Team:Queens Canada/Results

MhLap Dextran Binding

The first step in assessing the feasibility of using dextran to crosslink M. hydrocarbonoclasticus to our biofilm was to test the ability of this bacterium’s sugar binding domain to bind dextran. Luckily, Tyler Vance of Dr. Davies’ lab already had the sugar-binding MhLap region III cloned into pET-28 for overexpression in E. coli. We expressed, then purified this construct using a Ni-NTA column. Purified MhLap Region III fractions were pooled. We then set up a simple affinity chromatography binding assay. A Pasteur pipette was filled half-way with sephadex G-200 slurry. Purified MhLap was then added, followed by five washes with buffer. We then washed twice with 5 mg/mL dextran solution. Finally, the column was washed twice with EDTA to nonspecifically elute all protein. The collected fractions can be seen in the following SDS gels. A significant amount of protein was eluted by dextran, shown by the MhLap band in the D2 lane. The dextran solution added would act as a competitor for Sephadex binding. This suggests MhLap does indeed bind dextran. The large band eluted by EDTA in E2 is also MhLap. This suggests MhLap binding to the Sephadex solid phase is strong enough that aqueous dextran is insufficient for full elution.

Fig 1. MhLap dextran binding experiment gels. From left to right, the lanes contain flow through (FT), buffer washes 1-5, dextran (5 mg/mL) washes 1-2, and 1M EDTA washes 1-2.

FITC-Dextran Binding M. hydrocarbonoclasticus Cells

Following the promising binding of recombinant MhLap region III to dextran, we sought to test if the MhLap expressed on the surface of M. hydrocarbonoclasticus cells could also bind dextran. To do this, used fluorescently-labeled FITC-dextran and microfluidics. We grew overnight cultures of M. hydrocarbonoclasticus in marine broth, pelleted the cells, then incubated the cells with FITC-dextran solution. The cells were then washed twice with buffer to remove any unbound FITC-dextran. We then injected the cells into a microfluidic chip and imaged them with both brightfield and fluorescent microscopy. When the brightfield and fluorescent images were overlaid, we observed that the majority of the fluorescent dots corresponded in location to bacterial cells. There were some bacteria that failed to fluoresce, indicating imperfect dextran binding or heterogenous expression of the MhLap adhesin appedange.

Fig 2. M. hydrocarbonoclasticus dextran binding microfluidics. Brightfield, fluorescence, and overlaid images are shown at 10 x magnification.

Purification of GFP-SpyCatcher

We successfully expressed and purified GFP-SpyCatcher. Unfortunately, due to DNA synthesis difficulties and time constraints, we were not able to submit this part as a BioBrick. Nonetheless, were excited when we saw the results of our hard work glow green under UV light! This protein fusion will be very useful for proof-of-concept experiments, providing a way to visualize successful bonding of SpyCatcher onto a CsgA-SpyTag scaffold.

Fig 3. Purified GFP-SpyCatcher The tube on the left is buffer as a negative control, and the tube on the right is our GFP-SpyCatcher (~3.5 mg/mL).