Team:UCSF/Violacein Results

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<h3red>Production of Violacein by an <i>E. coli</i> Co-Culture
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<p>
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<br><regulartext>
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Before we started our experiments, we obtained a violacein standard from Sigma-Aldrich,<a href="http://www.sigmaaldrich.com/catalog/product/sigma/v9389?lang=en&region=US">Item:V9389-1MG </a><span>. We used this standard to measure the absorbance of violacein at varying concentrations, obtaining the standard curve shown below. Full wavelength scans were obtained, but as shown by previous iGEM teams (<a href="https://2009.igem.org/Team:Cambridge/Project/Violacein">Cambridge 2009 </a>) the maximum absorbance of violacein is seen at 575nm and is reported here. <br>
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<img align="left" style="margin-bottom:8px; width:755px;height:410px; padding:0;" src="https://dl.dropbox.com/u/24404809/iGEM%202012/igem%202012%20website%20photos/violacein/100212vio_std_cf.jpg">
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<regulartext> During our experiments we worked with several different strains of <i>E. coli</i>, each containing plasmids with different violacein constructs:
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<ul>
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<regulartext>
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<li>Strain 1: pcdfDuet+VioABE</li>
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<li>Strain 2:pcdfDuet+VioDC</li>
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<li>Strain 3: pcdfDuet:VioABE+VioDC (full operon)</li>
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<img align="left" style="margin-right:78px;margin-bottom:28px; width:655px; padding:0;" src="https://dl.dropbox.com/u/24404809/iGEM%202012/igem%202012%20website%20photos/violacein/VioPlates.jpg" <br>
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<P>
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<regulartext>
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While other teams and papers have reported that extracting pigment from cultures is the best way to perform analysis and quantification, several different solvents have been reported. The 2009 Cambridge iGEM team used acetone to extract, so we tested extraction of violacen from Strain 3 (full violacein operon) using acetone as well as methanol and ethanol. <p>
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<img align="left" style="margin-bottom:8px; width:755px;height:410px; padding:0;" src="https://dl.dropbox.com/u/24404809/iGEM%202012/igem%202012%20website%20photos/violacein/100112op_solvents.jpg">
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<regulartext>
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Our results show that ethanol was the best solvent to use for extraction and so the rest of our extractions were performed in ethanol. The details of growth and sample analysis can be found on our protocols page. <p><p><p>
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<regulartext>
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<h3red>Violacein Mono- and Co-Culture Wavelength Scans:</h3red>
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<br>
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Strain 1, which only has the first half of the enzymes necessary to produce violacein is still able to produce a green pigment. When the pigment is extracted from cells, the wavelength scan shown in dark blue is obtained.
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<br>
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Strain 2, which has the second half of the violacein pathway, produces no pigment. This is expected because without the first half of the pathway, no pigment can be produced.
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<br>
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Strain 3, which contains the entire violacein operon produces a wavelength scan similar to our standard obtained from Sigma-Aldrich, with violacein having a maximum absorbance near 575nm.
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<br><b>
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Co-Culture: Strain 1 and Strain 2 grown together (red line) produce a wavelength scan that indicates production of violacein. </b>
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<img align="left" style="margin-top:18px; margin-left:48px;margin-right:78px;width:755px;height:410px; padding:0;" src="https://dl.dropbox.com/u/24404809/iGEM%202012/igem%202012%20website%20photos/violacein/100112vio_wavelengths.jpg">
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<br>
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<h3red>Violacein Mono- and Co-Culture Extractions:</h3red>
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<br>
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<regulartext> When monocultures of strain 1 was grown, a dark green pigment was clearly observed. As seen in the plate above, strain 2 did not have any pigment on its own. However, when co-cultures of strain 1 and strain 2 were grown and the pigment extracted a purple pigment was seen and based on wavelength scans (above) appears to be violacein.
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<img align="left" style="margin-top:18px;margin-bottom:18px; margin-left:318px;margin-right:288px;width:255px;height:150px; padding:0;" src="https://dl.dropbox.com/u/24404809/iGEM%202012/igem%202012%20website%20photos/violacein/Extracts.png"><p>
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<br>
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<h3red>Addressing Efficiency and Metabolic Burden</h3red><br>
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Using linear interpolation (from the maximum absorbance at 575nm, wavelength scans above) we found that the co-culture produces 3.6199 mg/ml violacein and the total operon produces 3.5945mg/ml. While this amount is nearly equal, we believe that the production of violacein could be optimized such that it would produce much more violacein than the monoculture.
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<br><p>
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The growth rates of strains decrease when inducer is added, as shown in the left graph. By  comparing the rate of induced and uninduced growth, we can see how the cells are affected by production of their respective enzymes, and therefore how high the metabolic burden is. In the bar graph, Strain1+Strain2 (coculture) clearly is least affected by metabolic burden, showing that splitting a metabolic pathway does seem to lessen metabolic burden. <br> <p>
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<img align="left" style="margin-top:18px; margin-left:118px;margin-right:78px;width:700px;height:350px; padding:0;" src="https://dl.dropbox.com/u/24404809/iGEM%202012/igem%202012%20website%20photos/violacein/vio_growth_diff_vert.jpg">

Latest revision as of 04:06, 4 October 2012

Production of Violacein by an E. coli Co-Culture


Before we started our experiments, we obtained a violacein standard from Sigma-Aldrich,Item:V9389-1MG . We used this standard to measure the absorbance of violacein at varying concentrations, obtaining the standard curve shown below. Full wavelength scans were obtained, but as shown by previous iGEM teams (Cambridge 2009 ) the maximum absorbance of violacein is seen at 575nm and is reported here.
During our experiments we worked with several different strains of E. coli, each containing plasmids with different violacein constructs:

  • Strain 1: pcdfDuet+VioABE
  • Strain 2:pcdfDuet+VioDC
  • Strain 3: pcdfDuet:VioABE+VioDC (full operon)

    • While other teams and papers have reported that extracting pigment from cultures is the best way to perform analysis and quantification, several different solvents have been reported. The 2009 Cambridge iGEM team used acetone to extract, so we tested extraction of violacen from Strain 3 (full violacein operon) using acetone as well as methanol and ethanol.

    Our results show that ethanol was the best solvent to use for extraction and so the rest of our extractions were performed in ethanol. The details of growth and sample analysis can be found on our protocols page.

    Violacein Mono- and Co-Culture Wavelength Scans:
    Strain 1, which only has the first half of the enzymes necessary to produce violacein is still able to produce a green pigment. When the pigment is extracted from cells, the wavelength scan shown in dark blue is obtained.
    Strain 2, which has the second half of the violacein pathway, produces no pigment. This is expected because without the first half of the pathway, no pigment can be produced.
    Strain 3, which contains the entire violacein operon produces a wavelength scan similar to our standard obtained from Sigma-Aldrich, with violacein having a maximum absorbance near 575nm.
    Co-Culture: Strain 1 and Strain 2 grown together (red line) produce a wavelength scan that indicates production of violacein.
    Violacein Mono- and Co-Culture Extractions:
    When monocultures of strain 1 was grown, a dark green pigment was clearly observed. As seen in the plate above, strain 2 did not have any pigment on its own. However, when co-cultures of strain 1 and strain 2 were grown and the pigment extracted a purple pigment was seen and based on wavelength scans (above) appears to be violacein.


    Addressing Efficiency and Metabolic Burden
    Using linear interpolation (from the maximum absorbance at 575nm, wavelength scans above) we found that the co-culture produces 3.6199 mg/ml violacein and the total operon produces 3.5945mg/ml. While this amount is nearly equal, we believe that the production of violacein could be optimized such that it would produce much more violacein than the monoculture.

    The growth rates of strains decrease when inducer is added, as shown in the left graph. By comparing the rate of induced and uninduced growth, we can see how the cells are affected by production of their respective enzymes, and therefore how high the metabolic burden is. In the bar graph, Strain1+Strain2 (coculture) clearly is least affected by metabolic burden, showing that splitting a metabolic pathway does seem to lessen metabolic burden.

    Retrieved from "http://2012.igem.org/Team:UCSF/Violacein_Results"