Difference between revisions of "Part:BBa K1036001"

(→‎Experimental Data)
(→‎Experimental Data)
 
(7 intermediate revisions by the same user not shown)
Line 6: Line 6:
 
<body>
 
<body>
  
−
<i>ndh</i> is the coding gene for NADH dehydrogenase II (<a href="http://www.ncbi.nlm.nih.gov/gene/8183397">NCBI</a>), a non-proton-translating single subunit membrane bond enzyme important in maintaining a redox-balanced dinucleotide pool. NADH dehydrogenase II is the preponderant respiratory source of H<sub>2</sub>O<sub>2</sub> as well as O<sub>2</sub><sup>-</sup>. In fact, substantial H<sub>2</sub>O<sub>2</sub> was formed when quinoneless vesicles containing only NADH dehydrogenase II were incubated with NADH.<br/>Earlier studies estimated that the formation rate of intracellular O<sub>2</sub><sup>-</sup> was about 10<sup>-5</sup> M S<sup>-1</sup> by extrapolation of O<sub>2</sub><sup>-</sup> yield from this enzyme. And NADH dehydrogenase II formed about 4 O<sub>2</sub><sup>-</sup> per 10 molecules of H<sub>2</sub>O<sub>2</sub> both during respiration and in the quinoneless membranes.
+
<i>ndh</i> is the coding gene for NADH dehydrogenase II (<a href="http://www.ncbi.nlm.nih.gov/gene/8183397">NCBI</a>), a non-proton-translating single subunit membrane bond enzyme important in maintaining a redox-balanced dinucleotide pool. NADH dehydrogenase II is the preponderant respiratory source of H<sub>2</sub>O<sub>2</sub> as well as O<sub>2</sub><sup>-</sup>. In fact, substantial H<sub>2</sub>O<sub>2</sub> was formed when quinoneless vesicles containing only NADH dehydrogenase II were incubated with NADH.<br/>
 +
Earlier studies estimated that the formation rate of intracellular O<sub>2</sub><sup>-</sup> was about 10<sup>-5</sup> M S<sup>-1</sup> by extrapolation of O<sub>2</sub><sup>-</sup> yield from this enzyme. And NADH dehydrogenase II formed about 4 O<sub>2</sub><sup>-</sup> per 10 molecules of H<sub>2</sub>O<sub>2</sub> both during respiration and in the quinoneless membranes.<br/>
 +
In our oscillatoral circuit, <i>ndh</i> just like a "conductor" because this gene could express NDH-2 which is a membrane-bound respiratory enzyme that produces low levels of H<sub>2</sub>O<sub>2</sub> and superoxide. H<sub>2</sub>O<sub>2</sub> will trigger synchronized oscillation between colonies.
  
 
</body>
 
</body>
Line 14: Line 16:
 
<html>
 
<html>
 
<body>
 
<body>
−
<b>Construciton</b><br/>
+
The <i>ndh</i> gene was amplifed from the genome DNA of <i>E. coli</i> strain BL21(DE3) via PCR. Then RBS(<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034">BBa_B0034</a>), LVA-tag and terminator(<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_B0015">BBa_B0015</a>) were fused with <i>ndh</i> via fusion PCR becoming RBS-<i>ndh</i>-LVA-TT marked as <i><b>ndh</b></i>. The DH5α colony carried <i>ndh</i> gene was sequenced after TA-cloning.The Fig.1 showed the result of <i>ndh</i> gene fused with LVA-tag.<br/>  
−
<img src="https://static.igem.org/mediawiki/2013/6/67/XMU-China_ndh-fusion-pcr.png" width=800px; height=250px;/><br/><br/>
+
  
−
<b>SDS-PAGE</b><br/>
+
<img src="https://static.igem.org/mediawiki/2013/6/67/XMU-China_ndh-fusion-pcr.png" width=800px; height=250px;/><br/><br/>
−
To test the expression of <i>ndh</i> gene, an experiment was designed. The strain <i>E. coli</i> BL21(wide type) carried plasmid pSB4K5-<i>ndh</i> was cultured in LB medium with 25 μg/ml kanamycin under the condition of 37<sup>o</sup>C,200 rpm for 12 hours. Then the cultured medium was added into 100 volumes of flesh LB medium with 25 μg/ml kanamycin and cultured under the same condition. 10<sup>-3</sup> mM AHL was added into the medium after 2 hours and continued culturing for more 10 hours. The control was without AHL. The cell was harvested, washed twice by ddH<sub>2</sub>O and ran in SDS-PAGE.<br/><br/>
+
−
The result was showed in Fig.2 and <i>ndh</i> means without AHL and <i>ndh</i>+AHL means with AHL. The red and blue arrows showed the two up-regulation proteins, while the green arrow showed the down-regulation protein.According to the LC-MS-MS data, the band showed by the red arrow was the <i>ndh</i> gene.<br/><br/>
+
−
<img src="https://static.igem.org/mediawiki/2013/2/2e/XMU-China_SDS-PAGE-ndh.png" width=200px; height=350px;/>
+
 
</body>
 
</body>
 
</html>
 
</html>

Latest revision as of 13:04, 27 September 2013

the ndh gene coding for respiratory NADH dehydrogenase II

Description

ndh is the coding gene for NADH dehydrogenase II (NCBI), a non-proton-translating single subunit membrane bond enzyme important in maintaining a redox-balanced dinucleotide pool. NADH dehydrogenase II is the preponderant respiratory source of H2O2 as well as O2-. In fact, substantial H2O2 was formed when quinoneless vesicles containing only NADH dehydrogenase II were incubated with NADH.
Earlier studies estimated that the formation rate of intracellular O2- was about 10-5 M S-1 by extrapolation of O2- yield from this enzyme. And NADH dehydrogenase II formed about 4 O2- per 10 molecules of H2O2 both during respiration and in the quinoneless membranes.
In our oscillatoral circuit, ndh just like a "conductor" because this gene could express NDH-2 which is a membrane-bound respiratory enzyme that produces low levels of H2O2 and superoxide. H2O2 will trigger synchronized oscillation between colonies.

Experimental Data

The ndh gene was amplifed from the genome DNA of E. coli strain BL21(DE3) via PCR. Then RBS(BBa_B0034), LVA-tag and terminator(BBa_B0015) were fused with ndh via fusion PCR becoming RBS-ndh-LVA-TT marked as ndh. The DH5α colony carried ndh gene was sequenced after TA-cloning.The Fig.1 showed the result of ndh gene fused with LVA-tag.


Sequence and Features


Barcodes are discontinued, but one was appended to the sequence of this part. Composite parts using this part will include the barcode. More ...

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1360
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]