Team:Cornell/testing/project/wetlab/4/6

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Naphthalene Growth Assays

Naphthalene as a Sole Carbon Source

One method used to prove the function of the Nah operon indirectly was an attempt to grow E. coli on naphthalene as a sole carbon source. This test was done by adding naphthalene at 10 g/L to two salt solutions (M4 and only ammonium sulfate) to provide a constantly saturated solution of naphthalene. The solubility of naphthalene is 30 mg/L, therefore there would be solids present in the solution initially, disappearing as the cultures degraded the naphthalene currently in solution. Controls of M4 salts with no carbon source and LB media were used, as well as repeating all cultures with E. coli strain DH5%alpha; cells not containing naphthalene degrading plasmid. After three days, the OD600 of the naphthalene degrading cells remained at the initial amount, and the naphthalene present initially did not seem to decrease significantly. This is likely caused by an inability of E. coli to use any of the intermediates of the naphthalene degradation pathway as a carbon source.

Biosynthesis of Indigo

When the Nah7 operon is transformed into E. coli and expressed, the cells have the ability to produce indigo from tryptophan. Therefore, as an indirect proof of the Nah operon's function, several cultures were set up to determine if indigo was present when supplemented with 200 μM tryptophan. After one night, the cells did not appear to have been expressing the operon strongly enough for red fluorescent protein, which it was labeled with, to be detectable. After two nights, the red fluorescent protein could be seen, but an extraction with diethyl ether did not appear to remove any hydrophobic purple dye from the cultures, nor were purple spots visible on the plate.

Ensley, B., Ratzkin, B., Osslund, T., Simon, M., Wackett, L., & Gibson, D. (1983). Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science, 222(4620), 167-9.

Fig. 1. Pathway by which E. coli can produce indigo from tryptophan when expressing genes of the Nah7 operon. (Ensley et al., 1983)

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 					<h3>Biosynthesis of Indigo</h3>
-					It was discovered that when the Nah7 operon is transformed into <i>E. coli</i> and expressed, the cells have the ability to produce indigo from tryptophan. Therefore, as an indirect proof of the Nah operon's function, several cultures were set up to determine if indigo was present when supplemented with 200 &mu;M tryptophan. After one night, the cells did not appear to have been expressing the operon strongly enough for red fluorescent protein, which it was labeled with, to be detectable. After two nights, the red fluorescent protein could be seen, but an extraction with diethyl ether did not appear to remove any hydrophobic purple dye from the cultures, nor were purple spots visible on the plate.
+					When the Nah7 operon is transformed into <i>E. coli</i> and expressed, the cells have the ability to produce indigo from tryptophan. Therefore, as an indirect proof of the Nah operon's function, several cultures were set up to determine if indigo was present when supplemented with 200 &mu;M tryptophan. After one night, the cells did not appear to have been expressing the operon strongly enough for red fluorescent protein, which it was labeled with, to be detectable. After two nights, the red fluorescent protein could be seen, but an extraction with diethyl ether did not appear to remove any hydrophobic purple dye from the cultures, nor were purple spots visible on the plate.
 <br><br>
 Ensley, B., Ratzkin, B., Osslund, T., Simon, M., Wackett, L., & Gibson, D. (1983). Expression of naphthalene oxidation genes in <i>Escherichia coli</i> results in the biosynthesis of indigo. <i>Science</i>, 222(4620), 167-9.