Team:Cambridge/Project/Amplification

From 2009.igem.org

(Difference between revisions)
(Further Work - Cambridge 2009)
(The Threshold Device: ize -> ise)
Line 6: Line 6:
[[#Introduction | Introduction ]]
[[#Introduction | Introduction ]]
[[#Recreating Previous Work | Previous Work]]
[[#Recreating Previous Work | Previous Work]]
-
[[#Characterization | Characterization ]]
+
[[#Characterisation | Characterisation ]]
[[# | ]]
[[# | ]]
[[# | ]]
[[# | ]]
Line 28: Line 28:
===Further Work - Cambridge 2009===
===Further Work - Cambridge 2009===
-
We have two major goals - characterization and reconstruction.   
+
We have two major goals - characterisation and reconstruction.   
-
'''Characterization'''
+
'''Characterisation'''
-
First, we hope to characterize 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.
+
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.
[[Image:characterization.jpg]]
[[Image:characterization.jpg]]
Line 42: Line 42:
[[Image:thresholddevice3.jpg]] = [[Image:converter.jpg]]  
[[Image:thresholddevice3.jpg]] = [[Image:converter.jpg]]  
-
Our hope is that this well characterized library can be used by future iGEM teams to fit the needs of their projects.
+
Our hope is that this well characterised library can be used by future iGEM teams to fit the needs of their projects.
== Recreating Previous Work ==
== Recreating Previous Work ==
-
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 metabolize arabinose, making it an ideal host for arabinose titration experiments.
+
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.
'''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.
'''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.
Line 52: Line 52:
*graphs*
*graphs*
-
== Characterization ==
+
== Characterisation ==
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.
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.

Revision as of 14:32, 11 September 2009


The Threshold Device

Introduction

Cambridge 2007

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.

Construction07.jpg


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

Further Work - Cambridge 2009

We have two major goals - characterisation and reconstruction.

Characterisation

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.

Characterization.jpg

Reconstruction

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:

Thresholddevice3.jpg = Converter.jpg

Our hope is that this well characterised library can be used by future iGEM teams to fit the needs of their projects.

Recreating Previous Work

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.

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.

  • graphs*

Characterisation

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.

Results

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