Difference between revisions of "Part:BBa K4806219"
Line 11: Line 11:
  
  
−
<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>  
+
<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 CYP81A10V7 (<a href=" https://parts.igem.org/Part:BBa_K4806005">BBa_K4806005</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 (Ohkawa & Inui, 2015).</p>  
 
<br>
 
<br>
 
<h2>Construct</h2>
 
<h2>Construct</h2>
 
<p>
 
<p>
−
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp2d6-ha-construct.png">
+
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp81-ha-construct.png">
 
   <div class="unterschrift"><b>Fig.1 Construct design</b><br>
 
   <div class="unterschrift"><b>Fig.1 Construct design</b><br>
 
   This construct was designed using the modular cloning system (MoClo).</div>
 
   This construct was designed using the modular cloning system (MoClo).</div>
 
</p>
 
</p>
−
   <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>
+
   <p>The resistance cassette for spectinomycin is already built in the level 2 vector pMBS807 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>
  
 
<p><br></p>
 
<p><br></p>
Line 32: Line 32:
 
<html>
 
<html>
 
<h2>Results</h2>
 
<h2>Results</h2>
−
<p>We detected the expression of CYPCamC with HA-tag via immunoblotting.</p>
 
−
<p>
 
−
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp2d6-ha-wb.png">
 
−
  <div class="unterschrift"><b>Fig.2 Expression of CYPCamC with HA-tag</b><br>
 
−
  (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
 
−
</div>
 
−
</p>
 
−
<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.
 
−
</p>
 
−
<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>
 
−
</html>
 

Revision as of 09:04, 21 September 2023


CYP81A10V7 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick)


This composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of CYP81A10V7 (BBa_K4806005), 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 (Ohkawa & Inui, 2015).


Construct

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

The resistance cassette for spectinomycin is already built in the level 2 vector pMBS807 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 2427
    Illegal PstI site found at 2569
    Illegal PstI site found at 3191
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal PstI site found at 2427
    Illegal PstI site found at 2569
    Illegal PstI site found at 3191
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 530
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 2427
    Illegal PstI site found at 2569
    Illegal PstI site found at 3191
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 2427
    Illegal PstI site found at 2569
    Illegal PstI site found at 3191
    Illegal NgoMIV site found at 2274
    Illegal NgoMIV site found at 3636
    Illegal AgeI site found at 268
  • 1000
    COMPATIBLE WITH RFC[1000]


Results