Difference between revisions of "Part:BBa K1020005"

 
 
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<partinfo>BBa_K1020005 short</partinfo>
 
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The mechanism is that Alkane were presumably recognized by AlkR regulatory protein and their interaction triggered a conformation change of AlkR dimers which isomerizes the promoter-RNAP complex and lead to the activation of alkM gene. AlkR regulatory protein includes C-terminal DNA-binding domain(CTD) for promoter binding and N-terminal domain(NTD) for inducer recognition.
 
The mechanism is that Alkane were presumably recognized by AlkR regulatory protein and their interaction triggered a conformation change of AlkR dimers which isomerizes the promoter-RNAP complex and lead to the activation of alkM gene. AlkR regulatory protein includes C-terminal DNA-binding domain(CTD) for promoter binding and N-terminal domain(NTD) for inducer recognition.
 
Here, different from BBa_K1020004,we use a weaker consititutive promoter.
 
Here, different from BBa_K1020004,we use a weaker consititutive promoter.
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<html>
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<img src="https://static.igem.org/mediawiki/igem.org/6/62/0_%E5%89%AF%E6%9C%AC.jpg" width="700px"/>
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</html>
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We use different kinds of long chain alkane to characterize the part.
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<html>
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<img src="https://static.igem.org/mediawiki/2013/5/50/BBa_K1020005_4-2.png"/>
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</html>
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<html>
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<img src="https://static.igem.org/mediawiki/2013/3/3e/TJU-PROJECT-C3.jpg"/>
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</html>
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Then we test Octane(C8),Decane(C10),Dodecane(C12), Tetradecane(C14), Hexadecane(C16) and Octadecyl(C18) respectively and we use water as blank test.
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Strains are cultured in M9 medium for about 12 hours. Adjust the concentration of suspension culture until OD reaches 0.6, then take out 3mL and add 300μL. After that, induce for 24 hours.
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The respond to the different alkane is C8>C12>C14>C10>C16>control. Because of the low solubility of alkane especially the longer chain alkane, the response of Alk-Sensor may be influenced. So the response to the hexadecane is nearly to the control.
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Measure the ratio of fluorescent indensity (fluorescent indensity per OD) of cells, which is shown below:
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<img src="https://static.igem.org/mediawiki/igem.org/d/d1/PR_K1020005_1%E6%8D%95%E8%8E%B7.jpg"/>
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</html>
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From this diagram, we can clearly see that induced by saturated Octane(C8), ratio of fluorescent indensity is the strongest.
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Then we choose C8 and C14 for '''upper limits of detection'''. Because C8 is commonly used to characterize alkane-sensor in most references, and Pentadecane(C15) is our '''endogenous alkane''' producing module produce this kind of alkane.
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We culture the strains in 3mL LB medium until OD reaches 0.6,add C8 and C15 of a concentration range from100nmol/L to 1mmol/L , over-night cultured. 
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All of them are utilized to measure fluorescence intensity of cells.
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<html>
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<img src="https://static.igem.org/mediawiki/2013/d/d9/TJU-PROJECT-C5.jpg" width="700px"/>
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</html>
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The picture above is fluorescence intensity of cells for adding C8.
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<img src="https://static.igem.org/mediawiki/2013/6/60/TJU-PROJECT-C6.jpg" width="700px"/>
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The picture above is fluorescence intensity of cells for adding C15.
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'''Result''': With the increasing concentration of alkanes, ratio of fluorescent indensity is also increased. There is a positive correlation between concentration and ratio. Using logarithmic fitting curve, we find that with the increasing concentration of alkanes, the increase of ratio of fluorescent indensity becomes smooth, approaching its upper limit at the concentration of 10μmol/L C8 in LB medium, and approaching its upper limit at the concentration of 1μmol/L C15 in LB medium.
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For more details,please click <html><a href="http://2013.igem.org/Team:Tianjin/Project/Characterization">[here]</a></html>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 02:09, 29 October 2013

Alkane Marker--Device for detection of various alkanes

AlkR is a transcriptional regulator belonging to AraC/Xyls family, which could detect a broad range of alkanes and alkenes with carbon chain length from C7 to C36. It is said that AlkR is the only bioreporter that is able to detect alkane with carbon chain length greater than C18. The mechanism is that Alkane were presumably recognized by AlkR regulatory protein and their interaction triggered a conformation change of AlkR dimers which isomerizes the promoter-RNAP complex and lead to the activation of alkM gene. AlkR regulatory protein includes C-terminal DNA-binding domain(CTD) for promoter binding and N-terminal domain(NTD) for inducer recognition. Here, different from BBa_K1020004,we use a weaker consititutive promoter.



We use different kinds of long chain alkane to characterize the part.

Then we test Octane(C8),Decane(C10),Dodecane(C12), Tetradecane(C14), Hexadecane(C16) and Octadecyl(C18) respectively and we use water as blank test.

Strains are cultured in M9 medium for about 12 hours. Adjust the concentration of suspension culture until OD reaches 0.6, then take out 3mL and add 300μL. After that, induce for 24 hours.

The respond to the different alkane is C8>C12>C14>C10>C16>control. Because of the low solubility of alkane especially the longer chain alkane, the response of Alk-Sensor may be influenced. So the response to the hexadecane is nearly to the control.

Measure the ratio of fluorescent indensity (fluorescent indensity per OD) of cells, which is shown below:

From this diagram, we can clearly see that induced by saturated Octane(C8), ratio of fluorescent indensity is the strongest.

Then we choose C8 and C14 for upper limits of detection. Because C8 is commonly used to characterize alkane-sensor in most references, and Pentadecane(C15) is our endogenous alkane producing module produce this kind of alkane.

We culture the strains in 3mL LB medium until OD reaches 0.6,add C8 and C15 of a concentration range from100nmol/L to 1mmol/L , over-night cultured.

All of them are utilized to measure fluorescence intensity of cells.

The picture above is fluorescence intensity of cells for adding C8.

The picture above is fluorescence intensity of cells for adding C15.

Result: With the increasing concentration of alkanes, ratio of fluorescent indensity is also increased. There is a positive correlation between concentration and ratio. Using logarithmic fitting curve, we find that with the increasing concentration of alkanes, the increase of ratio of fluorescent indensity becomes smooth, approaching its upper limit at the concentration of 10μmol/L C8 in LB medium, and approaching its upper limit at the concentration of 1μmol/L C15 in LB medium.

For more details,please click [here]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1028
    Illegal NheI site found at 1051
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 752
    Illegal AgeI site found at 864
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1821