Endy:Screening plasmid/Terminator characterization/Protocols
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This is a work in progress, protocol is currently incomplete
Purpose
Characterize the invivo termination efficiency of a Biobricked terminator quickly and reliably.
Background
The terminators should be present in one of the PoPS based screening plasmids, this protocol refers to characterization of terminators in BBa_I13534 in CW2553+pJat8. pJat8 provides Gen resistance and the screening plasmid has Amp resistance.
The current characterization protocol follows the following steps: Day 1:
- Grow the cells overnight in a rich media to achieve density.
Day 2:
- Dilute back to return the cells to mid-log in M9/gly
- Innoculate experimental cultures of M9/gly/ara
Day 3:
- Harvest cells for FACS
- Run FACS
Procedure
- This protocol is fairly complicated. Each culture has a different growth rate, so ensuring that the intermediate dilution and experimental culture remain in log phase is difficult. Additionally, the final culture must match induction time (so that the fluorophore concentration reaches steady state) and growth time (so that the culture is dense enough to measure at the end of the experiment, but still in mid-log). My rough guidelines for timing is below:
Day 1
- Overnights -- These cells typically grow very slowly. I set up overnights early in the afternoon in a richer media (M9/glucose works reasonably well) and give them plenty of time to grow up.
- Controls
- Negative Control - CW2553/pJat8
- Empty Plasmid - This provides a baseline for calibrating GFP and RFP
Day 2
- Dilutions(AM): For high copy plasmids, I estimate a doubling time of 1.5 to 2 hours. I generally dilute back 100x and grow for 8-10 hours. For low copy plasmids, either shorten the growth time (5-6 hours rather than 8-10) or increase the dilution.
- Experimental cultures(PM): 12 hours induction seems to provide sufficient time for the fluorophores to reach steady state (14 hours was essentially unchanged). For a high copy plasmid, innoculating at OD 0.002 works reasonably well. Lower copy plasmids will need smaller innoculums (OD 0.0001-0.0005).
Day 3
- FACS Samples(AM): Take 1 ml aliquots
Analysis
Contact
- protocol based on screening plasmid protocols developed by Josh Michener
