Team:San Andres/Modeling

 

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Enzymes

During our investigation we sought the perfect enzyme to degrade the gluten, and we found:

• Prolyl Endopeptidase: The prolyl endopeptidase (PEP), is a class of serine-protease able to break peptide bonds following a PROLINE residue terminal carboxyl group. Were studying it its use as a therapeutic agent against celiac disease (CD), characterized by atrophy of the intestinal villi and inflammation. These reactions are attributed to peptides rich in PROLINE that are generated during the digestion of gluten of some cereals. This enzyme could be used as a nutritional supplement for individuals who suffer from celiac disease or during the processing of starches produced from cereals containing gluten. While it was a candidate for a possible treatment failed to meet expectations, because its activity is to a high pH, which is not suitable for an average digestive. Another reason was that degrades the gluten slowly, which would have resulted in a longer and inefficient treatment. 

• Kumamolisin As: It is the first known example of a collagenase derived from the family of the sedolisin. This operates at high temperatures and low pH levels. Its characteristics, together with those predicted are measured by comparison between a collagenase and a peptidase from serine, which are related to the enzyme preference, to thus Digest collagen as gluten.

• KumaMax (G319S, D358G, D368H, N281D): It is a mutation of the Kumamolisin As, which is designed to digest way more efficient gluten, because that can work at pH levels much more lower than the original enzyme (a pH of 4.0) which is excellent for the average digestive system. It was created by the team IGEM Washington 2011. Other advantages are:

1. It is resistant to high temperatures and acidity of the stomach.
2. It is heat stable, in others words, it is resistant to all changes in their physical and chemical structure.
3. It is easily repairable and creable.

   

Methodology 

The methodology consists of:

- For our Plasmid construction, we decided to take the method standard of Assembly biobrick, based on grafts and vectors, front and reverse.

- Then we cut the two genes and making them again an insert front, which we took to the vector of the terminator making as well as the genes a front vector.

- After making two front inserts, we proceed to start with two reverse inserts for where we cut the three genes (Kumamax, RFP, terminator) and we make them a reverse insert, and we took him to the inverse vector of the RBS.

- Finally we proceed to cut the four genes (RBS, Kumamax, RFP, terminator) as insert reverse, to take them to the inverse vector of the promoter, and we finished building our final plasmid, the "Kumamax Plux".