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From 2010.igem.org

The Project

The RMIT University iGEM project will attempt to create a peptide expression platform using Synthetic Biology. The recombinant production of protein is routine in science and industry. Despite protein expression being achievable, peptide expression is frequently too complicated or not economically viable; peptides are small and often contain very little – or even no secondary structure – making them highly susceptible to proteolysis and other cellular processes.

In the laboratory for research purposes, carrier proteins with affinity for a purification matrix are commonly employed, allowing “protection” of the peptide intracellularly and providing a defined bioprocess. There are several limitations to this technology that curtails the scale: Firstly, the process is extremely inefficient as it expends the feedstock and cellular metabolism to essentially produce large amounts of a worthless carrier protein (as much as 98% of the protein-peptide fused product by mass)., Secondly, affinity resins used to capture the carrier protein are expensive and rarely congenial to scalable chromatography unit operations such as expanded bed chromatography., Thirdly, there is a requirement to separate the peptide from the carrier protein that often utilises a protease. Proteases are expensive at elevated scales, and despite popular beliefs are actually inefficient with best estimates ranging in ratio yields of 1:40 – 1:200 for the desired product as a “good” outcome due to inefficiencies, non-specific cleavage or loss during purification. For these reasons there are very few commodity peptides made recombinantly or even by semi-synthetic methods, with solid-phase peptide synthesis remains the industry benchmark.

The RMIT University iGEM project poses the question: Can the bottlenecks of purification and proteolysis be removed from the workflow to economically produce a peptide and commodity carrier protein? In this project we proposemachine will aim to produce the thermo stable protein, Taqc polymerase may be used, as the a carrier molecule to allow for the production of a peptide that will be attached to the Taqc commodity enzyme. The peptide-Taq fusion will be attached by a thermolabile bond – an Asp/Pro bond in between the peptide and the Taq Polymerase, with the intention of releasing the peptide-Taq fusion products by thermolysis. The hypothetical bioprocess will involve heating the bacteria below the threshold of the thermolabile bond, then following clarification or polishing, heating above the threshold to separate the peptide from the Taq protein. The peptide and Taq may then be purified allowing two income streams from the manufacture.

Our project seeks to produce several unique parts for the registry: Firstly, a plasmid and system for producing Taq polymerase as a useful part for future iGEM projects undertaking PCR as part of their experiments. Secondly, an expression device will be provided to the registry that will allow peptides to be inserted easily and quickly by a process of l

The bacterial plasmid or the expression vector which was supplied by the RMIT synthetic biology lab, has been incorporated with a t7 promoter that is controlled by the Lac elements. The t7 was inserted into the Lac inducible promoter by removing the -35 and -10 elements, turning them into restriction sites. Primers were designed for a Quick Change Mutagenesis that would result in the -35 element being mutated into the AflII restriction site (c.ttaag), while the -10 element will be mutated into the BamH1 restriction site (g.gatcc). By removing these elements and inserting the t7 promoter, a new part for the biobrick registry has been created.

Through ligand independent cloning into the device to produce any peptide at will, or even allow the rapid and economical production of a library of peptides for drug design and screening. the desired peptide can be inserted into the plasmid with an engineered asp/pro bond in between the peptide and the Tac Polymerase. By heating the culture to 65°C, the bacteria will lyse and the Taq-peptide can be harvested. This is followed by further heating to break the thermoliable asp/pro bond that is holding the peptide and Taq together. Bioprocessing and process intensification can then follow.

Project Details

Creation of the Taq Polymerase Part

Making Vector

== Results ==