Team:Korea U Seoul/Project/Design Parts

     Our team aims to make 3 engineered bacteria in Rice Guardian project. In order to detect Ax21, we need to produce it. First, we created Ax21 producing bacteria. Second, we have to create an engineered E. coli which detects Ax21. Lastly we created an engineered E. coli which kills Xanthomonas oryzae KACC10331 in addition to detecting it, an improved bacterium. (still working on)

     Strain used for Rice Guardian was Xanthomonas oryzae KACC10331, a specie of Xanthomonas oryzae pv. oryzae populating in Korea. A Korean researcher named Ax21 detecting gene Col R and Col S, a different nomination of Rax R and Rax H which is commonly used. We used Xanthomonas oryzae KACC10331 and Col R, S instread of Xanthomonas oryzae pv oryzae and Rax R, H in our project. Even though Col R and Col S was used, Rax R and Rax H will be used interchangeably since they are more commonly used.

Figure.1: Plasmid construction of pAT – Ax21

     Ax21 is required for our engineered bacteria to detect Xanthomonas oryzae KACC10331. We tried to over-express functional Ax21 protein inside E. coli, but failed to purify soluble Ax21 protein. Thus we decided to display Ax21 on the membrane of E. coli. pAT is a vector designed in our laboratory to display any heterologous proteins on cell-surface of E. coli. This system uses a promoter which is regulated by arabinose. In presence of arabinose, a promoter will operate. In absence of arabinose, a promoter will not operate. By inserting ax21 gene inside pAT vector, Ax21 will be displayed on the membrane surface of E. coli .

     Construction of Ax21 detecting bacteria plasmid

Figure2: Plasmid construction of Rice Guardian