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

From 2012.igem.org

(Difference between revisions)
 
(15 intermediate revisions not shown)
Line 37: Line 37:
</li>
</li>
<li>
<li>
-
<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/reactors">Reactors</a>
+
<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a>
</li>
</li>
<li>
<li>
-
<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a>
+
<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a>
-
</li>
+
</li>
-
+
<li>
<li>
<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a>
<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a>
Line 72: Line 71:
<div class="row">
<div class="row">
<div class="nine columns">
<div class="nine columns">
-
<h3>Naphthalene as a Sole Carbon Source</h3>
+
<h3>Biosynthesis of Indigo</h3>
-
One method used to prove the function of the Nah operon indirectly was an attempt to grow <i>E. coli</i> 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 <i>E. coli</i> strain DH5&alpha; cells not containing any naphthalene degrading plasmid. After three days of the initial test, the OD600 of the naphthalene degrading cells remained at the initial amount, and the naphthalene present initially did not seem to decrease significantly. It is possible that using naphthalene as a sole carbon source is not possible due to an inability of <i>E. coli</i> to use any of the intermediates of the naphthalene degradation pathway as a carbon source.
+
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.
 +
<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.
</div>
</div>
<div class="three columns">
<div class="three columns">
-
<img src="http://2012.igem.org/wiki/images/b/b1/Napdeg.jpg">
+
<a href="http://igem.org/wiki/images/8/88/IMG_0149notswqua.jpg" rel="lightbox"><img src="http://igem.org/wiki/images/e/e3/IMG_0149_square.jpg"></a>
</div>
</div>
</div>
</div>
-
<div class="row last-ele">
+
<div class="row">
<div class="three columns">
<div class="three columns">
-
<img src="http://2012.igem.org/wiki/images/f/f0/Indigotest.jpg">
+
<a href="http://igem.org/wiki/images/a/aa/IMG_0133not.jpg" rel="lightbox"><img src="http://igem.org/wiki/images/9/95/IMG_0133square.jpg"></a>
</div>
</div>
<div class="nine columns">
<div class="nine columns">
-
<h3>Biosynthesis of Indigo</h3>
+
<h3>Testing and Results</h3>
-
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 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 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.
+
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.  
-
<br><br>
+
-
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.
+
-
<br><br>
+
</div>
</div>
</div>
</div>
Line 97: Line 94:
</div>
</div>
 +
<div class="twelve columns">
 +
<h3>References</h3>
 +
[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.
 +
<br></br>
 +
 +
</div>
</div>
</div>
</div>
</div>

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.