Team:Yale/Our Project/Methods/h2s production
experimental methods: h2s production
Our engineered E. coli produces hydrogen sulfide (H2S), which catalyzes mineral deposition and iron reduction depending on availability of metal ions in the growth medium. H2S reacts directly with metal ions in the bacterial environment. H2S production is induced by IPTG, which is incorporated into the plates the E.coli are grown on. When IPTG is used up, H2S production stops. This limiting step is beneficial so that H2S, a toxic substance, does not hinder E.coli growth.
The plates were prepared using a recipe for Triple Sugar Iron (TSI) agar. This agar is usually used to detect bacteria that ferment non-glucose sugars and produce H2S. The sugars were used for general identification purposes outside of hydrogen sulfide production as well as an indicator that the bacteria are still alive and oxidizing the sugars. Only ferrous sulfate and sodium thiosulfate are necessary to detect H2S. When bacteria couple thiosulfate reduction to oxidations, these iron ions serve as an indicator of H2S production by turning black upon formation of ferrous sulfide (FeS)
The original tests were conducted using agar slants in which E.coli were extracted using a sterile loop and stuck into the agar. Within a few days black spots were observed in the originally orange tubes, indicating the production of H2S. It was hypothesized that the anaerobic environment inside the agar tubes played a large role in the production of H2S. In addition, diffusion of the H2S gas is reduced in the solid media.
To further demonstrate ability of the engineered E.coli to produce H2S and to make more visually appealing use of the black that appears, plans were made to take advantage of the dark indicator to spell out “Yale” and create other school-spirited images. In order to facilitate the manipulation of E.coli, a thick liquid mixture of bacteria was made. Several different methods were adopted in order to get the same effect viewed in the slants on the petri dishes.
One of these methods involved cutting letters into the agar with a loop with E.coli. Another method devised was to inject E.Coli onto the petri dish using a syringe, and then later into the agar, in the formation of the desired design. There was also a sandwiching technique that was used in which E.coli were deposited onto the surface of the plate and another layer of already prepared agar was layered on top. These dishes were then sealed and placed in an anaerobic chamber in an incubator set at 37°C. For all these trials, bacteria succeeded in growing, but no H2S production was observed because no black spots were found on the plate.
This led to the hypothesis that the local concentration of H2S for bacteria embedded in the agar is much higher than those spread on the surface of the plates and that it was not anaerobic growth that allowed embedded bacteria to turn black versus plated bacteria.
The second trial to see if this hypothesis utilized a procedure very similar to the one used in the slants. Using the same liquid bacteria mixture, the bacteria were “tattooed” onto the petri dishes using a very thin loop. After each poke into the agar, the loop was dipped back in to the bacteria mixture to ensure a high concentration of E.coli for each prick into the agar. Again these dishes were sealed and placed into an incubator.
The tattooing of bacteria into the agar resulted in successful growth of bacteria and modest production of H2S as indicated by the presence of precipitated FeS.