Difference between revisions of "Part:BBa K1020012"

 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1020012 short</partinfo>
 
<partinfo>BBa_K1020012 short</partinfo>
  
 +
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.
 +
 +
<html>
 +
<img src=" https://static.igem.org/mediawiki/igem.org/6/62/11.jpg" width="700px"/>
 +
</html>
 +
 +
We add alkanes outside the cell to test the function of TetA:
 +
 +
Characterization:
 +
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.
 +
 +
Construct:
 +
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.
 +
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.
 +
 +
<html>
 +
<img src=" https://static.igem.org/mediawiki/igem.org/1/1e/DSC00002-1.jpg" width="700px"/>
 +
</html>
 +
 +
Result:
 +
Alkane producing E.coli can grow in tube but blank vector E.coli cannot grow.
 +
 +
Conclusion:
 +
Synthesized alkanes induce the expression of alkR, which enables E.coli to survive under the pressure of Tetracycline.
  
  

Revision as of 23:04, 27 September 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.

We add alkanes outside the cell to test the function of TetA:

Characterization: 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.

Construct: 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. 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.

Result: Alkane producing E.coli can grow in tube but blank vector E.coli cannot grow.

Conclusion: Synthesized alkanes induce the expression of alkR, 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