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 also made 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 detecing gene colR and colS, a different nomination of raxR and raxH. We used Xanthomonas oryzae KACC10331 and colR, S instread of Xanthomonas oryzae pv oryzae and raxR, H in our project.

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 (ref. 1). 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 cloning ax21 gene inside pAT vector, Ax21 will be displayed on the membrane surface of E. coli .

     Construction of Ax21 detecting bacterial plasmid

Figure 2. Plasmid construction of Rice Guardian

Figure 3. Unit module of logic gate

Figure 4. Experimental design of full adder circuit

      In binary full adder using bacterial logic gate system, our team has designed full adder circuit with 9 key parts and 5 logic gates - 2 XOR, 2 AND and OR gates. Because we designed the circuit to use a common signal transduction molecule, in this case C4 AHL. As a result, a common signal should be changed into input signals, 3OC6, 3OC12, product of hrpR and hrpS genes. The circuit is based on unit modules, containing steps to produce each input signal based on types of common signal molecule. After input signals are formed, step to breakdown a common signaling molecule is required to block possiblity that C4 AHL as signaling molecules pass through long path and reach 'gate' plate which could affect on concentration of output.
          Finally input signals dilute through path and reach 'gate' plate, deriving result in form of a common signaling molecule, C4 AHL again. By uniting primary input signal and output signal, it was enable to sesign the circuit with several modules with specific function as different logic gate. By arranging 5 modules, functioning as 2 XOR gates, 2 And gate and 1 OR gate in order, we constructed full adder circuit and were able to set a model and proceed simulation for the project.