Difference between revisions of "Part:BBa K2365037"

 
 
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===Usage and Biology===
  
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Trichothecene 3-O-acetyltransferase(AYT1)，an indispensable enzyme for the biosynthesis of trichothecenes, to catalyzes the transfer of an acetyl group from acetyl coenzyme A to the C3 hydroxyl moiety which will result in a switch from DON to 3ADON, so the toxicity will be reduced by 100-fold.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2365037 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K2365037 parameters</partinfo>
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====PCR and SDS-PAGE====
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Showing the size of the target gene.
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[[File:Don degrading enzyme 1.jpg|400px]]
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[[File:DON degrading enzyme SDS.jpg|400px]]
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In theory, the higher one of the two absorption peaks for BL21+DON group and DON group should be the same. In fact, the result in the figure qualifies our inference.(dont in view of the extraction efficiency).Compared with our control groups(BL21+DON and DON), there is a peak valley in DON’s absorption peak for our test group BL21+DON+IPTG, from which the DON degrading enzyme can be induced and then expressed. It proves that the degrading enzyme expressed in BL21 has a significant degrading effect.
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[[Image:Uv_don.png|thumb|center|500px|Fig3 shows that we use the MSD to analyze the degrading effects of our DON degrading enzyme]]
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We use the High Performance Liquid Chromatography(HPLC) to analyze the ability of acetylation of trichothecene 3-O-Acetyltransferase from Saccharomyces cerevisiae. The figure above shows data 1 representing the sample only with deoxynivalenol(DON) as contrast groups(sample 1a, 1b, 1b) and data 2 from our test groups(sample 2a, 2b, 2c). As we can apparantly see from the figure, there are three peaks. The peaks which appear at 2min and 4min represents the isomers of DON while the peak which appears at 3min represents DON. Due to the specifity of trichothecene 3-O-Acetyltransferase, its efficiency of acetylation on the isomers of DON is quite low. So we can not see an obvious decrease at 2min and 4min. However, at 3min the peak area of data1 is 4400 uV·min and that of data2 is 1486 uV·min and the ability of acetylation can reach as high as 66.23%. We preliminarily proved that trichothecene 3-O-Acetyltransferase from Saccharomyces cerevisiae works in the transformation from DON to 3ADON.
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[[Image:HLPC DON.jpg|thumb|center|500px|High Performance Liquid Chromatography(HPLC) analysis for acetylation ability of AYT1 protein]]

Latest revision as of 11:53, 31 October 2017


Trichothecene 3-O-acetyltransferase(AYT1)，an indispensable enzyme for the biosynthesis of trichothecenes, to catalyzes the transfer of an acetyl group from acetyl coenzyme A to the C3 hydroxyl moiety which will result in a switch from DON to 3ADON, so the toxicity will be reduced by 100-fold.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 213
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 306
    Illegal BglII site found at 318
    Illegal XhoI site found at 884
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

PCR and SDS-PAGE

Showing the size of the target gene.

Don degrading enzyme 1.jpg DON degrading enzyme SDS.jpg

In theory, the higher one of the two absorption peaks for BL21+DON group and DON group should be the same. In fact, the result in the figure qualifies our inference.(dont in view of the extraction efficiency).Compared with our control groups(BL21+DON and DON), there is a peak valley in DON’s absorption peak for our test group BL21+DON+IPTG, from which the DON degrading enzyme can be induced and then expressed. It proves that the degrading enzyme expressed in BL21 has a significant degrading effect.

Fig3 shows that we use the MSD to analyze the degrading effects of our DON degrading enzyme

We use the High Performance Liquid Chromatography(HPLC) to analyze the ability of acetylation of trichothecene 3-O-Acetyltransferase from Saccharomyces cerevisiae. The figure above shows data 1 representing the sample only with deoxynivalenol(DON) as contrast groups(sample 1a, 1b, 1b) and data 2 from our test groups(sample 2a, 2b, 2c). As we can apparantly see from the figure, there are three peaks. The peaks which appear at 2min and 4min represents the isomers of DON while the peak which appears at 3min represents DON. Due to the specifity of trichothecene 3-O-Acetyltransferase, its efficiency of acetylation on the isomers of DON is quite low. So we can not see an obvious decrease at 2min and 4min. However, at 3min the peak area of data1 is 4400 uV·min and that of data2 is 1486 uV·min and the ability of acetylation can reach as high as 66.23%. We preliminarily proved that trichothecene 3-O-Acetyltransferase from Saccharomyces cerevisiae works in the transformation from DON to 3ADON.

High Performance Liquid Chromatography(HPLC) analysis for acetylation ability of AYT1 protein