Team:Queens Canada/ChimeriQ

This year, our team is investigating new methods of increasing the efficiency of bioremediation and biosynthesis using modified bacteria. The development of the oil sands in Alberta, has resulted in the build up of toxic byproducts stored in massive tailings ponds. To help resolve these issues, our goals can be divided up into three main categories: the binding of pollutants, adhesion and aggregation of bacteria, and catalysis.

Most bacteria possess tail-like appendages called flagella, which can be genetically altered for novel functions. Each flagella is made up of a number of polymerizing proteins, often called flagellin. By making chimeric insertions in the variable domain of the flagellin, we can incorporate metal binding proteins, enzymes, adhesive proteins as well as scaffolding proteins to further extend the possible applications. To accomplish this, we can summarize the majority of our work into three main tasks:

• clone and modify the constant domains of the flagellin protein for making insertions using Biobricks and parts obtained from the wild.
• design a flexible, compatible cloning method for efficientlymaking chimeric insertions using Biobricks and other parts
• introduce binding and catalysis to the length of the flagella.

A potential application of this project would be to externally catalyze useful chemical reactions. The only real practical limitation would be the size of the amino acid insert. Prior research in the field of chimeric flagella has shown that inserts of around 300 amino acids can be inserted into the FliC D3 domain without affecting the tertiary structure of the flagella. Thus, the search for feasible pathways was limited to reactions with steps that involved the use of a single enzyme and were sufficiently small. A potential parts in the biobrick registry that fit this criteria was XylE (catechol 2,3-dioxygenase). This enzyme converts catechol into 2-hydroxymuconate semialdehyde, which conveniently provides a bright yellow product.

We also researched various pathways outside of the BioBrick registry for useful pathways and eventually settled on the enzyme Haloalkane dehalogenase. This enzyme is found in several organisms and refers to an enzyme the dehalogenates haloalkanes into alcohol and halides. Given that haloalkanes such as 1,2-dibromoethane are quite dangerous (Rated a 3 in the health section on the NFPA 704 scale [1]), this enzyme provides a useful means of cleaving these toxic products into safer products. We contacted various professors around the world, and were able to obtain the linB and the Rv2579 gene, which both encode the protein, but in Sphingomonas paucimobilis UT26 and Mycobacterium tuberculosis H37Rv, respectively.

These enzymes were incorporated into E. coli in a similar manner to the fluorescent proteins. Using PCR overlap extension, we were able to create the plasmid for LinB, Rv2579, and XylE from an RFP plasmid template. The PCR overlap extension products were digested using DpnI and transformed into E. coli. The colonies were plated. As expected there were several colonies on each plate, and using the fluorescence of the E. coli we were able to isolate which colonies potentially contained the plasmid by picking the colonies without fluorescence.