Endy:Screening plasmid

This page is a work in progress.

Introduction

Construction of engineered biological systems from collections of standard biological parts requires mechanisms for rapid and reliable characterization of parts. Additionally, the difficulty of rational part design necessitates library screening systems that can be employed in service of tuning part performance. Here we describe pSB1A10, a system for both characterizing and screening transcription-based parts based on their input / output function. We demonstrate the successful operation of this system by characterizing and tuning genetic inverters and transcriptional terminators.

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.

Screening Plasmid 1.0

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]

• Background
• Protocols
• Status
• Constructs

Characterization of Empty Screening Plasmid 1.0

• Endy:Screening plasmid/v1.0/Hairpin - characterize effectiveness of the hairpin to stabilize the mRFP1 transcript
• Endy:Screening plasmid/v1.0/RNAseE sites - characterize effectiveness of the RNAseE sites

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).

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.

• Protocols
• Analysis

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.

• Protocols
• Inverters
• Analysis
• /v1.0/Future work/

Inverter library screening via Screening Plasmid 1.0

To be added.

Screening Plasmid 1.5

This version of the screening makes use of an AHL-based induction system (<bbpart>F2620</bbpart>).

• Screening plasmid 1.5

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. Details can be found here:

• Screening Plasmid 2.0

Documents

• /Registry technical report/ (in progress)

References

1. Khlebnikov A, Skaug T, and Keasling JD. Modulation of gene expression from the arabinose-inducible araBAD promoter. J Ind Microbiol Biotechnol. 2002 Jul;29(1):34-7. DOI:10.1038/sj.jim.7000259 |
2. Smolke CD and Keasling JD. 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. DOI:10.1002/bit.10434 |