Difference between revisions of "Part:BBa K2984032"

 
 
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<partinfo>BBa_K2984032 short</partinfo>
 
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This part is composed of the PsaD promoter (BBa_K2984008), the ARS secretion signal (BBa_K2984000), the PETase enzyme (), a HA tag (BBa_K2984015), and the Rbcs2 terminator (BBa_K2984018). The part can be used to express and secrete the PETase enzyme in C. reinhardtii. The 3xHA tag can be used to purify and detect the PETase enzyme.  
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This vector is a part of the <a href="https://2019.igem.org/Team:Humboldt_Berlin/Part_Collection">Chlamy-HUB-Collection</a>. This part is composed of the <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2984008">PsaD promoter</a>, the <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2984000">ARS</a> secretion signal, the <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2984049">PETase enzyme</a>, a <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2984015">3xHA tag</a>, and the <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2984021">Rbcs2 terminator</a>. The part can be used to express and secrete the PETase enzyme in <i>C. reinhardtii</i>. The 3xHA tag can be used to purify and detect the PETase enzyme.
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<partinfo>BBa_K2984032 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2984032 SequenceAndFeatures</partinfo>
  
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==Characterization==
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<figure>
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<img src="https://2019.igem.org/wiki/images/6/63/T--Humboldt_Berlin--Konstrukt_5a.jpeg" alt="colonies_total" width="500">
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<figcaption>Fig. 1 - Image of a successful transformation in <i>E.coli</i> after ligation of the construct. The construct can then be isolated from <i>E.coli</i> to be transformed in <i>C. reinhardtii</i></figcaption>
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This construct was successfully transformed into <i>C. reinhardtii</i>. A colony PCR was made to amplify the transgene PETase out of the <i>C. reinhardtii</i> genome (Fig. 2). The expected band for the PETase on the agarose gel was of 885 bp. This proves, that this construct can be successfully transformed into <i>C. reinhardtii</i>.
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<img src="https://2019.igem.org/wiki/images/1/1b/T--Humboldt_Berlin--PETase_Gel.jpg" alt="PETase Gel" width="800">
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<figcaption>Fig. 2 - DNA fragment containing the PETase, amplified from <i>C. reinhardtii</i> through a colony PCR.</figcaption>
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Latest revision as of 22:29, 19 October 2019


L1c-PsaD-ARS-PETase-3xHA-RBCS2

This vector is a part of the Chlamy-HUB-Collection. This part is composed of the PsaD promoter, the ARS secretion signal, the PETase enzyme, a 3xHA tag, and the Rbcs2 terminator. The part can be used to express and secrete the PETase enzyme in C. reinhardtii. The 3xHA tag can be used to purify and detect the PETase enzyme.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1769
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1308
    Illegal PstI site found at 1769
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 4
    Illegal XhoI site found at 1032
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1769
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1769
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

colonies_total
Fig. 1 - Image of a successful transformation in E.coli after ligation of the construct. The construct can then be isolated from E.coli to be transformed in C. reinhardtii

This construct was successfully transformed into C. reinhardtii. A colony PCR was made to amplify the transgene PETase out of the C. reinhardtii genome (Fig. 2). The expected band for the PETase on the agarose gel was of 885 bp. This proves, that this construct can be successfully transformed into C. reinhardtii.

PETase Gel
Fig. 2 - DNA fragment containing the PETase, amplified from C. reinhardtii through a colony PCR.