Difference between revisions of "Part:BBa K4806219"

Revision as of 14:17, 18 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 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 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

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.

@@ Line 25: / Line 25: @@
 <h2>Sequence and Features</h2>
 </html>
-<partinfo>BBa_K4806205 SequenceAndFeatures</partinfo>
+<partinfo>BBa_K4806219 SequenceAndFeatures</partinfo>
-<partinfo>BBa_K4806205 parameters</partinfo>
+<partinfo>BBa_K4806219 parameters</partinfo>
 <html>
 <h2>Results</h2>
-<p>We confirmed that this construct is built correctly via agarose gel electrophoresis.</p>
+<p>We detected the expression of CYPCamC with HA-tag via immunoblotting.</p>
-<p>
-  <img class="agarose" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/2d6-ha.png">
-  <div class="unterschrift"><b>Fig.2 Test digest of CYP2D6 level 2 with HA-tag</b><br>
-We digested this level 2 MoClo part with the restriction enzymes <i>Sac</i>I and <i>Not</i>I.</div></p>
-<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>
-<p><br></p>
-<p>We tried to detect the expression of CYP2D6 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.3 Expression of CYP2D6 with FLAG-tag</b><br>
+   <div class="unterschrift"><b>Fig.2 Expression of CYPCamC with HA-tag</b><br>
-    (a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. 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
+    (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 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-HA antibody. The expression of CYP2D6 (~ 56 kDa) is not visible.
+<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>