Endy:Screening plasmid: Difference between revisions

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'''This page is a work in progress.'''
*[[test]]
'''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 [http://openwetware.org/index.php?filter=Endy:Screening_plasmid&feed=rss&title=Special:Recentchanges RSS feed].


==Introduction==
==Introduction==
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==Screening Plasmid 1.0==
==Current Experimental Work==
[[Image:ScreeningPlasmid1.0.PNG|600px|left|thumb|'''Design of Screening Plasmid 1.0:''' We are using the Pbad arabinose-inducible induction system <cite>Khlebnikov</cite> 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. <cite>Smolke</cite>
*[[Endy:Screening plasmid/Notebook]]
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*Construction of [[Endy:Screening plasmid/v1.5|Screening plasmid 1.5]] -- Andrzej
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*Construction of [[Endy:Screening plasmid/v1.0/Hairpin|hairpin test constructs]] -- jason
*[[Endy:Screening plasmid 1.0/Background|Background]]
*Development of [[Endy:Screening plasmid/RBS library|RBS library]] for tuning expression level -- jason
*[[Endy:Screening plasmid 1.0/Protocols|Protocols]]
*Characterization of [[Endy:Screening plasmid/v1.0|empty Screening Plasmid 1.0]] with different induction approaches. -- jason
*[[Endy:Screening plasmid status|Status]]
*[[Endy:Screening plasmid/Calibration beads|Measuring effectiveness of beads]] for normalizing flow cytometry data.
*[[Endy:Screening plasmid constructs|Constructs]]


===Characterization of Empty Screening Plasmid 1.0===
==Screening Plasmid 0.X==
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).  
*[[Endy:Screening plasmid/v0.X]]


 
==Screening Plasmid 1.0==
===Terminator Characterization via 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 [http://parts.mit.edu/registry/index.php/Part:pSB1A10 pSB1A10].'''
[[Image:TermDotPLot.JPG|left|300px|thumb|'''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.]]
*[[Endy:Screening plasmid/v1.0]]
 
[[Image:TermsCorrected.jpg|300px|thumb|Histogram of calculated termination efficiencies for each terminator. Note that B0025 is mostly off scale.]]
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*[[Endy:Terminator characterization/Protocols|Protocols]]
*[[Endy:Terminator characterization/Analysis|Analysis]]
 
===Inverter Characterization via Screening Plasmid 1.0===
 
[[Image:Q04740sp.PNG|thumb|400px|left|'''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.]]
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*[[Endy:Screening plasmid/Inverter characterization/Protocols|Protocols]]
*[[Endy:Screening plasmid/Inverter characterization|Inverters]]
*[[Endy:Screening plasmid/Inverter characterization/Analysis|Analysis]]


==Screening Plasmid 1.5==
==Screening Plasmid 1.5==

Latest revision as of 08:45, 13 November 2007

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

Screening Plasmid 0.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.

Screening Plasmid 1.5

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

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:

Documents

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 | PubMed ID:12080425 | HubMed [Khlebnikov]
  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 | PubMed ID:12402322 | HubMed [Smolke]

All Medline abstracts: PubMed | HubMed

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