Difference between revisions of "Part:BBa K4806002"

Revision as of 14:08, 13 September 2023

CYPCamC gene for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of CYPCamC (B3-B4). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promoter like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006), this level 0 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015). To detect the target protein a tag like HA-tag (BBa_K3002017) is recommended.

Constructs

Fig.1 Construct design
We designed 3 level 2 parts containing CYPCamC using the modular cloning system (MoClo).

Here are the links to the built constructs:

1. CYPCamC gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806216)
2. CYPCamC gene for expression in the mitochrondria for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806218)
3. CYPCamC tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806217)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the CYP2D6 coding sequence they contain a hygromycin resistance cassette (BBa_K4806100), either the βSAP(i)-promotor (AβSAP(i) (BBa_K4806013), the PAR-promotor (BBa_K3002010), or the PSAD-promotor (BBa_K4806010), the HA-tag (BBa_K3002017) for detection and the tRPL23-terminator (BBa_K3002006). Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (BBa_K4806014) and one the mtTP70C transit peptide to the mitochondria (BBa_K4806011)

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 281
Illegal PstI site found at 553
Illegal PstI site found at 1277
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 281
Illegal PstI site found at 553
Illegal PstI site found at 1277
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 281
Illegal PstI site found at 553
Illegal PstI site found at 1277
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 281
Illegal PstI site found at 553
Illegal PstI site found at 1277
1000
COMPATIBLE WITH RFC[1000]

Results

We detected the expression of CYPCamC with HA-tag (BBa_K4806216) 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.

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 <h2>Constructs</h2>
 <p>
-   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/bba-k4806002-cypcamc-fig1.png">
+   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cypcamc-bba-k4806002-fig1.png">
    <div class="unterschrift"><b>Fig.1 Construct design</b><br>
     We designed 3 level 2 parts containing CYPCamC using the modular cloning system (MoClo).
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 <p>We detected the expression of CYPCamC with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806216">BBa_K4806216</a>) via immunoblotting.</p>
 <p>
-   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/bba-k4806001-cyp2d6-fig2.png">
+   <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 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 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 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-FLAG antibody. The expression of CYP2D6 (~ 56 kDa) is 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>
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