Team:Queens/Results

Results

The following summarizes the results of the various aspects of this year's QGEM Project.

Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.

Fig. 1 SAA expression and secretion by E. coli cells. Cultures of E. coli cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein.

Our E. coli cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future.

References:

Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. New York Academy of Sciences. 82:183-189.
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. Journal of Biological Chemistry. Vol. 274, 11:7172-7181.

In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to E. coli cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process.

Fig. 2 Flowchart of PCR stitching for constructing the binding construct.

Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing.

Fig. 3 In silico model of the binding construct.

References:

Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Engineering. Vol. 12, 7:613-621.
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium Vibrio anguillarum. American Society for Microbiology. Vol. 74, 14: 4359-4365.

After our chassis has been bound to the site of the plaque for a period of time, it should begin to produce DNases and proteases. The DNases will function to sheer the bacterial genome, making it unable to proliferate in the blood; this functions as a safe-guard against bacteremia. The proteases serve to detach the chassis from the plaque site, so as to avoid a build-up of dead cell debris in an already inflamed area.

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