Difference between revisions of "Part:BBa K3002006:Experience"
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<I>Luca Langenberg</I>
 
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<p>We detected the expression of CYP3A4 with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806200">BBa_K4806200</a>) via immunoblotting.</p>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cyp3a4-ha.png">
 +
  <div class="unterschrift"><b>Fig.2 Expression of CYP3A4 with HA-tag</b><br>
 +
  (a)Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The enzyme CYP3A4 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 spectinomycin-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) is visible.
 +
</p>
 +
<p><br<p>
 +
<p>We detected the expression of CYP3A4 with FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806201">BBa_K4806201</a>) via immunoblotting.</p>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cyp3a4-flag.png">
 +
  <div class="unterschrift"><b>Fig.3 Expression of CYP3A4 with FLAG-tag</b><br>
 +
  (a)Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the FLAG-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The enzyme CYP3A4 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 FLAG-tagged VIPP1 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 spectinomycin-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-FLAG antibody. The expression of CYP3A4 (~ 57 kDa) is visible.
 +
</p>
 +
<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>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cyp3a4-tandem-por-wb.png">
 +
  <div class="unterschrift"><b>Fig.4 Expression of CYP3A4 tandem together with the POR with HA-tag</b><br>
 +
  (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
 +
  </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 CYP3A4 (~ 57 kDa) and the POR (~77 kDa) is visible.</p>
 +
<p><br></p>
 +
<p>We detected that the construct CYP3A4 with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806200">BBa_K4806200</a>) is correctly embedded within the membrane via freeze-thaw assay and immunoblotting.</p>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/freeze-thaw.png">
 +
  <div class="unterschrift"><b>Fig.6 Freeze thaw assay of CYP3A4 with HA-tag</b><br>
 +
  (a) Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag was designed. (b) The UVM4 strain was transformed with the construct in (a). An anti-HA antibody was used to detect the enzyme CYP3A4 (A). For control an anti-Cytf antibody was used to detect the membrane bound proteins and a CGE1-antibody for soluble proteins (B). 30 spectinomycin-resistant transformants were cultivated in TAP-medium and samples taken after. 3 days. For reference whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting. The other samples were treated according to protocol and analyzed also by SDS-PAGE and immunoblotting. The expression of CYP3A4 (~57 kDa) is visible in the pellet (P) and not in the supernatant (S) confirming the membrane intercalation. For reference, the UVM4 strain was used as a negative control.
 +
  </div>
 +
</p>
 +
<p>We were able to detect the expression of CYP3A4 within the pellet, demonstrating that our CYP is correctly embedded within the membrane.</p>
 +
<p><br></p>
 +
<p>We detected the expression of CYP2D6 with FLAG-tag via immunoblotting.</p>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cyp2d6-flag-wb.png">
 +
  <div class="unterschrift"><b>Fig.2 Expression of CYP2D6 with FLAG-tag</b><br>
 +
  (a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the FLAG-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The enzyme CYP2D6 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 FLAG-tagged VIPP1 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 paromomycin-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-FLAG antibody. The expression of CYP2D6 (~ 56 kDa) is visible.
 +
</p>
 +
<p><br></p>
 +
<p>We detected the expression of the POR with HA-tag via immunoblotting.</p>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/por-ha-wb.png">
 +
  <div class="unterschrift"><b>Fig.2 Expression of the POR with HA-tag</b><br>
 +
  (a)Level 2 MoClo construct for expression of the POR containing the HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The 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
 +
</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 the POR (~ 77 kDa) is visible.
 +
</p>
 +
<p><br></p>
 +
<p>We detected the expression of the POR with FLAG-tag via immunoblotting.</p>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/por-flag-wb.png">
 +
  <div class="unterschrift"><b>Fig.2 Expression of the POR with FLAG-tag</b><br>
 +
  (a)Level 2 MoClo construct for expression of the POR containing the FLAG-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The 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 FLAG-tagged VIPP1 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-FLAG antibody. The expression of the POR (~ 77 kDa) is visible.
 +
</p><p><br></p>
 +
<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/level-0/fig2-cypcamcbba-k4806002.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><p><br></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>
 +
<p><br></p>
 +
<p>We detected the expression of CYP9Q3 with HA-tag via immunoblotting.</p>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp9q3-con-neu.png">
 +
<img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp9q3-wb1-neu.png">
 +
<img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp9q3-wb2.png">
 +
<img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp9q3-wb3.png">
 +
  <div class="unterschrift"><b>Fig.2 Expression of CYP9Q3 with HA-tag</b><br>
 +
  (a)Level 2 MoClo construct for expression of the enzyme CYP9Q3 containing the HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The enzyme CYP9Q3 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 spectinomycin-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 CYP9Q3 (~ 59 kDa) is visible.
 +
</p>
 
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Revision as of 07:41, 12 October 2023


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K3002006

User Reviews

UNIQa05ca78256267920-partinfo-00000000-QINU

BBa_K3002006 5 Not understood Luca Langenberg

We detected the expression of CYP3A4 with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806200">BBa_K4806200</a>) via immunoblotting.

<img class="bild" src="cyp3a4-ha.png">

Fig.2 Expression of CYP3A4 with HA-tag
  (a)Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag was designed (see Fig.1 for part description) 
 (b) Picture of resulting western blot. The enzyme CYP3A4 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 spectinomycin-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) is visible.

<br<p> <p>We detected the expression of CYP3A4 with FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806201">BBa_K4806201</a>) via immunoblotting.

<img class="bild" src="cyp3a4-flag.png">

Fig.3 Expression of CYP3A4 with FLAG-tag
  (a)Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the FLAG-tag was designed (see Fig.1 for part description) 
 (b) Picture of resulting western blot. The enzyme CYP3A4 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 FLAG-tagged VIPP1 were used as a negative and positive control, respectively.

For detection the UVM4 strain was transformed with the construct in (a). 30 spectinomycin-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-FLAG antibody. The expression of CYP3A4 (~ 57 kDa) is visible.

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.

<img class="bild" src="cyp3a4-tandem-por-wb.png">

Fig.4 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.


We detected that the construct CYP3A4 with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806200">BBa_K4806200</a>) is correctly embedded within the membrane via freeze-thaw assay and immunoblotting.

<img class="bild" src="freeze-thaw.png">

Fig.6 Freeze thaw assay of CYP3A4 with HA-tag
  (a) Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag was designed. (b) The UVM4 strain was transformed with the construct in (a). An anti-HA antibody was used to detect the enzyme CYP3A4 (A). For control an anti-Cytf antibody was used to detect the membrane bound proteins and a CGE1-antibody for soluble proteins (B). 30 spectinomycin-resistant transformants were cultivated in TAP-medium and samples taken after. 3 days. For reference whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting. The other samples were treated according to protocol and analyzed also by SDS-PAGE and immunoblotting. The expression of CYP3A4 (~57 kDa) is visible in the pellet (P) and not in the supernatant (S) confirming the membrane intercalation. For reference, the UVM4 strain was used as a negative control.

We were able to detect the expression of CYP3A4 within the pellet, demonstrating that our CYP is correctly embedded within the membrane.


We detected the expression of CYP2D6 with FLAG-tag via immunoblotting.

<img class="bild" src="cyp2d6-flag-wb.png">

Fig.2 Expression of CYP2D6 with FLAG-tag
  (a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the FLAG-tag was designed (see Fig.1 for part description) 
 (b) Picture of resulting western blot. The enzyme CYP2D6 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 FLAG-tagged VIPP1 were used as a negative and positive control, respectively.

For detection the UVM4 strain was transformed with the construct in (a). 30 paromomycin-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-FLAG antibody. The expression of CYP2D6 (~ 56 kDa) is visible.


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

<img class="bild" src="por-ha-wb.png">

Fig.2 Expression of the POR with HA-tag
  (a)Level 2 MoClo construct for expression of the POR containing the HA-tag was designed (see Fig.1 for part description) 
 (b) Picture of resulting western blot. The 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 the POR (~ 77 kDa) is visible.


We detected the expression of the POR with FLAG-tag via immunoblotting.

<img class="bild" src="por-flag-wb.png">

Fig.2 Expression of the POR with FLAG-tag
  (a)Level 2 MoClo construct for expression of the POR containing the FLAG-tag was designed (see Fig.1 for part description) 
 (b) Picture of resulting western blot. The 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 FLAG-tagged VIPP1 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-FLAG antibody. The expression of the POR (~ 77 kDa) is visible.


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

<img class="bild" src="fig2-cypcamcbba-k4806002.png">

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.


We detected the expression of CYP9Q3 with HA-tag via immunoblotting.

<img class="bild" src="cyp9q3-con-neu.png"> <img class="bild" src="cyp9q3-wb1-neu.png"> <img class="bild" src="cyp9q3-wb2.png"> <img class="bild" src="cyp9q3-wb3.png">

Fig.2 Expression of CYP9Q3 with HA-tag
  (a)Level 2 MoClo construct for expression of the enzyme CYP9Q3 containing the HA-tag was designed (see Fig.1 for part description) 
 (b) Picture of resulting western blot. The enzyme CYP9Q3 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 spectinomycin-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 CYP9Q3 (~ 59 kDa) is visible.

UNIQa05ca78256267920-partinfo-00000002-QINU