Difference between revisions of "Part:BBa K1020012"
 
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We add alkanes outside the cell to test the function of TetA:
 
  
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Characterization:
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To verify the our Alk-Selector will produce more tetracycline resistance when more alkane is produced, we cotransform the Alk-Selector and an alkane producing modular to the E. coli BL21. The control strain just had Alk-Selector. These two strains were cultured in M9 media with 12ug/ml tetracycline. After 48h, we can find the growth of strain with alkane producing modular is higher than the control just as picture below showed.
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Construct:Strains of No.60 are cultured in LB medium for about 12 hours. Add 12μg/mL Cm and 12μg/mL Tc respectively. After that, inoculate into 3 ml LB medium for an overnight cultures at 37 ℃ with 220rpm shaking.
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Construct:
 
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Left: No.60: TetA downstream of Palkm and AlkR downstream of J23103, are cloned into vector PSB1C3 and there’s a alkane producing vector PSB3K3.
 
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Right: No.61: We construct a blank vector PSB3K3 without NPDC and AAR as blank control, and the other vector PSB1C3 is the same as No.60.
 
  
 
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<img src=" https://static.igem.org/mediawiki/igem.org/1/1e/DSC00002-1.jpg" width="700px"/>
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<img src=" https://static.igem.org/mediawiki/2013/b/bf/TJU-project-D4-2.jpg" width="400px"/>
 
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Result:  
 
Result:  
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Alkane producing E.coli can grow in tube but blank vector E.coli cannot grow.
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Alkane producing E.coli can grow in tube but blank vector E.coli cannot grow.'''It means that our Alk-Selector can work for endogenous alkanes under the pressure of special tetracycline concentration.'''
  
 
Conclusion:
 
Conclusion:
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Synthesized alkanes induce the expression of alkR, which enables E.coli to survive under the pressure of Tetracycline.
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Synthesized alkanes induce the expression of tetA gene, which enables E.coli to survive under the pressure of Tetracycline.
  
  

Latest revision as of 02:03, 29 October 2013

Alkane Selector for intracellular alkane production

We put the resistance gene encoding tetA under the regulation of PalkM. When PalkM-RNAP complex isomerizes due to the interaction between alkanes and alkR, downstream TetA gene will express. Then, TetA will transport tetracycline out of the cell so that the intracellular concentration of tetracycline is relatively stable. and the growth of cells will not be inhibited. Thus, we can turn alkane molecules into the output of the growth rate of cells.


To verify the our Alk-Selector will produce more tetracycline resistance when more alkane is produced, we cotransform the Alk-Selector and an alkane producing modular to the E. coli BL21. The control strain just had Alk-Selector. These two strains were cultured in M9 media with 12ug/ml tetracycline. After 48h, we can find the growth of strain with alkane producing modular is higher than the control just as picture below showed.


Result: Alkane producing E.coli can grow in tube but blank vector E.coli cannot grow.It means that our Alk-Selector can work for endogenous alkanes under the pressure of special tetracycline concentration.

Conclusion: Synthesized alkanes induce the expression of tetA gene, which enables E.coli to survive under the pressure of Tetracycline.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 346
    Illegal NheI site found at 1544
    Illegal NheI site found at 1567
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 492
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 518
    Illegal NgoMIV site found at 886
    Illegal NgoMIV site found at 1046
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2337