Difference between revisions of "Part:BBa K819005"
 
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<partinfo>BBa_K819005 short</partinfo>
 
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Lux operon genes (from [https://parts.igem.org/Part:BBa_K325909:Design BBa_K325909]) and related RBS are placed under T7 promoter (from [https://parts.igem.org/Part:BBa_I712074 BBa_I712074] ). Cells transformed with this part can produce blue luminescence while no exogenous substrate is needed.<br/><br/>
  
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When introducing synthetic DNA into a cell, it is desirable that the encoded processes be functionally distinct from host processes. Phage polymerases are a means to control orthogonal transcription and are one of the most used tools in genetic engineering. Specifically, T7 RNA polymerase (RNAP) has been shown to function in a variety of hosts, including most bacteria, plant chloroplasts and mammalian cells. One advantage of T7 promoter is low basal expression, for it tightly inactive in the absence of the polymerase.<br/><br/>
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<a href="/Part:BBa_K819005">Summary</a>
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<a href="/Part:BBa_K819005:Designing">Design</a>
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Therefore, T7 promoter separates sensing/circuitry functions from pathways/actuation. It is encoded in genetically distinct regions from other circuits, enabling its driving upon the expression of phage T7 polymerases. Luxbrick under T7 promoter is modular to form a interface between luxbrick and other systems. Also, when transformed into BL21 cells, it can be induced with IPTG to reach a high expression level. It is great improvement regarding time saving and cost efficiency.
  
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<b>Summary</b>
 
  
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This part, which is a fusion protein, serves as <b>an ultra sensitive photoreceptor</b> (which can sense light as weak as moonlight) and will induce a <b>light-dependent repression</b> of genes under specific promoters like [https://parts.igem.org/wiki/index.php?title=Part:BBa_K819002 RecA408 promoter] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K819017 SulA408 promoter].
 
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For the photosensor domain of this fusion protein, we chose VVD protein, the smallest photosensor protein known, which forms a rapidly exchanging dimer when excited by blue light. For the physiological function domain, we chose LexA protein, a bacteria transcription inhibitor, whose DNA binding domain rigorously requires help of the dimerization domain for its DNA binding activity. We fused these two separate domains together to form a fusion protein, which we termed our "Luminesensor". Both DNA binding domain and dimerization domain of this luminesensor was mutated at specific sites for better bio-orthogonality and higher on-off ratio.(to see the <b>design</b> page for details, please click [https://parts.igem.org/Part:BBa_K819005:Designing here])
 
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We then characterized this luminesensor in different ways and all the results showed that this luminesensor could response to light quite well. (to see the <b>characterization</b> page for details, please click [https://parts.igem.org/Part:BBa_K819005:Characterization here])
 
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To learn more details about this luminesensor and the related project by Peking 2012 iGEM team, please visit our [http://2012.igem.org/Team:Peking wiki]
 
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===Usage and Biology===
 
===Usage and Biology===
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To note that the incubating temperature should be no higher than 30<sup>o</sup>C, or the heavy Lux complex can easily aggregate. Optimum incubating conditions provided by Peking iGEM 2012: 250 rpm, 22oC, good ventilation after induction(final concentration of IPTG: round 0.5 mM).
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BL21 cells harboring T7-lux operon induced with IPTG at 0.5 mM is shown in the photo<br/><br />
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K819008 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K819008 parameters</partinfo>
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Latest revision as of 14:06, 20 October 2012

Constitutive LuxBrick Generator

Lux operon genes (from BBa_K325909) and related RBS are placed under T7 promoter (from BBa_I712074 ). Cells transformed with this part can produce blue luminescence while no exogenous substrate is needed.

When introducing synthetic DNA into a cell, it is desirable that the encoded processes be functionally distinct from host processes. Phage polymerases are a means to control orthogonal transcription and are one of the most used tools in genetic engineering. Specifically, T7 RNA polymerase (RNAP) has been shown to function in a variety of hosts, including most bacteria, plant chloroplasts and mammalian cells. One advantage of T7 promoter is low basal expression, for it tightly inactive in the absence of the polymerase.

Therefore, T7 promoter separates sensing/circuitry functions from pathways/actuation. It is encoded in genetically distinct regions from other circuits, enabling its driving upon the expression of phage T7 polymerases. Luxbrick under T7 promoter is modular to form a interface between luxbrick and other systems. Also, when transformed into BL21 cells, it can be induced with IPTG to reach a high expression level. It is great improvement regarding time saving and cost efficiency.


Usage and Biology

To note that the incubating temperature should be no higher than 30oC, or the heavy Lux complex can easily aggregate. Optimum incubating conditions provided by Peking iGEM 2012: 250 rpm, 22oC, good ventilation after induction(final concentration of IPTG: round 0.5 mM). BL21 cells harboring T7-lux operon induced with IPTG at 0.5 mM is shown in the photo



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3014
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2012
    Illegal XhoI site found at 2842
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4401
    Illegal BsaI.rc site found at 1410
    Illegal SapI.rc site found at 4726