pBAD synthetic promoter library

As a tool for expressing lethal proteins in E. coli we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the DTU iGEM team from 2010.

Methods

Experimental procedure

1. Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.
2. USER cloning to add RFP downstream of promoter.
3. Colonies were plated.
4. Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected.
5. Wells were inoculated from overnight cultures of each of the selected colonies
6. One plate was run with arabinose, and another plate was run without arabinose.

Data analysis

Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished).

The growth rate, mu, was estimated to be 1.28 for all wells on all plates since we expect each strain to grow at the same rate.

A time window representing exponential growth was selected (between 1 and 4.5 hours).

Results

Example of use

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See also "Hello World project".