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 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. The first enzyme involved in the system is the restriction endonuclease (REase). It is specific to a single DNA sequence that is typically four to eight nucleotides in length and is usually palindromic. When the REase recognizes this sequence, it will cleave both strands of the DNA at the backbone, resulting in “blunt” or “sticky” ends, depending on the exact function of the specific enzyme. When a bacterial cell is infected by a virus, the REase will bind to and cut its recognition sequence if present in the virus DNA; thereby, hindering or stopping the effects of the virus on the host. But 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 is specific to the same site as its corresponding REase, but instead of cutting the DNA at this site, it tags the DNA with a methyl group, which sterically inhibits the binding of the REase. 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.

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 an infection of Plasmodium falciparum and caused approximately 660,000 deaths in 2010 according to a World Health Organization report [1]. Chloroquine, an antimicrobial drug, is widely used to treat malaria; however, an increasing prevalence of chloroquine resistance has complicated malaria treatment.

This increased resistance is linked to 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 [2]. The same mutation in pfmdr-1 may also be involved in lumefantrine resistance and serves as a marker for mefloquine vulnerability [3].

In order to treat malaria, the infecting bacteria must be tested for the presence of these mutations. One possible way of testing for and monitoring the spread of malaria strains is by digestion with the enzyme ApoI[4]. ***HOW DOES THIS WORK****.

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.