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

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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/reactors">Reactors</a>
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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a>
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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a>
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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a>
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<h3>Biosynthesis of Indigo</h3>
<h3>Biosynthesis of Indigo</h3>
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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.
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Past work indicates that when the <i>nah</i> operon from the NAH7 <i>Pseudomonas</i> plasmid is heterologously expressed in <i>E. coli</i>, the cells have the ability to synthesize indigo from tryptophan [1]. Therefore, as indirect proof that our <i>nah operon</i> BioBrick has activity in our engineered strains, we set several cultures to determine whether indigo was present when cultures were 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.
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<br><br>We are in the process of isolating the four coding regions which make up the naphthalene dioxygenase complex from the operon and <a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">BioBrick-ing</a> this genetic part so that future researchers have access an indigo-producing BioBrick.
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<h3>Testing</h3>
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<h3>Testing and Results</h3>
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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.  
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Cultures of DH5&alpha; containing the <i>nah</i> operon were started in LB broth with 200 &mu;M tryptophan and 100 mg/L 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.  
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<h3>References</h3>
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Ensley, B., Ratzkin, B., Osslund, T., Simon, M., Wackett, L., &amp; 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.
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[1] Ensley, B., Ratzkin, B., Osslund, T., Simon, M., Wackett, L., &amp; 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.
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Latest revision as of 02:38, 27 October 2012

nah Operon Expression

Biosynthesis of Indigo

Past work indicates that when the nah operon from the NAH7 Pseudomonas plasmid is heterologously expressed in E. coli, the cells have the ability to synthesize indigo from tryptophan [1]. Therefore, as indirect proof that our nah operon BioBrick has activity in our engineered strains, we set several cultures to determine whether indigo was present when cultures were 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.

We are in the process of isolating the four coding regions which make up the naphthalene dioxygenase complex from the operon and BioBrick-ing this genetic part so that future researchers have access an indigo-producing BioBrick.

Testing and Results

Cultures of DH5α containing the nah operon were started in LB broth with 200 μM tryptophan and 100 mg/L 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

[1] 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.