Team:Korea U Seoul/Project/Protocols Results

Ax21 was over-expressed in E. coli BL21(DE3) under T7 promoter at 25℃ for 7hrs.

Figure 1. SDS-PAGE analysis of Ax21 in E. coli BL21(DE3)

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 IPTG induction); 5 and 6, E. coli transformed with pAT-ax21 (IPTG induction); 1,3 and 5, soluble part; 2,4 and 6, insoluble part. 0.5mM IPTG was used for induction.

     Rice Guardian project was to build an engineered E. coli which detects Xanthomonas oryzae KACC10331. To make our project more ‘synthetic’, we decided to make Ax21 producing E. coli . In order to find out whether Rice Guardian detects Ax21 and produces mRFP, co-culturing two cell types (Rice Guardian and Ax21 producing bacteria) was conducted. After cell density reaches up to OD₆₀₀ 1.0, its fluorescence level was measured. Data of our experiment are listed below.

Table1: Fluorescence and cell density of co-cultured media by 7 hours.

*pAT: Cell-surface display vector designed by our laboratory.
*Rice Guardian: engineered E. coli which expresses RaxR, RaxH, and mRFP.
*pAT-ax21: ax21 inserted pAT vector.

     pAT + 0% arabinose is a control group. It has only pAT cells. It was to measure basal level of fluorescence produced by non-RFP product. We chose pAT + 0% arabinose as a control group because its cell has own fluorescence due to some proteins and other cellular components. Thus, we subtracted its fluorescence value from other sample when we get “calculated” value. Its fluorescence is also dependent on cell density so fluorescence was divided by OD value.

     Though initially inoculated at 1:1 ratio, there are possibilities that the growth rate of E. coli cells with different functions 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 kind 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.

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

Figure 2. 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 3. 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 4. 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 5. 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 6. 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 7-1. Gate function

Figure 7-2. 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 8. The resulting plots in the various conditions

     As you can see in the Fig 7, 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.

     Rice Guardian project was to build an engineered E. coli which detects Xanthomonas oryzae KACC10331. To make our project more ‘synthetic’, we decided to make Ax21 producing E. coli . In order to find out whether Rice Guardian detects Ax21 and produces mRFP, co-culturing two cell types (Rice Guardian and Ax21 producing bacteria) was conducted. After cell density reaches up to OD₆₀₀ 1.0, its fluorescence level was measured. Data of our experiment are listed below.

Table1: Fluorescence and cell density of co-cultured media by 7 hours.

*pAT: Cell-surface display vector designed by our laboratory.
*Rice Guardian: engineered E. coli which expresses RaxR, RaxH, and mRFP.
*pAT-ax21: ax21 inserted pAT vector.

     pAT + 0% arabinose is a control group. It has only pAT cells. It was to measure basal level of fluorescence produced by non-RFP product. We chose pAT + 0% arabinose as a control group because its cell has own fluorescence due to some proteins and other cellular components. Thus, we subtracted its fluorescence value from other sample when we get “calculated” value. Its fluorescence is also dependent on cell density so fluorescence was divided by OD value.

     Though initially inoculated at 1:1 ratio, there are possibilities that the growth rate of E. coli cells with different functions 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 kind 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.