Team:Yale/Our Project/Methods/h2s production

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experimental methods

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Our engineered E. coli produces hydrogen sulfide (H2S), which catalyzes copper deposition and iron reduction depending on availability of metal ions in the growth medium. H2S reacts directly with metal ions in the bacterial environment. The hydrogen sulfide production is promoted by IPTG. IPTG 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, which is toxic, 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 aren't necessary in the recipe. Only ferrous sulfate and sodium thiosulfate are necessary to detect H2S production. These iron ions that serve as an indicator of H2S production by turning black upon contact with H2S when bacteria couple thiosulfate reduction to oxidations.

The composition of the plates were:

500 mL water

1.5 g Meat Extract (we got this from Steitz lab last time)

1.5 g Yeast Extract

7.5 g Pancreatic Digest of Casein

2.5 g Protease Peptone

0.5 g Dextrose

0.5 g Lactose

0.5 g Sucrose

0.26 g Ferrous Ammonium Sulfate (hexahydrate)

2.5 g NaCl

0.15 g Sodium Thiosulfate

6 g Agar

12 mg Phenol Red

+ Ampicillin

The original tests were conducted using agar slants in which E.coli were extracted using a 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.

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. Several different methods were adopted in order to get the same effect viewed in the slants on the petri dishes.

In order to facilitate the manipulation of E.coli, a solution was made using:

10 microliters of IPTG

200 microliters of S.O.C. (a medium that promotes growth)

cultures of E.coli

This mixture was then vortexed to create a thick liquid of bacteria.

One of these methods was to cut the letters into the agar with E.coli solution on a loop. Another method devised was to inject E.Coli onto the petri dish and, then later into the agar, in the formation of the desired design. There was also a sandwiching tecnique that was used in which the 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 embedded bacteria 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 was true 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 liquid to ensure a high concentration of E.coli for each prick into the agar. Again these dishes were sealed and placed into an incubator.

[results to be reported]