UGA iGEM Collaboration
Over the summer, we were able to collaborate with the University of Georgia Atlantic iGEM team. UGA iGEM aimed to shift the most commonly used chassis in synthetic biology, E. Coli, to Methanococcus maripaludis, a model organism for methanogens. The rationale behind the shift in chassis stems from the inefficiency presented by E. coli. E. coli use expensive feedstocks such as sugars and oftentimes produces CO2, a waste product that wreaks havoc on the environment. The benefit of using methanogens can be observed in their autotrophic metabolism, as well as their ability to produce highly valued biochemicals such as methane.
Our study consisted of measuring the ribosome binding site specificity in Methanococcus maripaludis. UGA iGEM sent cells tagged with mCherry carrying over 30 point mutations in the ribosomal binding site, which we then measured with a Tecan plate reader. The methanogens were grown using mCherry just upstream of the ribosome binding site, were lysed, and resuspended in PIPES buffer, which then released mCherry proteins in the cell extracts. The cell extracts were then subjected to oxygenation in an effort to activate the fluorophore.
The 30 cell extracts (1A...10A, 1B...10B, 1C...10C) were subjected to an excitation wavelength of 562 nm, an emission wavelength of 650 nm, an excitation bandwidth of 40 nm, and an emission bandwidth of 75 nm. 10 flashes were used with a gain of 50 using a dichroic 630 mirror to measure the following levels:
Once the relative fluorescence levels were measured, a BCA protein assay was then used to measure the protein concentrations. The standard curve was produced using absorbance and concentration from the BCA protocol. A standard curve was generated, which allowed for a fluorescence/protein concentration graph to be made.
The absorbances were then measured for all thirty cell extracts. Those values were entered into the standard curve, which yielded the following data:
From obtaining the concentrations in each of the wells, we plotted the fluorescence against the concentration in an effort to see a correlation.