Endy:Screening plasmid

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Introduction

Schematic of the screening plasmid design.
Schematic of the screening plasmid design.


The screening plasmid is designed to enable fast characterization of PoPS-based parts and devices. It consists of 4 components: (1) Tunable input (2) Input measurement (3) Part/Device insertion site (4) Output measurement.

Screening Plasmid 1.0

Design of Screening Plasmid 1.0: We are using the Pbad arabinose-inducible induction system [2] 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. [3]
Design of Screening Plasmid 1.0: We are using the Pbad arabinose-inducible induction system [2] 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. [3]


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.
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.
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.
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.


Screening Plasmid 2.0

We are in the process of designing a new version of the screening plasmid to account for some of the shortcomings of the previous version. An early list of possible improvements:

  1. Brighter FPs (in particular RFP is very dim)
    • Emerald & tdTomato look pretty promising
  2. Lower copy plasmid
  3. New Inducible Expression System
    • Propionate-inducible induction system, has a larger dynamic range than pBAD and doesn't require a specialized strain. (i.e. no knockouts of native systems needed.)
  4. It looks like MC4100 is missing the propionate metabolism genes and so wouldn't work with this system, these are the two genes in K12 are homologous to the prpBCDE operon:
    • 349236..350405, 350439..351890 Unfortunately, there is a 98kb deletion from the MC4100 (a K12 derivative) genome from 274723-371962, so it's missing these genes.
    • However, I'm not tied to MC4100, would rather get this working in K12 and then we could mnakybe make some progress on doing directed deletions of K12 as planned in the Biobricks Standard Strain.

References

[2] Khlebnikov et al,Modulation of gene expression from the arabinose-inducible araBAD promoter. J Ind Microbiol Biotechnol. 2002 Jul;29(1):34-7.

[3] Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon. Biotechnol Bioeng. 2002 Dec 30;80(7):762-76.


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