Team:WITS-South Africa/machine1 design

Lac/AraC Promoter

The Lac/Ara-1 promoter will be a synthetic fusion promoter comprised of the operator from the arabinose operon and the Lac promoter from the lac operon. The AraC protein is constitutively expressed and binds to the arabinose operon in the absence of the arabinose sugar. When arabinose is present, or an isomer thereof, it induces a conformational change in AraC thus preventing it from binding the operon and thus allowing transcription. Since IPTG is an isomer of β-galactosidase, it will induce the same conformational effect on AraC thus inducing transcription. In this way, the exogenous addition of IPTG will serve as a proxy for the HPV virus and induce the transcription cassette 1. Furthermore, as illustrated by Lutz and Bujard, the degree of induction is influenced by both IPTG and arabinose. Hence, promoter activity can be regulated and finely tuned by the addition of varying concentrations of IPTG and arabinose. The Lac/AraC promoter will be synthesized by primer-primer annealing and subsequent PCR elongation.

The PlcR regulon in Bacillus thuringiensis and Bacillus cereus houses approximately 15 genes required for the production of many virulence factors. The activation of this regulon is under the control of the PlcR peptide which binds a region within the promoter known as the PlcR box. The papR gene - which is found within the PlcR regulon - encodes a 48 amino acid peptide that is crucial for the binding to and subsequent activation of the PlcR box. The PapR pro-peptide is secreted from the cell (via the SecA pathway) following translation, cleaved extra-cellularly and the resulting pentapeptide is re-imported into the cell via an oligopeptide permease (a protease ubiquitous to the extra-cellular matrix of Gram-positive bacteria). Once inside the cell, PapR allows PlcR to bind to the PlcR box thus activating the regulon. Due to the presence of the plcR gene within the regulon, PlcR positively regulates its own transcription. The PlcR regulon within Bacillus anthracis houses a gene that codes for a PlcR-PapR fusion protein which was found to strongly induce transcription of the native plcR gene. It has been shown, by Pomerantsev and colleagues, that binding to the PlcR box and activation of the PlcR regulon is achieved by the expression of a hetrologous PlcR-PapR fusion protein. So as to ectopically express both PlcR and PapR in Lactobacillus gasseri, a sequence coding for the PlcR-PapR fusion protein will be derived from bioinformatic analysis of the native sequence in Bacillus anthracis. This will be implemented due to the inherent compactness of the fusion protein as well as ease of transportability when inserting into a foreign bacteria. The plcR-papR sequence will be synthesized by Gene Art and furthermore, the sequence will be optimized by an online tool.

So as to assess the degree of activation of cassette 1, a variant of the Yellow Fluorescent Protein, Venus, has been included in the construct. So as to obtain quantitative data as to the efficacy of IPTG in inducing the activation of cassette 1, a host of fluorometric analysis will be performed on a cassette-containing colony. Venus, which possesses a known excitation:emission characteristic, will be used as a reporter protein to quantify the degree of transcription (by proxy of fluorescence) with use of a fluorometer. Although venus is a ubiquitous fluorescent protein found in the laboratory, specialised sequences will be ordered from Gene Art. Furthermore, the sequences will be codon optimized so as to produce the greatest protein yield and fluorometric activity in Lactobacillus gasseri.