Endy:Screening plasmid

This project is in progress -- results described are preliminary. If you would like to keep up to date with this work, please subscribe to the RSS feed.

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.

Current Experimental Work

• Endy:Screening plasmid/Notebook
• Construction of Screening plasmid 1.5 -- Andrzej
• Construction of hairpin test constructs -- jason
• Development of RBS library for tuning expression level -- jason
• Characterization of empty Screening Plasmid 1.0 with different induction approaches. -- jason
• Measuring effectiveness of beads for normalizing flow cytometry data.

Screening Plasmid 0.X

• Endy:Screening plasmid/v0.X

Screening Plasmid 1.0

This is the current working version of the screening plasmid. It is available from the MIT Registry of Standard Biological Parts as pSB1A10.

• Endy:Screening plasmid/v1.0

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 |