Knight:Evolving Reshmaverters

Goal

Try to use the screening plasmid to tune the promoters (or RBSs) of Reshmaverters to get an appropriate transfer curve. In particular the current version is not easily repressed.

Stages

1. Design/Order/DS promoter library & Construct screening plamid + RBS/Rep/Term
2. Insert promoter librarty into screening plasmid w/ inverter and screen on FACS
3. Design/Order/DS RBS library & Construct screening plasmid + Rep/Term/Promoter
4. Insert RBS librarty into screening plasmid w/ inverter and screen on FACS

Note: If 1/2 work then great, but probably a good idea to start 3 in parallel with 2 just in case.

Stage 1

Promoter Library

Main consideration will be the spacing of the operator sites around the -10 and -35 regions of the promoter. We will not be altering the operator sequence since is is already optimized for maximum binding affinity. Also, we will not be altering the -10 or -35 regions since we do not want to weaken the promoter. (e.g. we want the HIGH output state to remain at the same level, we just want to improve the ability of the repressor to bind and shut down output when necessary)

1. Design X libraries with different spacings (other variations, too?)
2. Order libraries plus primers for DSing
3. DS libraries and cut X/P for BB suffixing
4. Mix libraries together (some combinations anyway)

Construction of Screening Plasmid

Should build two constructs:

1. I13534 + RBS/Rep/Term (do Resmaverters have BB#'s?)
2. I13534 + RBS/Rep/Term/Promter
   • This will serve as a control to compare to the library distribution. In particular, we are interested in the output level from the promoter, since we want to maintain that level of output in the mutants we select via FACS. (although, we will be removing the -14 TG, correct?)
3. Digest I13534 + RBS/Rep/Term with X/P to enable suffixing of the promoter libraries.

Stage 2

Insert promoter library

1. Ligate the mixed libraries with the screening plasmid
2. Transform and grow under high induction (3e-5%)
   • We'll start with high induction since that is where the original inverter is having trouble.
   • Notes: Dilute the 1ml tranformation mix into 50ml of the induction media and grow overnight, check density in AM and allow to grow until PM if necessary. It may take awhile for cells to grow up depending on the tranformation efficiency. When the cells are sufficiently dense (OD 0.1-0.4), then put 1ml at 4C in the tubes for FACS, push the cap down to lock it to prevent evaporation until the MOFLO run. (<1.5days prefereably, if longer make a glycerol and spin down and resuspend in PBS on the day of the MOFLO run)
3. Sort for cells that express a