Endy:Screening plasmid/v1.0

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Design

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]
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]


Controls

  • <bbpart>I13522</bbpart> - constitutive GFP
  • <bbpart>I13521</bbpart> - constitutive RFP
  • <bbpart>I13540</bbpart> - inducible GFP
  • <bbpart>I13520</bbpart> - inducible RFP
  • <bbpart>V1023</bbpart> - negative (CW2553/pJAt8)

Characterization of Empty Screening Plasmid 1.0

To ensure our screening system was operating as expected we measured the expression levels of GFP and mRFP1 when the plasmid did not contain a part (e.g. input equals output).

Measurement of Parts and Devices using Screening Plasmid 1.0

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.


Inverter library screening via Screening Plasmid 1.0

To be added.


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