Difference between revisions of "Team:Harvard BioDesign/Platform"

Strains and Assays

Before we could begin engineering the Fim system (link to Ben's description of the operon) we needed to establish a positive and negative baseline for the experiments we'd use to categorize our constructs. We selected two strains from the Yale's Keio collection (link) of nonessential gene knockouts which are each missing one of the recombinases that comprises the fim “switch” (link to Ben's background). We hypothesized based on the literature that a knockout for the fimB recombinase (Keio strain name JW4276) will not produce pili because fimE is biased to switch in the direction of “on” to “off” (switchAndInteractionfimEandB). When fimB is absent, fimE would switch the operon “off”. Contrarily, a knockout for fim E (Keio name- JW4276) will overproduce pili because fimB switches both “on” to “off” and “off” to “on”, so the operon will be “on” more than it would if both were present.

To confirm this hypothesis we used an agglutination assay (protocol here link to protocol) from the literature which is standard for measuring the expression of Type 1 Pili (Klemm, Schembri, Stentebjerg-Olesen, Hasman, Hasty 1998).

This protocol tests whether a culture of E. coli cells can agglutinate--clump together--a substrate which contains the binding partner (link to background) of fimH, mannose sugar. We chose to try to clump together S. cervisiae (baker's yeast) because it expresses mannose on its cell surface, is easy to grow in lab, and has been used in this assay before (Eshdat, Speth, Jann 1981).
We found that the fimE KO we hypothesized would overproduce type 1 pili indeed agglutinated the yeast, as indicated by a white clump at the bottom of the tube. The fim B KO samples showed no sign of clumping and were completely opaque:

To confirm these results, we examined equal volumes of OD standardized (protocol link) agglutinated samples from either strain under light microscopy. Slides were prepared according to the slide preparation protocol (link) and to visualize the bacteria and yeast, we stained the slides according to the Gram Stain protocol (link).
These photos are representative of the morphology of each sample. The large purple circular cells are S. cerevisiae and the small pink cocci are E. coli:

At this point we were confident in the agglutination assay’s ability to detect the presence of pili via mannose-mediated adhesion. However, we hypothesized that our future modifications of the fimH adhesin might lead to weakened mannose binding strength even while the pili were still properly assembling. We required a diagnostic assay that would detect whether type 1 Pili were present on the cell surface even when fimH no longer bound to mannose. So we conducted extensive literature review and developed the (Pili Purification Protocol link) with the aim of a rapid test to provide a “yes” or “no” answer to this question.

We first tested our assay on our control strains, growing both the overproducer and null strain overnight in LB liquid culture, then OD standardizing (protocol) to ensure our results were not dependent on variable cell growth. Then a volume of each sample was spun down at low speed (so as not to lyse the cells), washed once in DPBS to remove secreted proteins, and resuspended in a smaller volume of DPBS to concentrate the signal in later steps. These samples were then heat treated for 20 minutes at 60°C to shear-off the pili from the cell surface and spun down again at low-speed so the cells would pellet but the sheared pili would remain in solution. This pili-containing supernatant was then denatured using formic acid, boiled in 1x Lamelli’s buffer and run according to the SDS-PAGE protocol (link). The resulting gels were stained according to the R-250 Coomassie protocol (link) and compared at the molecular weight of Type 1 Pili’s structural subunit protein, fimA, which is most abundant. We hypothesized that the pili overproducing fimE would show a strong band at this weight compared to the null fimB strain. The resulting gel is below:

Caption: “A band is present at the expected weight in the purified pili of the fimE overproducer. We tried testing the purification with and without formic acid acid denaturation. The fimA band is stronger in the formic acid treated sample which indicates that the pili are insoluble and need to be broken up for detection”

Indeed, we see a dark band at 16.5 kD, the molecular weight of fimA, but only for the fimE overproducing strain, not for our negative control. This shows our pili purification method gives a clear signal to detect pili production, and can be used to characterize our modified Type 1 Pili and by future teams using our system. With functional assays and positive and negative controls, we felt confident that we had the tools necessary to begin engineering control over pili expression.