Difference between revisions of "Part:BBa K2984032"
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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 C. reinhardtii. The 3xHA tag can be used to purify and detect the PETase enzyme. | + | 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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<img src="https://2019.igem.org/wiki/images/6/63/T--Humboldt_Berlin--Konstrukt_5a.jpeg" alt="colonies_total" width="500"> | <img src="https://2019.igem.org/wiki/images/6/63/T--Humboldt_Berlin--Konstrukt_5a.jpeg" alt="colonies_total" width="500"> | ||
| − | <figcaption>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</figcaption> | + | <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"> | ||
| + | <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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1769
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1308
Illegal PstI site found at 1769 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4
Illegal XhoI site found at 1032 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1769
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1769
- 1000COMPATIBLE WITH RFC[1000]
Characterization
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.
