Difference between revisions of "Part:BBa K2631000"

 
 
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<partinfo>BBa_K2631000 short</partinfo>
 
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ADTZ, which is also called a&#64258;atoxin oxidase (AFO), is the &#64257;rst enzyme identi&#64257;ed to bed able to degrade AFB1. It can be isolated from intracellular extracts and show a strong affinity with AFB1. As a potential degradation enzyme candidate in our project.
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The construct contains the coding sequence of ADTZ.  ADTZ is also called a&#64258;atoxin oxidase (AFO), is the &#64257;rst enzyme identi&#64257;ed to be able to degrade AFB1(1). It can be isolated from intracellular extracts and show a strong affinity with AFB1. As a potential degradation enzyme candidate in our project. In order to express soluble ADTZ in E．coli，the DNA coding ADTZ was inserted into the vector pMAL-c5x to produce the fusion protein of maltose-binding protein (MBP) and ADTZ.  
  
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2631000 parameters</partinfo>
 
<partinfo>BBa_K2631000 parameters</partinfo>
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<h3>Expression of MBP-ADTZ</h3>
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<p>Firstly the vector pMAL-c5x-ADTZ was transformed into E.coli BL21 Gold DE(3). Then an overnight culture of the bacteria was inoculated into LB broth containing 2g/L glucose and 100mg/L ampicillin. After 2h culture at 37 centigrade and 200rpm, recombinant protein production was induced by addition of 0.3 mM IPTG at 20 centigrade and 200rpm for 24h. The cell was collected by centrifugation. The pellet was resuspended in H2O about an OD600 of 30. Then the cell was disrupted by sonication. The results showed that most of highly expressed MBP-ADTZ(119kDa) was soluble(Fig. 1).</p>
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<img src="https://static.igem.org/mediawiki/2018/1/1e/T--BFSUICC-China--Collaboration1.jpeg" >
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<h3>Degradation of aflatoxin B1 by MBP-ADTZ</h3>
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<p>The degradation reaction of aflatoxin B1 was performed in a final volume of 700 ul composed of 150ul H2O, 350 ul buffer(100mM Na2HPO4, 50mM citric acid, 0.4mg/L aflatoxin B1, pH6.0) and 200ul crude MBP-ADTZ obtained after sonication. The mixture was incubated in the dark at 30 centigrade without shaking for 0, 3, 6, and 12 h. After incubation, aflatoxin B1 was extracted three times with 700ul chloroform. After the chloroform was evaporated under nitrogen gas, the samples were dissolved in 140ul acetonitrile and analyzed by HPLC. HPLC analysis was performed using Diamonsil C18 column (250×4.6 mm). The mobile phase was acetonitrile/water (45:55, v/v) at a flow rate of 1 ml/min and the sample temperature was 28 centigrade. The detection wavelength was 360 nm. The result showed that MBP-ADTZ was able to degrade AFT B1 obviously(Fig.2).</p>
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<img src="https://static.igem.org/mediawiki/2018/6/63/T--BFSUICC-China--Part.png" >
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<h4>Reference</h4>
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<p>(1)Xu,T., Xie,C., Yao,D., Zhou,C.Z. and Liu,J. (2017). Crystal structures of Aflatoxin-oxidase from Armillariella tabescens reveal a dual activity enzyme. Biochem. Biophys. Res. Commun. 494(3-4), 621-625.</p>

Latest revision as of 11:17, 12 October 2018


ADTZ Enzyme

The construct contains the coding sequence of ADTZ. ADTZ is also called aflatoxin oxidase (AFO), is the first enzyme identified to be able to degrade AFB1(1). It can be isolated from intracellular extracts and show a strong affinity with AFB1. As a potential degradation enzyme candidate in our project. In order to express soluble ADTZ in E．coli，the DNA coding ADTZ was inserted into the vector pMAL-c5x to produce the fusion protein of maltose-binding protein (MBP) and ADTZ.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1556
    Illegal BglII site found at 1979
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Expression of MBP-ADTZ

Firstly the vector pMAL-c5x-ADTZ was transformed into E.coli BL21 Gold DE(3). Then an overnight culture of the bacteria was inoculated into LB broth containing 2g/L glucose and 100mg/L ampicillin. After 2h culture at 37 centigrade and 200rpm, recombinant protein production was induced by addition of 0.3 mM IPTG at 20 centigrade and 200rpm for 24h. The cell was collected by centrifugation. The pellet was resuspended in H2O about an OD600 of 30. Then the cell was disrupted by sonication. The results showed that most of highly expressed MBP-ADTZ(119kDa) was soluble(Fig. 1).


<img src="T--BFSUICC-China--Collaboration1.jpeg" >

Degradation of aflatoxin B1 by MBP-ADTZ

The degradation reaction of aflatoxin B1 was performed in a final volume of 700 ul composed of 150ul H2O, 350 ul buffer(100mM Na2HPO4, 50mM citric acid, 0.4mg/L aflatoxin B1, pH6.0) and 200ul crude MBP-ADTZ obtained after sonication. The mixture was incubated in the dark at 30 centigrade without shaking for 0, 3, 6, and 12 h. After incubation, aflatoxin B1 was extracted three times with 700ul chloroform. After the chloroform was evaporated under nitrogen gas, the samples were dissolved in 140ul acetonitrile and analyzed by HPLC. HPLC analysis was performed using Diamonsil C18 column (250×4.6 mm). The mobile phase was acetonitrile/water (45:55, v/v) at a flow rate of 1 ml/min and the sample temperature was 28 centigrade. The detection wavelength was 360 nm. The result showed that MBP-ADTZ was able to degrade AFT B1 obviously(Fig.2).

<img src="T--BFSUICC-China--Part.png" >

Reference

(1)Xu,T., Xie,C., Yao,D., Zhou,C.Z. and Liu,J. (2017). Crystal structures of Aflatoxin-oxidase from Armillariella tabescens reveal a dual activity enzyme. Biochem. Biophys. Res. Commun. 494(3-4), 621-625.