Difference between revisions of "Part:BBa K316003:Experience"
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<partinfo>BBa_K316003 AddReview 5</partinfo>
 
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<I>Imperial College iGEM 2010</I>
 
<I>Imperial College iGEM 2010</I>
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*The spectra (figure 1) showed that in XylE transformed cells, a broad peak appears at about '''380nm'''. The absorbance at this particular wavelength is by the product of the C2,3O reaction which is called 2-hydroxymuconic semialdehyde and is what causes the yellow output.
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[[Image:Spectra of Xyle cells.jpg|thumb|center|400px|Spectra of cultures of cells expressing the XylE and cells negative for the XylE gene. Note the broad peak in the spectra of Xyle transformed cells, which is centered around 380nm.]]
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<span class='h3bb'><big>'''Absorbtion spectrum assay'''</big></span>
  
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[[Image:Spectra of Xyle cells.jpg|thumb|right|350px|Spectra of cultures of cells expressing the XylE and cells negative for the XylE gene. Note the broad peak in the spectra of Xyle transformed cells, which is centered around 380nm.]]
  
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*data that delineate the course of the reaction in terms of yellow product production over time at various catechol concentrations. The results of one of these assays is presented in the figure below. In order to extract data that will allow characterization of the kinetic parameters of catechol dioxygenase enzyme our lab team proposes purification of the protein from cell lysate, '''several fold dilution''', and in vitro characterization.
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The spectra showed that in XylE transformed cells, a broad peak appears at about '''380nm'''. The absorbance at this particular wavelength is by the product of the C2,3O reaction which is called 2-hydroxymuconic semialdehyde and is what causes the yellow output.
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[[Image:HMS prod. over time curve for xylE.jpg|thumb|center|300px|Graph shows production of HMS (yellow product) over time after catechol addition at time 0 minutes. Different curves represent different catechol concentration added to the cell cultures.]]
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<span class='h3bb'><big>'''2-hydroxymuconate semialdehyde production rate'''</big></span>
  
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[[Image:XylE M9 Growth (600).jpg|thumb|right|350px|O.D. at 600 over 3h for XylE-transformed Top10 cells in presence of different catechol concentrations, growing in M9 medium.]]
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[[Image:HMS prod. over time curve for xylE.jpg|thumb|right|350px|Graph shows production of HMS (yellow product) over time after catechol addition at time 0 minutes. Different curves represent different catechol concentration added to the cell cultures.]]
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[[Image:CMR M9 Growth (600).jpg|thumb|right|350px|O.D. at 600 over 3h for CMR-transformed Top10 cells in presence of different catechol concentrations, growing in M9 medium.]]
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*Growth with Catechol
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(LB) The addition of catechol had distinctive effects on the XylE expressing cells growing in LB medium. While at 0% catechol growth-behavior did not show a significant change (dark blue), even the lowest concentration of 0.25% catechol appeared to drastically reduce cell-survival (red). In contrast, CMR-control cells did not change their growing behavior in the presence of catechol. From this we conclude that in LB medium, the breakdown product of catechol, 2-hydroxymuconic semialdehyde, has a lethal effect on E. coli.
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(M9) Cells growing in M9 medium appeared more resistant to the effects of catechol. Even though absorbance at 380 nm increased significantly in well containing XylE expressing cells, indicating strong turnover of Catechol by C2,3O (4), Catechol did not appear to influence growing behavior in generalizable fashion: while a significant increase in the variance in the sample could be determined. Such was not observed for CMR expressing cells exposed to the same conditions. However, we were not able to establish a clear trend of growing-behavior in the presence of catechol as in case of the LB – XylE samples.  
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Data that delineate the course of the reaction in terms of yellow product production over time at various catechol concentrations. The results of one of these assays is presented in the figure below. In order to extract data that will allow characterization of the kinetic parameters of catechol dioxygenase enzyme our lab team proposes purification of the protein from cell lysate, '''several fold dilution''', and in vitro characterization.
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<span class='h3bb'><big>'''Cytotoxicity of Catechol'''</big></span>
  
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'''Growth with Catechol'''
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(LB) The addition of catechol had distinctive effects on the XylE expressing cells growing in LB medium. While at 0% catechol growth-behavior did not show a significant change (dark blue), even the lowest concentration of 0.25% catechol appeared to drastically reduce cell-survival (red). In contrast, CMR-control cells did not change their growing behavior in the presence of catechol. From this we conclude that in LB medium, the breakdown product of catechol, 2-hydroxymuconic semialdehyde, has a lethal effect on E. coli.
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|[[Image:XylE LB Growth (600) 2.jpg|thumb|left|350px|O.D. at 600 over 3h for XylE-transformed Top10 cells in presence of different catechol concentrations, growing in LB medium.]]
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[[Image:CMR LB Growth (600).jpg|thumb|right|350px|O.D. at 600 over 3h for CMR-transformed Top10 cells in presence of different catechol concentrations, growing in LB medium.]]
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(M9) Cells growing in M9 medium appeared more resistant to the effects of catechol. With absorbance at 380 nm increased significantly in well containing XylE expressing cells, indicating strong turnover of Catechol by C2,3O (4), Catechol showed strong influence on growing behavior in generalizable fashion: increasing concentrations of catechol progressively inhibited cell population growth. However, the overall effect of the breakdown product HMS appeared less severe than compared with the LB – XylE samples.
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|[[Image:XylE M9 Growth (600).jpg|thumb|left|350px|O.D. at 600 over 3h for XylE-transformed Top10 cells in presence of different catechol concentrations, growing in M9 medium.]]
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[[Image:CMR M9 Growth (600).jpg|thumb|right|350px|O.D. at 600 over 3h for CMR-transformed Top10 cells in presence of different catechol concentrations, growing in M9 medium.]]
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<span class='h3bb'><big>'''C23O kinetics'''</big></span>
  
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[[Image:Grapfit curve 1.jpg|thumb|right|400px]]
  
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*The Michaelis-Menten curve was delineated by non-linear regression analysis using GraFit software tool. The calculated Km is 0.71mM catechol (with a Vmax of 3.37 in O.D. arbitrary units for this dilution of cell lysate).  
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The Michaelis-Menten curve was delineated by non-linear regression analysis using GraFit software tool. The calculated Km is 0.71mM catechol (with a Vmax of 3.37 in O.D. arbitrary units for this dilution of cell lysate).
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[[Image:Grapfit curve 1.jpg|thumb|center|500px]]
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Revision as of 00:29, 28 October 2010

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K316003

  • The enzymatic reaction catalysed by C2,3O is an ideal output signal for our engineered bacterial detector and it can also serve as a very useful reporter gene.
  • Catechol, the substrate of C2,3O, is colourless. However within seconds of its addition, the colonies/liquid cultures of XylE-expressing cells become yellow, indicating production of a product which absorbs light in the visible spectrum


User Reviews

UNIQfaf2331b4dce8ba5-partinfo-00000000-QINU

•••••

Imperial College iGEM 2010

Absorbtion spectrum assay

Spectra of cultures of cells expressing the XylE and cells negative for the XylE gene. Note the broad peak in the spectra of Xyle transformed cells, which is centered around 380nm.

The spectra showed that in XylE transformed cells, a broad peak appears at about 380nm. The absorbance at this particular wavelength is by the product of the C2,3O reaction which is called 2-hydroxymuconic semialdehyde and is what causes the yellow output.

2-hydroxymuconate semialdehyde production rate

Graph shows production of HMS (yellow product) over time after catechol addition at time 0 minutes. Different curves represent different catechol concentration added to the cell cultures.

Data that delineate the course of the reaction in terms of yellow product production over time at various catechol concentrations. The results of one of these assays is presented in the figure below. In order to extract data that will allow characterization of the kinetic parameters of catechol dioxygenase enzyme our lab team proposes purification of the protein from cell lysate, several fold dilution, and in vitro characterization.

Cytotoxicity of Catechol

Growth with Catechol (LB) The addition of catechol had distinctive effects on the XylE expressing cells growing in LB medium. While at 0% catechol growth-behavior did not show a significant change (dark blue), even the lowest concentration of 0.25% catechol appeared to drastically reduce cell-survival (red). In contrast, CMR-control cells did not change their growing behavior in the presence of catechol. From this we conclude that in LB medium, the breakdown product of catechol, 2-hydroxymuconic semialdehyde, has a lethal effect on E. coli.

O.D. at 600 over 3h for XylE-transformed Top10 cells in presence of different catechol concentrations, growing in LB medium.
File:CMR LB Growth (600).jpg
O.D. at 600 over 3h for CMR-transformed Top10 cells in presence of different catechol concentrations, growing in LB medium.

(M9) Cells growing in M9 medium appeared more resistant to the effects of catechol. With absorbance at 380 nm increased significantly in well containing XylE expressing cells, indicating strong turnover of Catechol by C2,3O (4), Catechol showed strong influence on growing behavior in generalizable fashion: increasing concentrations of catechol progressively inhibited cell population growth. However, the overall effect of the breakdown product HMS appeared less severe than compared with the LB – XylE samples.

O.D. at 600 over 3h for XylE-transformed Top10 cells in presence of different catechol concentrations, growing in M9 medium.
O.D. at 600 over 3h for CMR-transformed Top10 cells in presence of different catechol concentrations, growing in M9 medium.

C23O kinetics

Grapfit curve 1.jpg

The Michaelis-Menten curve was delineated by non-linear regression analysis using GraFit software tool. The calculated Km is 0.71mM catechol (with a Vmax of 3.37 in O.D. arbitrary units for this dilution of cell lysate).

UNIQfaf2331b4dce8ba5-partinfo-00000002-QINU