Difference between revisions of "Part:BBa K4806216"

 
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<partinfo>BBa_K4806216 short</partinfo>
 
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4806216 SequenceAndFeatures</partinfo>
 
  
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<p>This composite part contains the AβSAP(i)-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>), the coding sequence of CYPCamC (<a href=" https://parts.igem.org/Part:BBa_K4806002">BBa_K4806002</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)<sup>*</sup> for detection and the tRPL23-terminator (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>. This part is codon-optimized for <i>Chlamydomonas reinhardtii</i> and was built as part of the CYPurify Collection. This level 2 part leads to expression and potential detoxification of specific chemicals (Bell <i>et al</i>., 2001).</p>
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<h2>Construct</h2>
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  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cypcamc-ha-construct.png">
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  <div class="unterschrift"><b>Fig.1 Construct design</b><br>
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  This construct was designed using the modular cloning system (MoClo).</div>
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  <p>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). </p>
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<h2>Sequence and Features</h2>
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<partinfo>BBa_K4806216 SequenceAndFeatures</partinfo>
  
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===Functional Parameters===
 
 
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<h2>Results</h2>
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<p>We detected the expression of CYPCamC with HA-tag via immunoblotting.</p>
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<p>
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  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp2d6-ha-wb.png">
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  <div class="unterschrift"><b>Fig.2 Expression of CYPCamC with HA-tag</b><br>
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  (a)Level 2 MoClo construct for expression of the enzyme CYPCamC containing the HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The enzyme CYPCamC 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>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 CYPCamC (~ 47 kDa) is visible.
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</p>
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<h2>Contribution</h2>
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<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 14:25, 18 September 2023


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


This composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of CYPCamC (BBa_K4806002), 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. This level 2 part leads to expression and potential detoxification of specific chemicals (Bell et al., 2001).


Construct

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

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 1023
    Illegal PstI site found at 1295
    Illegal PstI site found at 2019
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal PstI site found at 1023
    Illegal PstI site found at 1295
    Illegal PstI site found at 2019
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 530
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1023
    Illegal PstI site found at 1295
    Illegal PstI site found at 2019
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1023
    Illegal PstI site found at 1295
    Illegal PstI site found at 2019
    Illegal NgoMIV site found at 3040
    Illegal AgeI site found at 268
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of CYPCamC with HA-tag via immunoblotting.

Fig.2 Expression of CYPCamC with HA-tag
(a)Level 2 MoClo construct for expression of the enzyme CYPCamC containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYPCamC 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 CYPCamC (~ 47 kDa) is visible.

Contribution

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