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)

See Knight:Evolving Reshmaverters/Promoter library design.

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% arabinose)
   • We'll start with high induction since that is where the original inverter is having trouble.
3. Collect 1ml samples for FACS at 4C, store long term samples at -80C
   • 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)
4. Sort for cells on MOFLO with HIGH GFP and LOW RFP - the sorting box can be defined somewhat arbitrary, but try to keep it at extremes because there is a good deal of noise even in clonal populations. Can sort into media containing low (1e-6% arabinose) induction, and then dilute into 5ml or more depending on time of day, number of cells sorted, etc. Want to avoid the cells growing to stationary, but they should grow at least 12-14 hrs to hit SS.
5. Collect 1ml samples for FACS at 4C, store long term samples at -80C
6. Sort for cells on MOFLO with LOW GFP and HIGH RFP (RFP level should be equivalent to the control of I13534 + RBS/Rep/Term/Promter under low induction.
7. Grow library in 5ml media with high induction (or no induciton depending on whether you are doing another round of sorting)
   • At this point can plate library and test individual mutants, or continue rounds of sorting ad neaseum. Might be smart to plate the library on plates containing 3e-5% arabinose and pick colonies that aren't red.

Stage 3/4

Repeats of 1/2 except prefixing with an RBS library. Also, need to pick our "favorite" promoter since only get to use one.