"
Team:Korea U Seoul/Project/Protocols Results
From 2012.igem.org
Result : Rice Guardian
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.
B. Co-culture of Rice guardian with Ax21 displaying E. coli
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.
C. Growth rate of individual cell kind
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.
