Difference between revisions of "Part:BBa K4806106"
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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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<p>We detected the expression of CYP3A4 tandem together with the POR with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806214">BBa_K4806214</a>) via immunoblotting.</p>
 
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   <img class="agarose" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/2d6-ha.png">
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   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cyp3a4-tandem-por-wb.png">
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   <div class="unterschrift"><b>Fig.2 Test digest of the POR level 1 with HA-tag</b><br>
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   <div class="unterschrift"><b>Fig.2 Expression of CYP3A4 tandem together with the POR with HA-tag</b><br>
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We digested this level 2 MoClo part with the restriction enzymes <i>Not</i>I and <i>Bam</i>HI.</div></p>
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  (a)Level 2 MoClo construct for expression of the enzyme CYP3A4 tandem together with the POR containing the HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The enzyme CYP3A4/POR is marked by a black arrow, the white arrow marks a cross reaction of antibodies. For reference, the UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplast 50S protein L5 (RPL5) were used as a negative and positive control, respectively
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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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<p>For detection the UVM4 strain was transformed with the construct in (a). 30 hygromycin-resistant transformants were cultivated in TAP medium and samples were taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody. The expression of CYP3A4 (~ 57 kDa) and the POR (~77 kDa) is visible.</p>  
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<h2>Contribution</h2>
 
<h2>Contribution</h2>
 
<p>The <sup>*</sup> marked parts were not created by us. Our results can be found on the experience page of each part.</p>
 
<p>The <sup>*</sup> marked parts were not created by us. Our results can be found on the experience page of each part.</p>
 
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Revision as of 10:41, 2 October 2023


POR gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick)


This level 1 composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of the POR (BBa_K4806003), the HA-tag (BBa_K3002017)* for detection and the tRPL23-terminator (BBa_K3002006)*. This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection.


Construct

Fig.1 Construct design
This construct was designed using the modular cloning system (MoClo).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1353
    Illegal PstI site found at 2613
    Illegal PstI site found at 2673
    Illegal PstI site found at 3332
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal NheI site found at 2538
    Illegal PstI site found at 1353
    Illegal PstI site found at 2613
    Illegal PstI site found at 2673
    Illegal PstI site found at 3332
    Illegal NotI site found at 2978
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 530
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1353
    Illegal PstI site found at 2613
    Illegal PstI site found at 2673
    Illegal PstI site found at 3332
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1353
    Illegal PstI site found at 2613
    Illegal PstI site found at 2673
    Illegal PstI site found at 3332
    Illegal NgoMIV site found at 4261
    Illegal AgeI site found at 268
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of CYP3A4 tandem together with the POR with HA-tag (BBa_K4806214) via immunoblotting.

Fig.2 Expression of CYP3A4 tandem together with the POR with HA-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 tandem together with the POR containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP3A4/POR is marked by a black arrow, the white arrow marks a cross reaction of antibodies. For reference, the UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplast 50S protein L5 (RPL5) were used as a negative and positive control, respectively

For detection the UVM4 strain was transformed with the construct in (a). 30 hygromycin-resistant transformants were cultivated in TAP medium and samples were taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody. The expression of CYP3A4 (~ 57 kDa) and the POR (~77 kDa) is visible.


Contribution

The * marked parts were not created by us. Our results can be found on the experience page of each part.