Team:Cornell/project/wetlab/results/nah operon

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Revision as of 21:56, 26 October 2012

nah Operon Expression

Biosynthesis of Indigo

When the nah operon from the nah7 plasmid 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 tryptophan.

E. coli contains the enzyme tryptophanase, which splits the indole group from the peptide backbone. Naphthalene dioxygenase, from the nah operon, then creates a compound which will spontaneously eliminate water and be air oxidized into indigo as the final product.
The naphthalene dioxygenase gene will be isolated from the operon and created into a biobrick for future use of the enzyme in the synthesis of indigo from tryptophan or indole.

Testing and Results

Cultures of DH5α containing the nah operon were started in LB broth with 200 μM tryptophan and 1x ampicillin. Six days after the initial cultures were started, the liquid culture appeared purple. After extracting with chloroform, the organic phase was a light purple color. Testing is being done to determine if this purple compound is indigo.

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

References

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.

@@ Line 73: / Line 73: @@
 				<div class="nine columns">
 					<h3>Biosynthesis of Indigo</h3>
-					When the <i>nah7</i> 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 <i>nah</i> operon's function, several cultures were set up to determine if indigo was present when supplemented with 200 &mu;M tryptophan. <br><br><i>E. coli</i> contains the enzyme tryptophanase, which splits the indole group from the peptide backbone. Naphthalene dioxygenase, from the <i>nah</i> operon, then creates a compound which will spontaneously eliminate water and be air oxidized into indigo as the final product.
+					When the <i>nah</i> operon from the <i>nah7</i> plasmid 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 <i>nah</i> operon's function, several cultures were set up to determine if indigo was present when supplemented tryptophan. <br><br><i>E. coli</i> contains the enzyme tryptophanase, which splits the indole group from the peptide backbone. Naphthalene dioxygenase, from the <i>nah</i> operon, then creates a compound which will spontaneously eliminate water and be air oxidized into indigo as the final product.
+<br>The naphthalene dioxygenase gene will be isolated from the operon and created into a biobrick for future use of the enzyme in the synthesis of indigo from tryptophan or indole.
 				</div>
 				<div class="three columns">
@@ Line 85: / Line 86: @@
 				<div class="nine columns">
 					<h3>Testing and Results</h3>
-					After one day of initial testing, 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 days, the red fluorescent protein could be seen, but an extraction with chloroform did not appear to remove any hydrophobic purple dye from the cultures, nor were purple spots visible on the plate. Six days after the initial cultures were started, the liquid culture appeared purple. After extracting with chloroform, the solution was a light purple color. Chemical tests to determine if the compound was indigo were unsuccessful.
+					Cultures of DH5&alpha; containing the <i>nah</i> operon were started in LB broth with 200 &mu;M tryptophan and 1x ampicillin. Six days after the initial cultures were started, the liquid culture appeared purple. After extracting with chloroform, the organic phase was a light purple color. Testing is being done to determine if this purple compound is indigo.
 				</div>
 			</div>