ICSynBio:Wetlab Results
Analysis of Chassis Compatibility
E. coli containing toggle switch constructs were cultured, and toggle switch plasmids were extracted and purified using the protocol and reagents from the QIAGEN Miniprep Spin Kit. The chassis to be tested (∆lacI/∆araC E. coli cells) were also cultured and transformed into chemically competent cells. The purified toggle switch constructs were then transformed into the competent ∆lacI/∆araC E. coli cells, and this was cultured. A plate reader assay was conducted in order to evaluate chassis compatibility in BL21 and ∆lacI/∆araC E. coli strains. The cultures and inducers were transferred to their respectable wells. The plate reader was programmed to measure absorbance at 600nm wavelength every 11 minutes for 9 hours. The excitation wavelengths were set at 485 and 590nm, and emission wavelengths were set at 528 and 645nm for measuring GFP and RFP respectively. For an exhaustive list of all experiments see Appendix G.
The expected results are as follows:
- No inducer: Constituently expresses GFP
- IPTG: Transition to RFP+/GFP- state
- Arabinose: transition to an RFP+/GFP- state* due to induced expression of mflon protease
- pECJ3 + pZA16mflon: fast rate of switching
- pECJ3B + pZA16mflon: decreased rate of switching due to weaker degradation tag
- pECJ3D + pZA16mflon: even more decreased rate of switching due to even weaker degradation tags
