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.

• 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.

Through the learning phase we researched intensively on the full flagella construct and what was required to incorporate protein infusions. As stated before, the D3 domain in the flagella construct has no real function, and can therefore be replaced with a protein of our choice. Through PCR overlap extension we would incorporate GFP and RFP into our constructs. To prove that it PCR overlap extension would worked, we would incorporate this PCR product containing the full fliC construct with no linkers with the GFP replacing the D3 domain (this was labelled nFFGFP - no linker Full Flic GFP construct) into the T7 promotor J04500 vector that contained the chloramphenicol resistance through digestion/ligation processes. We grew these ligations overnight and expected colonies.

As expected there were several colonies on each plate, to prove that the ligations worked only the colonies with green fluorescence were used for liquid colonies. Unfortunately we did not have access to an electron microscope, therefore we could not visually see the proteins on the flagella. Researching various flagellar isolation protocols , we developed two tested protocols to isolate the flagella containing GFP, which can be found in the protocols section. We also had a batch of J04500 controlled bacteria made to act as the control that would not contain protein infusions.

Essentially these two protocols would shear the flagella off of the cells using blender and heat techniques. The cell debris would be removed by centrifuge, and the sheared flagella would be collected as pellets. These isolated pellets when observed under the fluorescent microscope did glow green. Since there is a possibility that some cell debris would be present in the flagella pellets, we decided to run SDS-PAGE to see if there was a different between the controlled bacteria that would only contain flagella but not protein infusions. What we predicted was that at there should be a protein band at 70 kDa, which would be due to the GFP protein infused on the full fliC construct which would not be found on the control bacteria. Through running a few SDS- pages and comparing the control vs. fluorescent flagella bacteria, there was a distinct darker band at 70 kDa that was not found in the control. This means that it is likely that the flagella pellets in our nFFGFP (no linker Full Flic GFP construct) contained GFP proteins, which the J04500 bacteria did not.

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.