Team:CU-Boulder/Project/Kit/RestrictionEnzymes

***In the process of editing***

The main focus of our project was to create the constructs and purification methods necessary to produce and isolate the restriction enzyme EcoRI. EcoRI is commonly used for research and is also one of the primary restriction endonucleases used in the Biobrick standard. Synthesizing a plasmid containing the gene for EcoRI and producing a simple purification method would provide a cheaper option for obtaining the enzyme.

Bacteria lack an immune system so rely on restriction-modification (R-M) systems for defense against foreign viruses. When the bacterial cell is infected, a restriction endonuclease (REase) recognizes a specific sequence on the virus DNA and cleaves the strand(s); however, this process is not specific to host or virus DNA. To protect their own genetic information, bacterial cells also produce a methyltransferase (MTase). This enzyme shares sequence specificity with the REase and works to methylate the DNA of the host cell at the site which prevents the REase from cleaving here. In type I R-M systems, the REase and MTase are apart of a single enzyme whereas in the simpler, type II system, they are found as two individual enzymes. When isolated, the REase from a type II system can be used independently of the methylase to digest DNA for analysis and synthesis making these enzymes a valuable tool in synthetic biology.

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.

In order to produce restriction enzymes in vivo, it is necessary to protect the host DNA in order to avoid auto-restriction and cell death. This is possible by expressing an excess of the MTase corresponding to the REase that is being produced within the cell. Since REases form dimers (whereas MTases are monomeric) in their active form there is a natural lag phase before enzymatic activity is observed. This also implies a 2:1 excess of active MTase within the cell if both enzymes are expressed at the same rate. We intend to create a construct that expresses both enzymes on the same promoter in order to test if MTase can effectively protect the cell's DNA through this mechanism alone. It is likely that this will not provide the MTase with the advantage needed to sufficiently protect the host DNA from auto-restriction. It may be possible for the MTase to out compete the REase by simply expressing the MTase on a strong constitutive promoter and the MTase on a weak constitutive promoter. Alternatively, it should be possible to express the MTase on a constitutive promoter and use an inducible promoter system to delay the expression of the REase until the host genome is protected.

In addition to successfully producing restriction enzymes, it is also necessary to separate the MTase away from the REase for these enzymes to be functional for the purposes of synthetic biology. (add more about protein purification)

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.