Difference between revisions of "Part:BBa K228009"
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<partinfo>BBa_K228009 short</partinfo>
 
<partinfo>BBa_K228009 short</partinfo>
  
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This part contains AraC protein generator(<partinfo>BBa_K228008</partinfo>) and Pbad promoter(<partinfo>BBa_I13453</partinfo>). The expression of any coding sequence following the Pbad promoter is repressed constitutively by AraC protein and induced by L-arabinose.
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==='''Description'''===
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We have constructed the inducement system composed of L-Arabinose inducible Pbad promoter and constitutively expressed AraC regulatory protein. When arabinose is absent, the AraC protein binds to and represses Pbad very efficiently. Thus, the transcription of any downstream coding gene is restricted. On the other hand, the import of arabinose could change the conformation of AraC protein, successfully prevent it repressing Pbad promoter and activate the transcription of downstream coding gene, such as GFP.  
  
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This promoter can be placed upstream of a GFP gene to character its induction curve. We cultured the bacteria until the value of OD600 reached 0.4 and then added different concentration of arabinose. The process of inducement lasted two hours. We used flowcytometry to test the fluorescence in order to acquire the induction curve, which is shown in the below graph:
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This part is constructed for two purposes. First, we coupled two parts, an AraC protein pand a Pbad promoter, in order to characterize the latter one (Part <partinfo>BBa_I13453</partinfo>) and obtain more information about this promoter. We place a GFP coding gene(Part <partinfo>BBa_E0840</partinfo>) downstream of Pbad promoter to construct a report system. If arabinose is added to the culture, the expression of GFP will be triggered and green fluorescence can be tested. We have tested the function of this promoter according to concentration gradient and time scale, as is shown below. Secondly, we use this part as a sensor in our AND gate system, which works very efficiently.
  
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[[Image:PKU_AraC.jpg|600px]]
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The direction of AraC coding sequence is opposite compared to the Part <partinfo>BBa_C0080</partinfo>. We have PCRed <partinfo>BBa_C0080</partinfo> with primers which include standard enzyme-cutting sites. The aim of this reversion is to avoid unexpected expression leaking of downstream sequences.
  
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==='''Performance'''===
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{|cellpadding=5
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|Experiment
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|-
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|Transfer Fuction
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|}
  
  

Revision as of 10:05, 21 October 2009

AraC protein(reversed sequence) and Pbad promoter

Description

We have constructed the inducement system composed of L-Arabinose inducible Pbad promoter and constitutively expressed AraC regulatory protein. When arabinose is absent, the AraC protein binds to and represses Pbad very efficiently. Thus, the transcription of any downstream coding gene is restricted. On the other hand, the import of arabinose could change the conformation of AraC protein, successfully prevent it repressing Pbad promoter and activate the transcription of downstream coding gene, such as GFP.

This part is constructed for two purposes. First, we coupled two parts, an AraC protein pand a Pbad promoter, in order to characterize the latter one (Part BBa_I13453) and obtain more information about this promoter. We place a GFP coding gene(Part BBa_E0840) downstream of Pbad promoter to construct a report system. If arabinose is added to the culture, the expression of GFP will be triggered and green fluorescence can be tested. We have tested the function of this promoter according to concentration gradient and time scale, as is shown below. Secondly, we use this part as a sensor in our AND gate system, which works very efficiently.

The direction of AraC coding sequence is opposite compared to the Part BBa_C0080. We have PCRed BBa_C0080 with primers which include standard enzyme-cutting sites. The aim of this reversion is to avoid unexpected expression leaking of downstream sequences.

Performance

Experiment
Transfer Fuction


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1274
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1214
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]