Difference between revisions of "Part:BBa K1541017"

Revision as of 11:25, 25 October 2014

rhlI with optimized RBS

N-butyryl-L-Homoserine lactone or short C4-HSL

This part contains the part C0170 (RhlI), which is an autoinducer synthesis protein that produces N-butyryl-L-HSL (C4-HSL) which binds to RhlR (C0171), obtained from Pseudomonas aeruginosa. Since this part was used in a low-copy vector, RBS engineering was performed to increase expression levels of this enzyme and thus also improve the production of C4-HSL.

Usage and Biology

Quorum Sensing

RhlI is the autoinducer synthetase from Pseudomonas aeruginosa, which can synthesize the molecule N-butyryl-L-HSL (C4-HSL). C4-HSL can bind to the regulatory protein RhlR (C0171). Once C4-HSL is bound to RhlR it can activate the promoters R0071 or I14017.

RBS Engineering

The ribosomal binding site (RBS) is a sequence on the mRNA crucial for the translation initiation step in prokaryotes, which highly influences protein levels. The influence of the RBS sequence also depend of the genetic context and identical RBS sequences in different contexts can result in different protein expression levels^[1]. The [http://salis.psu.edu/software/RBSLibraryCalculatorSearchMode RBS Calculater] provided by the Salis Lab was used to find an optimized RBS for the genetic context of rhlI. The calculator provides a value for the translation initiation rate (TIR), which was shown to highly correlate with protein expression levels^[1].

This optimized RBS was compared to the case where the RBS B0034 is placed upstream of rhlI (compare I9026). The TIR for rhlI with upstream placed B0034 was calculated as approximately 1500 while the optimized construct reaches a TIR of approx. 684200, which is an increase of more than 450 times.

Experimental Validation

Setup

Results

Figure 1

Figure 2

Sequence and Features