Endy:Screening plasmid

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'''This page is a work in progress.'''
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*[[test]]
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'''This project is in progress -- results described are preliminary.'''
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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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[[Image:ScreeningPlasmidSchematic.PNG|500px|left|thumb|Schematic of the screening plasmid design.]]
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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.
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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.  
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[[Image:ScreeningPlasmidSchematic.PNG|500px|left|thumb|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.]]
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==Screening Plasmid 1.0==
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[[Image:ScreeningPlasmid1.0.PNG|600px|left|thumb|'''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]
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*[[Endy:Screening plasmid 1.0/Background|Background]]
 
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*[[Endy:Screening plasmid protocols|Protocols]]
 
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*[[Endy:Screening plasmid status|Status]]
 
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*[[Endy:Screening plasmid constructs|Constructs]]
 
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===Terminator Characterization via Screening Plasmid 1.0===
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==Current Experimental Work==
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[[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.]]
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*[[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
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*Development of [[Endy:Screening plasmid/RBS library|RBS library]] for tuning expression level -- jason
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*Characterization of [[Endy:Screening plasmid/v1.0|empty Screening Plasmid 1.0]] with different induction approaches. -- jason
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*[[Endy:Screening plasmid/Calibration beads|Measuring effectiveness of beads]] for normalizing flow cytometry data.
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[[Image:TermsCorrected.jpg|left|300px|thumb|Histogram of calculated termination efficiencies for each terminator.  Note that B0025 is mostly off scale.]]
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==Screening Plasmid 0.X==
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*[[Endy:Screening plasmid/v0.X]]
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*[[Endy:Terminator characterization/Protocols|Protocols]]
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*[[Endy:Terminator characterization/Analysis|Analysis]]
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===Inverter Characterization via Screening Plasmid 1.0===
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==Screening Plasmid 1.0==
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'''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].'''
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*[[Endy:Screening plasmid/v1.0]]
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[[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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==Screening Plasmid 1.5==
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This version of the screening makes use of an AHL-based induction system (<bbpart>F2620</bbpart>). 
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*[[Endy:Inverter characterization/Protocols|Protocols]]
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*[[Endy:Screening plasmid/v1.5|Screening plasmid 1.5]]
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*[[Endy:Screening plasmid inverters|Inverters]]
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==Screening Plasmid 2.0==
==Screening Plasmid 2.0==
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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:
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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:  
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#Brighter FPs (in particular RFP is very dim)
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*[[Endy:Screening plasmid 2.0|Screening Plasmid 2.0]]
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#*Emerald & tdTomato look pretty promising
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#Lower copy plasmid
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==Documents==
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#New Inducible Expression System
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*[[/Registry technical report/]] (''in progress'')
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#*[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16269719&query_hl=2&itool=pubmed_docsum 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.)
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#**[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16333863&itool=pubmed_DocSum  more details of this system]
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#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:
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#*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=49175990&db=Nucleotide&from=349236&to=350405&view=gbwithparts  349236..350405], [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=49175990&db=Nucleotide&from=350439&to=351890&view=gbwithparts 350439..351890] Unfortunately, there is a [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=12618467 98kb deletion] from the MC4100 (a K12 derivative) genome from 274723-371962, so it's missing these genes.
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#*However, I'm not tied to MC4100, would rather get this working in K12 and then we could maybe make some progress on doing directed deletions of K12 as planned in the [[Standard E. coli Strain for BioBricks|Biobricks Standard Strain]].
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==References==
==References==
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[2] Khlebnikov et al,Modulation of gene expression from the arabinose-inducible araBAD promoter.  J Ind Microbiol Biotechnol. 2002 Jul;29(1):34-7.
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<biblio>
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#Khlebnikov pmid=12080425
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[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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#Smolke pmid=12402322
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</biblio>
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__NOTOC__
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Current revision

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.

Contents

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.
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. . pmid:12080425. PubMed HubMed [Khlebnikov]
  2. Smolke CD and Keasling JD. . pmid:12402322. PubMed HubMed [Smolke]
All Medline abstracts: PubMed HubMed
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