Team:Imperial College London/M1
From 2009.igem.org
Please delete as completed.
Module 1 Feedback from todays session:
1) Genetic circuit - isn't this chemoinduction?!
2) Too much text - break it up and add relevant pictures
3) More detail on the stats for the 2 enzymes
4) Results should include all the enzyme kinetics stuff
5) Rebalance Chemoinduction and Module 1!!!
6) If no data exists - say that as the wiki is being frozen we haven't added the data but will have it in time for the Jamboree.
7) Have a rationale section.
8) Add what teams can reuse from this module.
9) Have a conclusion of the page at the end - couple of lines.
Contents |
Module 1: Protein Production
Protein Production
Overview
The E.ncapsulator has been designed to produce and deliver protein biopharmaceuticals to the intestine. <b>Module 1 encompasses the protein production phase of the system. The E.ncapsulator is engineered to allow the production of any protein or short chain peptide (collectively called polypeptides).
Rationale
In order to perform this function successfuly, the polypeptide (amino acid polymer) must be synthesised at a rate that will be sufficient to facilitate its accumulation inside the cytoplasm of the cell. With our generic design, it is possible to synthesise any polypeptide, so we have a thoroughly reusable system.
About the difference between enzymes and peptides.
Theory
Engineering a cell to produce a protein
Engineering The E.ncapsulator to produce proteins is quite simple. DNA is transcribed into mRNA which is in turn translated into protein. By knowing the amino acid sequence of the polypeptide of interest and working backwards, this can be converted into a DNA sequence coding for production of the protein. In our case this has been developed further by optimising the DNA sequence for E.coli. By combining this coding sequence the other necessary genetic components, we engineer the chassis to manufacture the protein.
Genetic circuit
This is the remaining part of the Module 1 genetic circuit
Polypeptide Showcase
To demonstrate The E.ncapsulator's versatility, we have chosen to showcase it with both enzymes and peptides. These two classes of polypeptide have very different properties that we have considered and catered for in The E.ncapsulator's design.
Enzyme Production
One of the challenges involved in enyzme production is the need for resistance to the proteases found within the small intestine. Although The E.ncapsulator is capable of delivering the enyzmes safely through the harsh environment in the stomach, upon release into the small intestine, the cells would be susceptible to breakdown by the proteases naturally found in the gut environent. For this reason, we have used protease resistant forms of the enyzmes to be produced.We have chosen two enzymes to showcase The E.ncapsulator's protein production module. These are:
- Cellulase - an enzyme that breaks down the tough fibrous molecule cellulose into cellobiose. In delivering cellulase to the gut we aim to increase the nutritional value of food consumed.
About cellulase and our proposed solution
- Phenylalanine Hydroxylase - an enzyme that breaks down the amino acid phenylalanine into tyrosine. In delivering this enzyme to the gut we aim to provide a treatment to those suffering from the metabolic condition, phenylketonuria.
About the genetic condition phenylketonuria, and PAH and how we modified it
Peptide Production
The delivery and production of short chain peptides is a different challenge altogether. All peptides when synthesised always start with the amino acid methionine. If synthesised directly, this can mean that the peptide no longer has the same bioactivity. The body naturally has a mechanism by which larger polypeptides are degraded into smaller functional peptides. Using this mechanism, we have designed a universal adapter for short chain peptide production and delivery, by which any peptide can be produced and delivered to the gut.
About our unique strategy for universal manufacture of peptides
To demonstrate this, we have chosen to showcase a short chain peptide, opiorphin:
- Opiorphin - is a small pentapeptide (5 amino acids) that is naturally produced by the body, and plays a role in pain relief and as an anti depressant. By delivering this small peptide into the gut, we hope to offer a natural alternative to the other more addictive drugs.
About opiorphin, and the remarkable effects of this small pentapeptide
Results
Wet Lab
Dry Lab
Conclusion
Engineering of bacteria to produce proteins is by no means a new development. However, due to the way in which we have designed our chassis to manufacture a protease resitant strains of the enzymes, as well as the unique method for the production and delivery of small peptides, we believe that <b>The E.ncapsulator offers a novel approach to polypeptide production.
Project Tour
Module 1 Contents