Difference between revisions of "Part:BBa K1036000"
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<partinfo>BBa_K1036000 short</partinfo>
 
<partinfo>BBa_K1036000 short</partinfo>
  
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The ''ndh'' gene codes for NDH-2, an enzyme that generates
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=Description=
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This part is made up of a quorum sensing promoter (<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_F2621">BBa_F2621</a>) and <i>ndh</i> gene with a LAV-tag. Under control of the quorum sensing promoter, <i>ndh</i> gene will express periodically as the promoter is periodically activated and repressed. Since the <i>ndh</i> gene is described detailed in Part: <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1036001">BBa_K1036001</a>, here is a detailed description of quorum sensing promoter. Firstly, we should be familiar with the quorum sensing mechanism, the <i>luxI</i> gene is at low expression level and produces LuxI protein that synthesizes a kind of acyl-homoserine lactone (AHL), which is a small molecule that can diffuse across the cell membrane and mediate intercellular coupling when it reaches the threshold as enough biomass accumulated. AHL will bind intracellular protein LuxR, which is also consecutively produced by <i>luxR</i> gene. The LuxR-AHL complex can activate the <i>luxI</i> promoter, and the positive feedback loop is built. However, since in this circuit the <i>luxI</i> gene behind the <i>luxI</i> promoter is changed into <i>ndh</i> gene (with LVA-tag), so it could only be activated by AHL produced by <i>luxI</i> gene on another plasmid, which contains Part: <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1036003">BBa_K1036003</a>.<br/><br/>
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This year our team designed a synchronous oscillator which contained three main parts. The first part was pSB1C3-<i>gfp</i>-<i>luxI</i> which part was <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1036003">BBa_K1036003</a> with pSB1C3 backbone. The second one was pSB3T5-<i>aiiA</i> which part was <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K546001">BBa_K546001</a> with pSB3T5 backbone. The last one was pSB4K5-<i>ndh</i> which part was <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1036000">BBa_K1036000</a> with pSB4T5 backbone.
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=Usage and Biology=
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=Experimental Data=
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<b style="font-size:17px">Construction</b><br/>
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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 biobrick <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_F2621">BBa_F2621</a>(located in 2012-Plate 2-21F) digested by <i>Eco</i>R & <i>Xba</i>I and the <i><b>ndh</b></i> digested by <i>Eco</i>R & <i>Spe</i>I were ligated together by T4 ligase.The Fig.1 showed the result of pSB1A2-<i>ndh</i> construction and the changed backbone pSB4K5.<br/> <br/>
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<img src="https://static.igem.org/mediawiki/2013/2/25/BBa_K1036000-Fig.1.png" width=750px; height=250px;/><br/>
  
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The global synchronization mechanism is comprised of two modes of communication that work on different scales. The quorum-sensing machinery (LuxI, AiiA) uses an acyl-homoserine lactone (AHL) to mediate intracolony synchronization. In our device, the degree to which neighbouring colonies are able to influence each other via AHL diffusion is negligible owing to the high media channel flow rates. Instead, we engineered the cells to communicate via gas exchange by placing a copy of the gene coding for NADH dehydrogenase II (ndh) under the control of an additional lux promoter. NDH-2 is a membrane-bound respiratory enzyme that produces low levels of H2O2 and superoxide. As H2O2 vapour is able to pass through the 25-mm oxygen-permeable polydimethylsiloxane (PDMS) walls that separate adjacent colonies, periodic production of NDH-2 yields periodic exchange of H2O2 between biopixels. When H2O2 enters the cell, it transiently changes its redox state, interacting with our synthetic circuit through the native aerobic response control systems, including ArcAB, which has a binding site in the lux promoter region. Under normal conditions, ArcAB is partially active so lux is partially repressed. In contrast, oxidizing conditions triggered by H2O2 inactivate ArcAB, relieving this repression. Each oscillatory burst promotes firing in neighbouring colonies by relieving repression on the lux promoter. This constitutes an additional positive feedback that rapidly synchronizes the population.
 
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We investigated the effects of catalase and superoxide dismutase (SOD) to probe the nature of H2O2 communication. When a population of synchronized colonies was exposed to a step increase of 200Uml21 catalase, an enzyme that rapidly degrades extracellular H2O2, synchronization was broken and colonies continued to oscillate individually (Supplementary Fig. 3). As the cell membrane is impermeable to catalase, asynchronous colony oscillations confirm that communication between colonies depends on external H2O2 whereas oscillations within a colony do not. Conversely, when we enhanced the rate of superoxide conversion to H2O2 by expressing sodA from an additional lux promoter, colonies quickly fired in a spatial wave and failed to oscillate further despite no changes to growth rate or cell viability. BecauseH2O2 is produced internal to the cell, this confirms that H2O2 is capable of escaping the cell and activating lux-regulated genes in neighbouring colonies via diffusion. The apparent higher output ofH2O2 bySODas compared to NDH-2 is probably due to its very high catalytic efficiency. Lastly, we observed synchronization between arrays of traps even when they were fluidically isolated but held in close proximity. These devices share no common fluid sources or channels, making communication by dissolved molecules like AHL impossible. Taken together, these results confirm that gaseousH2O2 is the mode of communication between oscillating colonies.
 
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=Protocol=
 
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Ndh with LVA tag is formed as a composite by the ligation of quorum sensing promoter luxR via digestion and ligation.
 
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=Experimental Data=
 
  
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SDS page, microfluid, and gel image of construction.
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<b style="font-size:17px">Expression</b><br/>
  
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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/>
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The result was showed in Fig.1 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. The band showed by the red arrow was assumed the <i>ndh</i> gene according to the protein size.<br/><br/>
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<img src="https://static.igem.org/mediawiki/2013/d/d8/BBa_K1036000-Fig.2.png" width=200px; height=350px;/>
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Latest revision as of 22:19, 27 September 2013

lux pL controlled luxR with lux pR controlled ndh (LVA-tag) coding for NADH dehydrogenase II

Description

This part is made up of a quorum sensing promoter (BBa_F2621) and ndh gene with a LAV-tag. Under control of the quorum sensing promoter, ndh gene will express periodically as the promoter is periodically activated and repressed. Since the ndh gene is described detailed in Part: BBa_K1036001, here is a detailed description of quorum sensing promoter. Firstly, we should be familiar with the quorum sensing mechanism, the luxI gene is at low expression level and produces LuxI protein that synthesizes a kind of acyl-homoserine lactone (AHL), which is a small molecule that can diffuse across the cell membrane and mediate intercellular coupling when it reaches the threshold as enough biomass accumulated. AHL will bind intracellular protein LuxR, which is also consecutively produced by luxR gene. The LuxR-AHL complex can activate the luxI promoter, and the positive feedback loop is built. However, since in this circuit the luxI gene behind the luxI promoter is changed into ndh gene (with LVA-tag), so it could only be activated by AHL produced by luxI gene on another plasmid, which contains Part: BBa_K1036003.

This year our team designed a synchronous oscillator which contained three main parts. The first part was pSB1C3-gfp-luxI which part was BBa_K1036003 with pSB1C3 backbone. The second one was pSB3T5-aiiA which part was BBa_K546001 with pSB3T5 backbone. The last one was pSB4K5-ndh which part was BBa_K1036000 with pSB4T5 backbone.

Experimental Data

Construction
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 biobrick BBa_F2621(located in 2012-Plate 2-21F) digested by EcoR & XbaI and the ndh digested by EcoR & SpeI were ligated together by T4 ligase.The Fig.1 showed the result of pSB1A2-ndh construction and the changed backbone pSB4K5.


Expression
To test the expression of ndh gene, an experiment was designed. The strain E. coli BL21(wide type) carried plasmid pSB4K5-ndh was cultured in LB medium with 25 μg/ml kanamycin under the condition of 37oC,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. 10-3 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 ddH2O and ran in SDS-PAGE.

The result was showed in Fig.1 and ndh means without AHL and ndh+AHL means with AHL. The red and blue arrows showed the two up-regulation proteins, while the green arrow showed the down-regulation protein. The band showed by the red arrow was assumed the ndh gene according to the protein size.

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 2544
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1101