Team:Cambridge/Project/Amplification

From 2009.igem.org

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{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!-->
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= A Reliable Amplification System =
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= Sensitivity Tuners =
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== Introduction ==
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==Previous Work==
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The Cambridge 2007 iGEM team developed a Pops amplifier system using phage activators and promoters.  The system works by using a Pops input to make an activator protein, as shown in the diagram from their wiki below, which then binds to a promoter and generates a Pops output.   
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Part of the iGEM mission is to build on previous projects.  The Cambridge 2007 iGEM team developed a PoPS amplifier system using phage activators and promoters.  The system works by using a PoPS input to make an activator protein, as shown in the diagram from their wiki below, which then binds to a promoter and generates a PoPS output.   
[[Image:amplifier07.jpg]]
[[Image:amplifier07.jpg]]
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In order to quantify the ratio between Pops in and Pops out, the team built the following construction on the high copy plasmid pSB1A2, with mRFP and GFP as Pops reporters and 15 total combinations of different activators and reporters.
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In order to quantify the ratio between PoPS in and PoPS out, the team built the following construction on the high copy plasmid pSB1A2, with mRFP and GFP as PoPS reporter.  They genenerated 15 total combinations of different activators and promoters.
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In addition to analysing the characteristics of the Cambridge 2007 constructs, we looked at [https://2009.igem.org/Team:Cambridge/Modelling models] of alternatives ways to alter the sensitivity of a promoter
[[Image:construction07.jpg]]
[[Image:construction07.jpg]]
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They successfully quantified the Pops amplification factors for each activator/promoter combination after arabinose induction.  However, after data analysis they pointed out two phenomena that required further investigation:
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They successfully quantified the PoPS amplification factors for each activator/promoter combination after arabinose induction.
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1. Cultures transformed with the amplifier constructs showed reduced growth which increased in severity with increased arabinose induction.  It was suspected that the activators were toxic to the cells at high concentrations.
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2. RFP was an unreliable reporter.  For some reason, RFP appears to be more stable when its gene is downstream of another gene in a polycistronic transcript compared to when its gene is directly downstream of a promoter. 
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The Cambridge 2009 iGEM team hopes to debug this system by concentrating on these two problems.
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== Project ==
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==Kit of Parts==
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===Starting off with 2007===
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The Cambridge 2009 team analyzed the properties of these constructs, paying particular attention to how adding phage activator and promoter combinations downstream of pBad altered the behaviour of pBad.  In particular, it caught our attention that the phage activator and promoter combinations changed the sensitivity of pBad.  We sought to thoroughly characterise each combination to generate a useful description of each so that future iGEM teams seeking to build biosensors can easily identify which combination is appropriate, depending on how they desire to manipulate their chosen promoter.  Further, we redesigned the 2007 team's constructs, removing the fluorescent reporter genes used for characterisation, as follows:
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We began by recreating the 2007 team's data to see if we would encounter the same problems.
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[[Image:thresholddevice3.jpg]] = [[Image:converter.jpg]]
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== The Experiments ==
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The table summarizes the Sensitivity Tuners we designed and characterised:
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== Results ==
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{| border="1"
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|+
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!  !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator
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|-
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! PF promoter
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| <partinfo>BBa_K274370</partinfo> || <partinfo>BBa_K274380</partinfo>||
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|-
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! PO promoter
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|<partinfo>BBa_K274371</partinfo>
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|<partinfo>BBa_K274381</partinfo>
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|<partinfo>BBa_K274391</partinfo>
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|-
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! PP promoter
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|
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|<partinfo>BBa_K274382</partinfo>
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|<partinfo>BBa_K274392</partinfo>
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|-
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! Psid promoter
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|<partinfo>BBa_K274374</partinfo>
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|<partinfo>BBa_K274384</partinfo>
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|<partinfo>BBa_K274394</partinfo>
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|-
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! PLL promoter
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|<partinfo>BBa_K274375</partinfo>
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|
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|<partinfo>BBa_K274395</partinfo>
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|}
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<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}

Latest revision as of 22:45, 21 October 2009


Sensitivity Tuners

Previous Work

Part of the iGEM mission is to build on previous projects. The Cambridge 2007 iGEM team developed a PoPS amplifier system using phage activators and promoters. The system works by using a PoPS input to make an activator protein, as shown in the diagram from their wiki below, which then binds to a promoter and generates a PoPS output.

Amplifier07.jpg


In order to quantify the ratio between PoPS in and PoPS out, the team built the following construction on the high copy plasmid pSB1A2, with mRFP and GFP as PoPS reporter. They genenerated 15 total combinations of different activators and promoters.

In addition to analysing the characteristics of the Cambridge 2007 constructs, we looked at models of alternatives ways to alter the sensitivity of a promoter

Construction07.jpg


They successfully quantified the PoPS amplification factors for each activator/promoter combination after arabinose induction.

Kit of Parts

The Cambridge 2009 team analyzed the properties of these constructs, paying particular attention to how adding phage activator and promoter combinations downstream of pBad altered the behaviour of pBad. In particular, it caught our attention that the phage activator and promoter combinations changed the sensitivity of pBad. We sought to thoroughly characterise each combination to generate a useful description of each so that future iGEM teams seeking to build biosensors can easily identify which combination is appropriate, depending on how they desire to manipulate their chosen promoter. Further, we redesigned the 2007 team's constructs, removing the fluorescent reporter genes used for characterisation, as follows:

Thresholddevice3.jpg = Converter.jpg

The table summarizes the Sensitivity Tuners we designed and characterised:

P2 ogr activator PSP3 pag activator phiR73 delta activator
PF promoter
PO promoter
PP promoter
Psid promoter
PLL promoter

Cambridge Sponsor Logo1.pngCambridge Sponsor Logo2.pngCambridge Sponsor Logo3.pngCambridge Sponsor Logo4.pngCambridge Sponsor Logo5.pngCambridge Sponsor Logo8.pngCambridge Sponsor Logo6.pngCambridge Sponsor Logo7.pngCambridge Sponsor Logo9.pngCambridge Sponsor Logo10.pngCambridge Sponsor Logo11.pngCambridge Sponsor Logo12.pngCambridge Sponsor Logo14.pngCambridge Sponsor Logo13.pngCambridge Sponsor Logo15.pngCambridge Sponsor Logo16.pngCambridge Sponsor Logo17.pngCambridge Sponsor Logo18.pngCambridge Sponsor Logo19.pngBmglab.jpg

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