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Go Fish - Targeted Capturing of Members of the Microbiome

GRAPHICS

Glycans on a cell surface Most glycans are found on the surface of the cell membrane and are important in modulating or mediating a wide variety of events in cell – cell interaction in a variety of cell types.	Roles of the human microbiome The microbiome plays a number of crucial roles within the human body, including metabolism of therapeutics, development and training of the immune system, and promotion of angiogenesis.	qPCR amplification curve This is an example of a qPCR amplification curve. Initially, the levels of fluorescence are too low to for the increase to be detected. The fluorescence levels then increase exponentially before plateauing due to PCR resource limitation.
Glycan binding and capture protocol Describe your image here.	E. coli primer standard We generated a best fit line equation of y = -4.75x + 18.1. This corresponds to a conversion from Cq value to a bacteria concentration of [DNA] = 10^[-(Cq-18.1)/4.75].	S. epidermidis primer standard We generated a best fit line equation of y = -4.6x + 19.4. This corresponds to a conversion from Cq value to a bacteria concentration of [DNA] = 10^[-(Cq - 19.4)/4.6].
S. aureus primer standards Only two points were readable on the first two standard tests we performed for S. aureus (top left and right). The third standard generated a best fit line equation of y = -3.64x+26.1. This corresponds to a conversion from Cq value to a bacteria concentration of [DNA] = 10^[(-Cq - 26.1)/3.64].	EDTA test Samples where EDTA was added had little to no DNA present, indicating that the addition of EDTA does not increase binding efficiency.	Capture efficiency graph The percent binding values and number of bacteria captured were analyzed at different starting concentrations. As the starting concentration increases, the number of bacteria captured increases and the percent binding decreases.
Incubation temperature optimization The protocol was performed at 4°C, 20°C, and 37°C as the incubation temperatures. We calculated the percent binding for each condition and observed the highest level of binding in the 37℃ incubation.	Differential capture from co-culture A co-culture of E. coli. S. epidermidis, and S. aureus was tested for binding to Mannose, 2' Fucosyllactose, N-Acetylgalactosamine, and Fucose. Mannose and E. coli show the highest binding efficiency at 6.2%. All of the glycans besides GalNac show a higher affinity for E. coli than S. epidermidis.	Serial depletion from co-culture We prepared a culture of 46.3% S. aureus and 53.7% E. coli and depleted the culture with 7 treatments of the protocol. These results were obtained using the first standard for S. aureus, because the third standard resulted in an unreasonably high starting concentration of S. aureus. From this co-culture, we obtained an E.coli culture with negligible (undetectable) amounts of S. aureus.

Page Leader: Alexander Kasper

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