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!-->
{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!-->
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= The Threshold Device =
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= Sensitivity Tuners =
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==Previous Work==
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{{Template:Cambridgetemplatetop}}
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[[#Introduction | Introduction ]]
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[[#Recreating Previous Work | Previous Work]]
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[[#Characterisation | Characterisation ]]
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[[# | ]]
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[[# | ]]
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{{Template:Cambridgetemplatebottom}}
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== Introduction ==
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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.   
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===Cambridge 2007===
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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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[[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 reporter.  They genenerated 15 total combinations of different activators and promoters.
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.
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They successfully quantified the PoPS amplification factors for each activator/promoter combination after arabinose induction.
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===Further Work - Cambridge 2009===
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==Kit of Parts==
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We have two major goals - characterisation and reconstruction.   
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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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'''Characterisation'''
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First, we hope to characterise the Cambridge 2007 activator constructs with RFP and GFP reporters on low copy plasmids, looking at three major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.
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[[Image:characterization.jpg]]
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'''Reconstruction'''
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Our second goal is to build a library of devices following the pattern in the figure below, which can also be abstracted as a PoPS converter:
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[[Image:thresholddevice3.jpg]] = [[Image:converter.jpg]]  
[[Image:thresholddevice3.jpg]] = [[Image:converter.jpg]]  
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Our hope is that this well characterised library can be used by future iGEM teams to fit the needs of their projects.
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The table summarizes the Sensitivity Tuners we designed and characterised:  
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== Recreating Previous Work ==
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We began by recreating the 2007 team's data with some select amplifier constructs.  We have the advantage over the 2007 team in that we have a better plate reader that is able to take OD600 absorbance readings at the same time as taking RFP and GFP output readings.  For our transformations, we used the ''E. coli'' host strain BW27783.  This host strain constitutively expresses arabinose transporters and is unable to metabolise arabinose, making it an ideal host for arabinose titration experiments.
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'''Results''': The 2007 team's hopes for future work included investigating a problem they attributed to the toxicity of high levels of activator in the cell.  Overnight OD600 readings of cells transformed with their amplifier constructs indicated cell death.  However, these OD readings were conducted separately from their RFP and GFP output measurements.  The 2009 team gathered data on the plate reader capable of taking OD600 absorbance readings as well as RFP and GFP output readings; no OD600 readings suggested cell death due to toxicity.
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*graphs*
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== Characterisation ==
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We moved all 15 activator constructs onto pSB3K3, a low copy plasmid.  The standard promoter for 1 RPU, J69591, is also on pSB3K3 and has a GFP reporter, so we can make meaningful comparisons on the plate reader.
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== Results ==
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{| border="1"
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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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|}
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}
<!--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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