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Bioaccumulator device: Strong promoter + OmpA fused to Synthetic Phytochelatin + B0015

Overview

In the environment Hg has successive transformations which pose risks not only for microorganisms but also to macro fauna. However its known that some bacteria specie has mercury resistance, among them Serratia marcescens, Pseudomonas putida, Cupriavidus metallidurans and Entereobacter. Bacterial resistance to mercury occurs due to membrane protein expression that can act in Hg capture. Among those we can find the phytochelatin. These proteins have as main feature the interaction with heavy metals. Probably this occurs due to the great amount of cystein amino acid in this protein.

Description

The use of natural membrane proteins is already in place and serve as a tool to anchor heterologous proteins in a system called “cell surface display”. It presents a great potential for a variety of biotech uses. By this strategy target peptides could be anchored to antibodies production, biocatalizers, bioremediation and other uses. In heavy metal bioremediation its is showed that recombinant microorganisms with modified surface, enriched with metal chelant proteins are better to cope the adsorption of metallic ions.

There are several strategies to anchor peptides in the bacterial membrane. In this project we used the most abundant protein to do so, the E. coli outer membrane protein A (OmpA) – fused with synthetic phytochelatin to bioremediation of mercury metal,as represented in the image below. In 2000 a new series of peptides serving as heavy metal adsorbants was proposed by Bae and collaborators. The strategy consisted in the use of an analogous to a natural phytochelatin without the necessity of post-transdutional modifications to work without using enzymatic routes or precursor molecules to its, in other words: a gene a protein.

In previous competition we tested the efficiency of Metal Binding Peptide. In this project we design a system for Hg bioaccumulation by a synthetic phytochelatin anchored in the membrane protein OmpA of a host bacteria.

One of the pillar of our project is to design the sequences for our genetic modified systems and for that the most challenging step is to adjust preferential codons for our chassis. Glutamic acid (E) and cystein (C) show two different codons in E. coli: GAG and GAA for E; TGC and TGT for C. GAA 70% e códon GAG 30%; códon TGC 60% e códon TGT 40%.

Our synthetic phytochelatin (EC20 - Glu-Cis) optimized to E. coli has: I) 10 cystein amino acids being 12 codons TGC and 8 codons TGT; II) 10 glutamic acid amino acids, 14 codons GAA and 6 codons GAG. As described in the image below.

With the phytochelatin designed we decided to express it in membrane cell display. For that we incorporated the followed parts:

Promotor JK26 interacting with sigma factor RpoS or sigma factor S to express in the stationary phase in the cell growth. JK26 is a promotor for late phase (lag) described by Miksch et al in 2005 and has been the strongest one fwe tested.

Important to say in this construction, the promotor JK26 with NdeI site was calculated to - when bond to the Llp-OmpA-phytochelatin + terminator – have 9 base pairs in distance between Shine-Dalgarno and the mRNA initiation.

Lpp-OmpA (BBa_K103006) E. coli membrane protein is where our phytochelatin was bond;The double transcription terminator is the union of T1 from E. coli and TE from T7, denominated as BBa_B0015, available in the Registry.

Usage and Results

Sequence and Features