To produce a sucrose selection system for cloning and protein expression in E. coli without the need for antibiotics, toward a more accessible, equitable, and open biotech pipeline independent of exotic or regulated chemicals.
The ability to metabolize sucrose is not very common among laboratory strains of E. coli. By complimenting a wild type sucrose metabolism gene circuit in a simple plasmid, one can utilize this new ability as a means of selecting transformed cells via positive selection. Viability needs to be assessed since there will be some drawbacks including growth media requirements and overall transformation efficiency so lots of benchmarking to be done. The system can be expanded further by adding a second counterselection circuit by way of citrate metabolism. Typically, E. coli can metabolize citrate but only in anaerobic environments. The famous Richard Lenski LTEE experiment showed that spontaneous mutation to metabolize citrate in aerobic conditions is extremely rare, so a plasmid which compliments the necessary genes without oxygen inhibition of said expression would be a plausible selection system. Couple both sucrose and citrate in a Golden Braid cloning grammar and you get the Lemonade Cloning System. Antibiotic-free and globally accessible molecular cloning for everyone!
This is where I write my personal notes on the day to day of this project. Results will always be in the Experiments pages so this is more musings, thinking, drafting, and dreaming.