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From 2013.igem.org

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Project Abstract

Restriction Enzymes are necessary tools in synthetic biology, without them, biobrick assembly would be impossible. These restriction enzymes are also very pricey and prove to be a significant cost in labs everywhere. Because these enzymes are expensive, but necessary, experiments in synthetic biology are limited to companies and universities with high budgets. Here at the University of Colorado-Boulder, we aim to find a technology that lowers the costs of these restriction enzymes and open the field of synthetic biology to more people. Our solution to this problem we are trying to create is a "BetaBioBrick." Essentially we are trying to come up a with low cost method for labs to create their own restriction enzymes so they won't have to order from pricey suppliers. We attempting to do this by creating a BioBrick part that will house the genes for a restriction enzyme (EcoRI, XbaI, ApoI), their relative methylase, and if needed a method of purification.

Background

Restriction-modification (R-M) systems are used by prokaryotes (mostly bacteria) as a defense mechanism to protect themselves from infection of foreign DNA from viruses, such as bacteriophages, and can be thought of as the prokaryotic equivalent of the immune system. The function of an R-M system requires two independent enzymes that share a particular DNA sequence specificity: a restriction endonuclease (REase) which is used to digest foreign DNA, and a modification methyltransferase (MTase) which is used to protect the cell’s native DNA. Type II R-M systems are the simplest and most prevalent, and also produce REases (and MTases) which are highly predictable with regard to sequence specificity. These characteristics have enabled these enzymes to become valuable tools in synthetic biology for for the purposes of gene cloning and DNA analysis. Each REase and corresponding MTase recognize a specific sequence of DNA which is typically 4 to 8 nucleotides in length and is generally palindromic. The REase effectively cleaves the both strands of the DNA backbone at a specific position within this sequence, which can result in either “blunt” or “sticky” ends depending on the location of the cut site. These enzymes typically form homodimers and require an Mg2+ ion for enzymatic activity to take place. An MTase is used to tag the native DNA with a methyl group at the site of each specific sequence, which sterically inhibits the binding of the REase. These enzymes are typically monomeric and are necessary to protect the cells native DNA from REase activity. R-M system must be closely regulated by the cell in order to avoid auto-restiction and cell death in addition to over-modification, which could potentially interfere with genome function.

Biobrick Assembly Kit

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ApoI Malaria Test Kit

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