Team:Cambridge/Project/Amplification/Characterisation

From 2009.igem.org


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 (BBa_I0500), 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 BBa_K274370 BBa_K274380
PO promoter BBa_K274371 BBa_K274381 BBa_K274391
PP promoter BBa_K274382 BBa_K274392
Psid promoter BBa_K274374 BBa_K274384 BBa_K274394
PLL promoter BBa_K274375 BBa_K274395

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 BBa_J69591, 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.

Cambridge hillfunction.gif

Characterization3.jpg

For our characterisation 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 BBa_I746370 BBa_I746380BBa_I746390
PO promoter BBa_I746371 BBa_I746381 BBa_I746391
PP promoter BBa_I746372 BBa_I746382 BBa_I746392
Psid promoter BBa_I746374 BBa_I746384 BBa_I746394
PLL promoter BBa_I746375 BBa_I746385 BBa_I746395

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

Parameters of Fitted Curves

Construct Increase in Rate, a (RPU) Basal Rate, c (RPU) Switch Point, k (uM) Hill Coefficient, n
BBa_I746370 0.205 0.022 0.85 2.72
BBa_I746371 0.323 0.039 0.92 2.89
BBa_I746374 0.137 0.021 0.88 2.51
BBa_I746375 0.196 0.032 0.95 2.71
BBa_I746380 0.129 0.018 2.20 4.00
BBa_I746381 0.452 0.118 1.05 2.45
BBa_I746382 0.496 0.029 3.98 2.37
BBa_I746384 0.194 0.018 1.46 3.96
BBa_I746391 1.312 0.058 1.11 2.78
BBa_I746392 1.271 0.040 0.35 2.26
BBa_I746394 1.105 0.046 1.81 4.00
BBa_I746395 1.366 0.040 1.91 4.00

Sample Graph

Cambridge characterisation82.png

Discussion

Using the plate reader data, we were able to successfully characterise a wide range of Sensitivity Tuners. The switching point varies across the devices by a factor of ten, showing that the constructs would be suitable for their purpose.

The size of the response is shown to depend to a large extent on the activator. The choice of promoter has has less of an impact, although a discernible one.

The basal rate was consistent across the constructs, with the exception of BBa_I747381. A closer look at the data shows that the larger response only happened on one of the three cultures, so can be assumed anomolous.

The hill coefficients are largely within an accepted range, suggesting that 2 or 3 molecules of the activator are necessary to engage the promoter. In some cases, however, the resolution of concentrations was too small to achieve a sensible value for the hill coefficient. Here, the upper limit of the fitting script was reached; the parameter takes the value 4.00.

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