Team:Korea U Seoul/Project/Protocols Results

• Transform E. coli TOP10 with each plasmid and spread on agar plates: pick one colony from each plate and culture overnight in 3ml broth.
• Inoculate two different cells together at 1:1 ratio in 10ml broth
• Culture the cells untill OD₆₀₀ becomes 0.5 at 37 degree celcius.(approximately takes 3 hours)
• Induce 0.2% arabinose and culture at 25 degree celcius for 7h.
• Centrifuge 2ml of cultured cells and remove the supernatant.
• Wash the cells with 20mM NaCl for 2 times.
• Resuspend the cells to the final volume of 200ul and measure fluorescence.

Ax21 was displayed on the membrane of E. coli TOP10 at 25 degree celcius for 7hrs.

Figure 1. SDS-PAGE analysis of Ax21 in E. coli TOP10

Lanes: M, Molecular weight standards; 1 and 2, E. coli transformed with pAT empty vector; 3 and 4, E. coli transformed with pAT-ax21 (no induction); 5 and 6, E. coli transformed with pAT-ax21 (arabinose induction); 1,3 and 5, soluble part; 2,4 and 6, insoluble part. 0.2% arabinose was used for induction.

     Rice Guardian project was to build an engineered E. coli which detects Xanthomonas oryzae KACC10331. We made Ax21 producing E. coli to mimic Xanthomonas oryzae secreting Ax21 protein. In order to find out whether Rice Guardian detects Ax21 and produces mRFP, co-culturing two cell types (Rice Guardian and Ax21 producing E. coli ) was conducted. After cell density reaches up to OD₆₀₀ 1.0, its fluorescence level was measured (data below).

*pAT: Cell-surface display vector designed by our laboratory.
*pAT-ax21: ax21 cloned in pAT vector.
*R.G.: Rice Guardian, engineered E. coli which expresses ColR, ColS, and mRFP.

     The data show that when ax21 gene is lost or gene expression is not induced, rax promoter in Rice guardian expresses only basal level of mRFP (2nd, 3rd, and 4th bar). Only when E. coli carrying Ax21 surface-display system properly expresses Ax21, mRFP level in Rice Guardian cells doubles (5th bar). Our result is consistent with natural rax system in Xanthomonas oryzae.

     Though initially inoculated at 1:1 ratio, there are possibilities that the growth rate of E. coli cells carrying different plasmids might be different. Also, L-arabinose, which works as an inducer for Ax21 protein expression, may affect the growth rate of each cell kind. We cultured each cell independently with and without arabinose induction, and made each cell's growth curve. Based on this information, we decided the proportin of each cell kind from co-cultured cell mixture.

          Since Ax21 detecting process is not revealed completely, we couldn’t guarantee our synthetic E. coli would work as intended. However, through experimental analysis, we found out ColS acts directly on Ax21 protein, and ColRS is all E. coli needs to detect Ax21 outside cell membrane. Even though Rice Guardian we built can only determine the existence of Xanthomonas oryzae at current state, we are planning to upgrade our design to both detect, and kill Xanthomonas oryzae , which eventually matches the title of our project.

Binary full adder consists of five logic gates: two XOR gates, two AND gates and one OR gate.

Figure 3. Binary full adder

     Each logic gate can be expressed as below differential equation being composed of basal expression rate, regulation function and decay rate.

Figure 4. Differential equation on synthesis of a protein

     where z isprotein synthesis rate, α the basal transcription level, α + β the maximum synthesis rate. x and y are each proteins, f(x, y) is the regulation function of gene, and z is the decay rate. To describe transcriptional regulation of protein x and y mathematically, we implemented Hill equation.

Figure 5. Hill equation

     where f(x) is the probability which the operate is full, K is the Hill constant, l is Hill coefficient. Using above two equations, we constructed regulation functions on each logic gates such as AND, OR and XOR gates.

Figure 6. Assemble of Hill equation expressing each logic gate

     The following set of constants have been used : K = 0.5 μM, l = 3, α = 0.2 μM·min^-1, β = 1.0 μM·min^-1, μ = 1 min^-1. We solved differential equation on dz/dt to exponential equation on input concentrations of x, y and time t

Figure 7. solved differential equation

     Using this equation, we calculated the amount of protein with the course of time. Function f(x,y) indicates regulation of gene expression at each logic gate such as AND, OR, XOR gates at input concentration of protein x, y. To plot above equations, we used R which is one of statistical tools. We made two functions reflecting the equations described in figure 1 and 2.

Figure 8-1. Gate function

Figure 8-2. Full.adder function

     GATE() function receives four parameters including initial concentrations of x, y, time and type of logic gate such as AND, OR, XOR. The return value is the concentration of expressed protein as a result of transcriptional regulation. Full adder function operates whole binary full adder via 5 logic gates, two XOR gates, two AND gates and one OR gate. Return values are sum and carry as in the electrical logic circuit. To visualize result of Full adder function, we plotted the concentration of expressed protein along with time.

Figure 9. The resulting plots in the various conditions

     As you can see in the Fig 9, eight possible combinations of three signals (A, B, C) produced different results of sum and carry. The concentration of sum is indicated by red line and that of carry is indicated by blue one. These graphs fairly reflect the theoretical logic gates. We consider 0.5 as a boundary between on and off of the binary signal.

          In this research, we designed microbial logic gates including AND, OR, XOR and connected them to assemble binary full adder. To acheive this, we considered quorum sensing to trasmit molecular signal and diffusion rates of signal molecules. However, density dependent quorum sensing takes much time to perform experiment becuase of condition of laboratory. Thus, instead of doing experiment, we designed the mathematical model using Hill equation which describes transcriptional regulation of specific gene. We plotted the result of simulation and it succesfully refleced binary full adder. Based on the model, we planned to perform this experimentally for furthur study.