Go Fish - Targeted Capturing of Members of the Microbiome

We will design and optimize a protocol for bacterial capture and analysis using glycosylated magnetic beads. The bacterial strains used will be of the E. coli, S. epidermidis, and S. aureus species, and analysis of the bound bacteria will be done using Qubit fluorometric quantification and qPCR.

 

Glycan Immobilization on Beads

Bacteria express glycans on their surface, which could be utilized as a method for cell targeting by taking advantage of glycan:glycan affinity interactions. This method offers the possibility of being strain-specific, as glycans are differentially expressed between strains.

Protocol:

1. Isolate cell-surface glycans from strains of Staphylococcus, and make a glycan array to determine which glycan from each strain has the strongest affinity for its analog and offers the most specificity.

2. Create magnetic beads with the glycans that were found in step 1 aggregated in the polymer so that they are on the surface.

3. Test each bead on just the strain it is specific for.

4. Co-culture a variety of strains and test each bead design to determine specificity and efficiency.

 

qPCR Sorting Method

Using DNA extracted from our samples, qPCR can be run using primers specific to a bacteria strain in order to quantify the amount of DNA present of each strain type. This method can be used even for small amounts of DNA. Samples with higher amounts of DNA will increase in fluorescence faster than those with smaller amounts of DNA.

DNA will be extracted from the samples directly using bacteria genomic DNA extraction kit. The designed probe and primers set for the qPCR will be based on designed primers from the literature that have been proven to work for the strains we are using. The primers will be fluorescently labeled with SYBR green and amplified in cycles. The amount of the fluorescence released during amplification is directly proportional to the amount of amplified DNA. Fluorescence is monitored during the whole PCR process (along all 30 to 45 cycles). The higher the initial number of DNA molecules in the sample, the faster the fluorescence will increase during the PCR cycles.

To get standard curves and to confirm the presence of DNA in a sample we also use the quantification results of the DNA extraction on the Qubit machine. This gives us an estimate of the DNA concentration which can be used to create a standard used to determine the DNA in each sample with an unknown concentration. Additionally, it can confirm the absence of DNA in a sample if we use a higher enough volume in the sample and still do not get a concentration from Qubit.

 

Protocol:

 

1. Extract gDNA from the bacteria captured by the glycan beads.

2. Use designed bacterial species-specific primers for qPCR.

3. Put the gDNA, PCR reagents, and SYBR green into the qPCR plates.

4. qPCR is run in a thermal cycler with the capacity to illuminate each sample with a beam of light and detect the fluorescence emitted by the excited fluorophores.