From 2009.igem.org

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

We re-designed these constructs as follows...

...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. We moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with , the standard promoter.

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.

Reconstruction

Our second goal is to build a kif of parts following the pattern in the figure below, which can also be abstracted as a PoPS converter:

=

his kit of sensitivity tuners, characterised using the Cambridge 2007 contructs, can be used by future iGEM teams to fit the needs of their projects. Further, this kit of parts can be expanded in the future to include more phage activators and promoters that have yet to be added to the registry.

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.