Endy:Screening plasmid/v1.0/Update

Issues

General

• What are the most common things people end up needing to repair when they build their devices (or do everyone else's just work the first time)? (expression level, mRNA stability, protein function, RNA/DNA secondary structure, etc)
• Do people frequently use screens now to repair/build their devices? What sort of screens do they use? How can we incorporate more complicated screening and selection for tuning/constructing our devices?

Screening Plasmid specific

• need to move to lower copy if we want to test 2 inverters in series (o/w get a pretty big growth hit)
• Would like to move to an induction system that doesn't require co-transformation with a plasmid and accommodates more host strains (e.g. pBAD requires native arabinose transport to be knocked out).

Design

Schematic of the screening plasmid design. It consists of 4 components: (1) Tunable input (2) Input measurement (3) Part/Device insertion site (4) Output measurement.

Design of Screening Plasmid 1.0: We are using the Pbad arabinose-inducible induction system [1] as a tunable input. GFP is a measure of input and RFP is a measure of output. A Biobricks cloning site enables easy insertion of any Biobricks part. RNase E sites create independence between the mRNA stability of the device being screened and the mRNA stability of the fluorescent proteins. In particular, we suspect mRFP1 contains internal RNaseE cut sites and have added a hairpin 5’ of the coding region to slow degradation by RNase E. [2]

Terminator Characterization via Screening Plasmid 1.0

Characterization of 6 terminators from the Registry of Standard Biological Parts inserted into the Screening Plasmid. The black line is the best fit to the empty screening plasmid, and serves as a standard for 0% termination efficiency. Functional terminators should lie below the line, note that B0025 (red) is sometimes acting as a promoter.

Histogram of calculated termination efficiencies for each terminator. Note that B0025 is mostly off scale.

Inverter Characterization via Screening Plasmid 1.0

Characterization of Q04740: Dot plot of one replicate is shown in upper right. Mean RFP expression for 3 replicates is plotted against GFP showing characteristic inverter transfer curve.

Stuch in "HIGH output" state independent of input

Stuck in "LOW output" state independent of input

Screening an Inverter Library

Two rounds of screening on a library of inverters generated by mutagenic PCR

Functional mutant isolated from the library. Mutation was found to weaken the RBS (~35% as strong as the original)