Team:CU-Boulder/Project

Restriction enzymes are invaluable tools in the field synthetic biology, and are essential for carrying out BioBrick assembly. However, these enzymes also represent a significant portion of the cost associated with gene cloning and DNA analysis. Here at the University of Colorado-Boulder, our vision is to develop the parts and methods necessary to produce and purify these enzymes cheaply and reliably. In order to make our project successful, we will be creating the BioBrick constructs that contain the restriction enzymes we are trying to produce. In addition, we will be developing procedures that allow us to purify these enzymes cheaply and effectively. The end result of our project will be the completion of several RE prep kits which will contain all of the components necessary to create working stocks of several commonly used restriction enzymes, including EcoRI, XbaI, and PstI which are all part of the BioBrick standard.

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 usually palindromic. The REase effectively cleaves 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. This enzyme typically forms a homodimer and requires 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. This enzyme is typically monomeric and is 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.

"Paragraph about Assembly kit"

Malaria is caused by an infection of Plasmodium falciparum and resulted in approximately 660,000 deaths in 2010 according to a World Health Organization report [1]. The antimicrobial chloroquine has been widely used to treat malaria. However, increasing prevalence of chloroquine resistance has complicated malaria treatment. An essential step in deciding the optimal course of treatment is determination of the most effective antimicrobial. A mutation in one of the membrane transporters of Plasmodium falciparum (pfmdr-1) where a tyrosine residue is replaced by an arginine residue and a mutation of the transporter pfcrt where lysine is replaced by threonine have been linked to increased resistance of chloroquine [2]. The same mutation in pfmdr-1 has also been suggested as playing a role in lumefantrine resistance as well as serving as a marker for mefloquine vulnerability [3] . Restriction digest with the enzyme Apo I can be used to detect the presence of these two mutations. Assays based on restriction digest have been suggested as potential ways of monitoring the spread of antimicrobial resistant malaria strains [4]. Our team is working to develop a means to allow inexpensive production of Apo I in resource limited conditions. Readily available restriction enzymes may serve to make restriction enzyme assay viable for determination of antimicrobial resistance in malaria.

1. Organization WH: World Malaria Report 2012. 2012.

2. H.H. Abruquah FYB, S.C.K. Tay, and B.W.L. Lawson: Resistance-Mediating Polymorphisms of Plasmodium Falciparum Among Isolates from Children with Severe Malaria in Kumasi, Ghana. Ghana Medical Journal 2010, 44:52-58.

3. Laufer MTaM: Resistance to Antimalarial Drugs: Molecular, Pharmacologic, and Clinical Considerations. Pediatric Research 2009, 65:64-70.

4. Durand R, Huart V, Jafari S, Le Bras J: Rapid Detection of a Molecular Marker for ChloroquineResistant Falciparum Malaria. Antimicrobial Agents and Chemotherapy 2002, 46:2684-2686.