Team:RMIT Australia/Project
From 2010.igem.org
(→Overall project) |
(→The Project) |
||
Line 408: | Line 408: | ||
== '''The Project''' == | == '''The Project''' == | ||
- | < | + | <p> |
The RMIT University iGEM project will attempt to create a peptide expression | The RMIT University iGEM project will attempt to create a peptide expression | ||
platform using Synthetic Biology. The recombinant production of protein is routine in | platform using Synthetic Biology. The recombinant production of protein is routine in | ||
Line 415: | Line 415: | ||
contain very little – or even no secondary structure – making them highly susceptible | contain very little – or even no secondary structure – making them highly susceptible | ||
to proteolysis and other cellular processes. | to proteolysis and other cellular processes. | ||
- | < | + | <p> |
In the laboratory for research purposes, carrier proteins with affinity for a purification | In the laboratory for research purposes, carrier proteins with affinity for a purification | ||
matrix are commonly employed, allowing “protection” of the peptide intracellularly | matrix are commonly employed, allowing “protection” of the peptide intracellularly |
Revision as of 01:26, 16 July 2010
![](https://2010.igem.org/Image:RMIT_logo.jpg)
![](https://static.igem.org/mediawiki/2008/e/ef/Navi_bg.gif)
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.
<p>
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