Difference between revisions of "Part:BBa K4806205"

Revision as of 13:50, 18 September 2023

CYP2D6 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick)

This composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of CYP2D6 (BBa_K4806001), 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 potential detoxification of specific chemicals (Ohkawa & Inui, 2015).

Construct

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

The resistance cassette for paromomycin 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 1098
Illegal PstI site found at 2454
Illegal PstI site found at 2887
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 249
Illegal PstI site found at 1098
Illegal PstI site found at 2454
Illegal PstI site found at 2887
Illegal NotI site found at 1604
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 530
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 1098
Illegal PstI site found at 2454
Illegal PstI site found at 2887
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 1098
Illegal PstI site found at 2454
Illegal PstI site found at 2887
Illegal NgoMIV site found at 1741
Illegal NgoMIV site found at 2090
Illegal NgoMIV site found at 3431
Illegal AgeI site found at 268
1000
COMPATIBLE WITH RFC[1000]

Results

We tried to detect the expression of CYP2D6 with HA-tag via immunoblotting.

Fig.2 Expression of CYP2D6 with FLAG-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the HA-tag was designed (see Fig.1 for part description)
(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

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.

Contribution

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

@@ Line 13: / Line 13: @@
 <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 CYP2D6 (<a href=" https://parts.igem.org/Part:BBa_K4806001">BBa_K4806001</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 potential detoxification of specific chemicals (Ohkawa & Inui, 2015).</p>
 <br>
-<h2>Constructs</h2>
+<h2>Construct</h2>
 <p>
-   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/bba-k4806001-cyp2d6-fig1.png"/">
+   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp2d6-ha-construct.png">
    <div class="unterschrift"><b>Fig.1 Construct design</b><br>
     This construct was degisned using the modular cloning system (MoClo).
-  </div>
 </p>
+  <p>The resistance cassette for paromomycin 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><br></p>
@@ Line 33: / Line 34: @@
 <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/level-0/cyp2d6-bba-k4806001-fig2.png">
+   <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 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.
+    (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
    </div>
 </p>