Difference between revisions of "Part:BBa K527002"
 
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<partinfo>BBa_K527002 parameters</partinfo>
 
<partinfo>BBa_K527002 parameters</partinfo>
 
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<html>We expressed the CYP and confirmed that a membrane preperation of BL21 cells were able to confirm trypthophane to an oxime with a TLC</p>
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<b><u> TLC </b></u>
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<table align="center">
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<tr>
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<td align="center">
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<table class="https://static.igem.org/mediawiki/2011/d/d7/Oxim_B1.jpg" align="center">
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<tr><td>
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<img src="https://static.igem.org/mediawiki/2011/d/d7/Oxim_B1.jpg"  width="300px"></td></tr>
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</table><br>
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</tr>
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</table>
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<br>
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<ul>
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<li> cB1: B1 with illegal restriction sites. <br>
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<li> User B1: B1 assembled with USER assembly method <br>
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<li> Tryptophane: Substrate for B1. <br>
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<li> Oxime: Reference oxime. <br>
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<br>
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</ul>
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As is observed from the TLC, the bond of the reference oxime has moved slightly shorter than the oximes produced by the cytochromes. The reference oxime was the oxime produced by CYP79A1 and would probably migrate a little different, but insignificant.
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<br>
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We believe that the difference in migration is due to the fact that the container used, caused a smiling effect on the TLC.  Because of limited time, we did not have time to reproduce data in another container, but we are convinced that if we used an other container, the reference oxime would verify the production of oximes produced by B1.
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<br>
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<br>
 +
<b><u>CO-measurement</b></u>
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<br>
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<br>
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A specific characteristic of p450's, is their absorbance of light at 450 nm in their active state. As a part of our characterization, we have performed CO-measurements on the purified p450 membrane fractions, which supposedly should contain a enriched amount of p450 enzyme. <br>
 +
CO (carbon monoxide) is added to a membrane sample, by blowing bubbles at approximately 1 bubble/sec in 60 seconds.
 +
The reason for doing this, is that the CO binds the heme group of cytochrome p450, which holds the p450 in its active conformation and shifts the absorbance from 420 to 450 nm. For the CO to bind, it is necessary to reduce the heme group with a strong reducing agent as dithionite.
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<br>
 +
Unfortunately, we did not observe a peak at 450 nm. We did although repeatedly observe peaks at 420. This is most probably something endogenous substance in <i>E.coli</i>. We did not perform a negative control, which could have indicated whether the 420 was only a result of endogenous compounds or the oxidized form of p450.
 +
<br>
 +
Although all active p450's theoretically should absorb at 450 nm in their active state, it is in some cases possible to have an active enzyme without observing the peak at 450 nm.
 +
<br>
 +
As the CO-measurement shows, there is no peak at 450 nm for the User assembled B1, but we did observe an oxime, indicating that the p450 in fact was active.
 +
<br><br>
 +
 +
<table align="center">
 +
<tr>
 +
<td align="center">
 +
<table class="https://static.igem.org/mediawiki/2011/c/c1/Co_m%C3%A5ling_B12.jpg" align="center">
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<tr><td>
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<img src="https://static.igem.org/mediawiki/2011/c/c1/Co_m%C3%A5ling_B12.jpg"  width="300px"></td></tr>
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</table><br>
 +
</tr>
 +
</table>
 +
<br><br>
 +
We have included a spectrum of an active p450 below. This is what we would have liked to observe.
 +
The different lines, are measurements at different times. It takes a little while for the dithionite to reduce the cytochrome. This is why there is an increase in the absorbance at 450 nm. Accordingly there is a decrease at 420 nm, as the cytochrome shifts to absorbing at 450 nm.
 +
Also, there is an increase and decrease at 420 and 450 nm, respectively, at the latest measurement. This is due to the enzyme being oxidized and returning to the inactive state.
 +
<br>
 +
 +
<table align="center">
 +
<tr>
 +
<td align="center">
 +
<table class="https://static.igem.org/mediawiki/2011/3/3e/Positivt_CO.jpg" align="center">
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<tr><td>
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<img src="https://static.igem.org/mediawiki/2011/3/3e/Positivt_CO.jpg"  width="300px"></td></tr>
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</table><br>
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</tr>
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</table>
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</html>
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===References===
 
===References===

Latest revision as of 20:26, 21 September 2011

Biosynthesis/Oximes/CYP79B1/Plug 'n' Play

This BioBrick BBa_K527002 encode the plant enzyme Cytochrome P450 CYP79B1. Cytochromes P450 are defined as heme-thiolate proteins featuring a particular spectral signature at 450 nm, thus the name. Despite the label as cytochromes, these proteins are not involved in electron transfers, but act as monooxygenases in a wide range of reactions such as epoxidation, N-dealkylation, O-dealkylation, S-oxidation and hydroxylation.

CYP79B1 is an N-hydroxylase converting L-Tryptophan into indole 3-acetaldoxime, the precursor of a cyanogenic glycoside . Oximes as the indole 3-acetaldoxime is generally toxic to fungi and is frequently used as a chemical fungicide. They function by inhibiting mitochondrial oxidases and thereby promote lipid peroxidation and the production of toxic reactive oxygen species.

BBa_K527002 is submitted in the plasmid BBa_K678023 due to the different assembly method (Plug 'n' Play) hence the missing prefix and suffix.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


We expressed the CYP and confirmed that a membrane preperation of BL21 cells were able to confirm trypthophane to an oxime with a TLC

TLC


  • cB1: B1 with illegal restriction sites.
  • User B1: B1 assembled with USER assembly method
  • Tryptophane: Substrate for B1.
  • Oxime: Reference oxime.

As is observed from the TLC, the bond of the reference oxime has moved slightly shorter than the oximes produced by the cytochromes. The reference oxime was the oxime produced by CYP79A1 and would probably migrate a little different, but insignificant.
We believe that the difference in migration is due to the fact that the container used, caused a smiling effect on the TLC. Because of limited time, we did not have time to reproduce data in another container, but we are convinced that if we used an other container, the reference oxime would verify the production of oximes produced by B1.

CO-measurement

A specific characteristic of p450's, is their absorbance of light at 450 nm in their active state. As a part of our characterization, we have performed CO-measurements on the purified p450 membrane fractions, which supposedly should contain a enriched amount of p450 enzyme.
CO (carbon monoxide) is added to a membrane sample, by blowing bubbles at approximately 1 bubble/sec in 60 seconds. The reason for doing this, is that the CO binds the heme group of cytochrome p450, which holds the p450 in its active conformation and shifts the absorbance from 420 to 450 nm. For the CO to bind, it is necessary to reduce the heme group with a strong reducing agent as dithionite.
Unfortunately, we did not observe a peak at 450 nm. We did although repeatedly observe peaks at 420. This is most probably something endogenous substance in E.coli. We did not perform a negative control, which could have indicated whether the 420 was only a result of endogenous compounds or the oxidized form of p450.
Although all active p450's theoretically should absorb at 450 nm in their active state, it is in some cases possible to have an active enzyme without observing the peak at 450 nm.
As the CO-measurement shows, there is no peak at 450 nm for the User assembled B1, but we did observe an oxime, indicating that the p450 in fact was active.




We have included a spectrum of an active p450 below. This is what we would have liked to observe. The different lines, are measurements at different times. It takes a little while for the dithionite to reduce the cytochrome. This is why there is an increase in the absorbance at 450 nm. Accordingly there is a decrease at 420 nm, as the cytochrome shifts to absorbing at 450 nm. Also, there is an increase and decrease at 420 and 450 nm, respectively, at the latest measurement. This is due to the enzyme being oxidized and returning to the inactive state.


References

  • Naur P, Hansen P H, Bak S, Hansen B G, Jensen N B, Nielsen H L, Halkier B A (2002). CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime. Archives of Biochemistry and Biophysics Vol. 409, 235–241.
  • Møller B L (2010). Dynamic Metabolons. Science Vol. 330, 1328-1329.