Difference between revisions of "Part:BBa K4806011"
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<h2>Results</h2>
 
<h2>Results</h2>
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<p>We confirmed that this construct is built correctly via agarose gel electrophoresis.</p>
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  <img class="agarose" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cypcamc-mito-agarose.png">
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  <div class="unterschrift"><b>Fig.2 Test digest of CYPCamC level 2 targeted to the mitochondria</b><br>
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We digested this level 2 MoClo part with the restriction enzymes <i>Nhe</i>I and <i>Eco</i>RV.</div></p>
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<p>The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.</p>
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Revision as of 12:53, 20 September 2023


mtTP70C-transit peptide for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the mtTP70C-transit peptide to the chloroplast (B2). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promotor like PAR like CYP3A4 (BBa_K3002010)* and a terminator like tRPL23 (BBa_K3002006)*, this level 0 part leads to expression of your target protein. To detect the target protein a tag like HA-tag (BBa_K3002017)* is recommended.


Constructs

Fig.1 Construct design
We designed 1 level 2 construct containing the mtTP70C-transit peptide using the modular cloning system (MoClo).


Here is the link to the built construct:

  • 1. CYPCamC gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806218)

This constructs was transformed into Chlamydomonas reinhardtii. Besides the mtTP70C-transit peptide mStop the construct contains the AβSAP(i)-promotor (BBa_K4806013), the CYPCamC coding sequence (BBa_K4806002), the HA-tag (BBa_K3002017) for detection and the tRPL23-terminator (BBa_K3002006)*. The resistance cassette for hygromycin is already built in the level 2 vector pMBS810 we are using. The usage of this vector allows the direct assembly of level 0 parts to level 2 constructs, facilitating the cloning time (Niemeyer & Schroda, 2022).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 2
    Illegal PstI site found at 183
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 2
    Illegal PstI site found at 183
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 198
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 2
    Illegal PstI site found at 183
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 2
    Illegal PstI site found at 183
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We confirmed that this construct is built correctly via agarose gel electrophoresis.

Fig.2 Test digest of CYPCamC level 2 targeted to the mitochondria
We digested this level 2 MoClo part with the restriction enzymes NheI and EcoRV.

The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.