter. It encodes β-1,4 udp-galactosyltransferase for lactose synthesis. In pGBKT7 we insert LALBA gene with CYC1 promoter as well,it encodes lactalbuminα as it can enhance the specificity of B4GalT1 We will also insert GalE gene with FIG1 promoter. It will produce UGP-glucose epimerase when fungal hormone receptor is activated.