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: It is a kind of serine protease capable of breaking peptide bonds following to the group of terminal carboxyl of a PROLINE residue. While he was a candidate for a possible treatment failed to meet expectations, because its activity is a high pH, which is not suitable for an average digestive. Another reason was that it degrades 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.

 

Metodology 

The methodology consists of cutting and pasting, with ligases and restriction enzymes as a "waterfall", for final assembly that will produce both proteins. The motive of this assembly is due to the fact that the promoter, the RBS and the terminator were too small, and if you take out them from their original plasmids, these are lost, so, simply sticking and peeling it off one by one, we arrived to a linear Assembly in the three corresponding plasmids (promoter, RBS and terminator).

At this time we are innovating ideas to add a circuit that will allow us in the future to obtain a method of detecting and quantifying the presence of gluten, which can also be checked by a commercial kit.