Team:Cambridge/Project/Amplification/Characterisation

From 2009.igem.org

Revision as of 02:34, 22 October 2009 by Awalbridge (Talk | contribs)


The Sensitivity Tuner

Introduction

The Cambridge 2007 iGEM team build 15 "amplifiers," constructs with RFP and GFP reporters that amplified the PoPS output of the promoter pBad/AraC (), as described below:

Amplifier07.jpg

We re-designed these constructs to be PoPS converters as follows...

Thresholddevice3.jpg

...and generated our own set of Sensitivity Tuners:

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

In order to characterize these phage activator/promoter constructs, we used the corresponding Cambridge 2007 amplifier as an illustration of how our Sensitivity Tuners alter the behaviour of pBad/AraC. These parts are very useful for characterisation as they contain fluorescent reporters; the parts we designed, which lack an input promoter and fluorescent reporters, are more useful parts for other iGEM teams to incorporate into their own projects.

Characterisation

For characterisation, we moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with , the standard promoter. We looked at a few major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.

Characterization3.jpg

For our characterization assay, we transformed the E. coli strain BW27783 with the Cambridge 2007 amplifiers (biobricks summarised in the table below):

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

The BW27783 strain is ideal for assays using arabinose as it expresses arabinose transporters in the membrane constitutively (rather than in response to the detection of arabinose) and is unable to metabolise arabinose. We used a standard assay for each construct. We characterised cultures transformed with the amplifier constructs in exponential phase at the following arabinose concentrations: 0, 0.1, 0.5, 1, 10, 50, 100, and 500 uM. The controls that we ran for each assay included LB, untransformed BW27783, and J59691, the standard promoter. With the data we gathered, we were able to update the Registry pages of the Cambridge 2007 amplifiers, and also illustrated the ability of our Sensitivity Tuners to modulate the transcriptional system downstream of pBad/AraC.

Data

Maximum Rates against Arabinose Concentrations

Construct Increase in Rate, a (RPU) Basal Rate, c (RPU) Switch Point, k (uM) Hill Coefficient, n
1.256 | 76.49 | 2.18
0.022 | 0.85 | 2.72
0.039 | 0.92 | 2.89
0.021 | 0.88 | 2.51
0.032 | 0.95 | 2.71
0.018 | 2.20 | 4.00
0.118 | 1.05 | 2.45
0.029 | 3.98 | 2.37
0.018 | 1.46 | 3.96
0.058 | 1.11 | 2.78
0.040 | 0.35 | 2.26
0.046 | 1.81 | 4.00
0.040 | 1.91 | 4.00

80

Cambridge maxrates1.jpg

81

Cambridge maxrates2.jpg

82

Cambridge maxrates3.jpg

84

Cambridge maxrates4.jpg

85

Cambridge maxrates5.jpg

90

Cambridge maxrates6.jpg

92

Cambridge maxrates7.jpg

94

Cambridge maxrates8.jpg

95

Cambridge maxrates9.jpg


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