Difference between revisions of "Part:BBa K4806002"

Revision as of 13:43, 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 (), or the PSAD-promotor (BBa_K4806010), either the HA-tag (BBa_K3002017) or the FLAG-tag (BBa_K4806012) for detection and the tRPL23-terminator (BBa_K3002006). Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (BBa_K4806014)

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 356
Illegal PstI site found at 1712
Illegal PstI site found at 2145
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 356
Illegal PstI site found at 1712
Illegal PstI site found at 2145
Illegal NotI site found at 862
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 356
Illegal PstI site found at 1712
Illegal PstI site found at 2145
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 356
Illegal PstI site found at 1712
Illegal PstI site found at 2145
Illegal NgoMIV site found at 999
Illegal NgoMIV site found at 1348
1000
COMPATIBLE WITH RFC[1000]

Results

We detected the expression of CYP2D6 with FLAG-tag (BBa_K4806206) via immunoblotting.

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.

@@ Line 24: / Line 24: @@
 <ul>
 <li>1. CYPCamC gene with HA-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806216">BBa_K4806216</a>)</li>
-<li>2. CYP2D6 gene for expression in the mitochrondria for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806218">BBa_K4806218</a>)</li>
+<li>2. CYPCamC gene for expression in the mitochrondria for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806218">BBa_K4806218</a>)</li>
-<li>5. CYP2D6 tandem for expression together with the POR for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806217">BBa_K4806217</a>)</li>
+<li>3. CYPCamC tandem for expression together with the POR for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806217">BBa_K4806217</a>)</li>
 </ul>
 </p>
 <p>
-    These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP2D6 coding sequence they contain an the paromomycin resistance cassette (<a href=" https://parts.igem.org/Part:BBa_K4806101 ">BBa_K4806101</a>), either the βSAP(i)-promotor (AβSAP(i) (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>) or the PSAD-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806010">BBa_K4806010</a>), either the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>) or the FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806012">BBa_K4806012</a>) for detection and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>). Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>)
+    These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP2D6 coding sequence they contain a hygromycin resistance cassette (<a href=" https://parts.igem.org/Part:BBa_K4806100">BBa_K4806100</a>), either the βSAP(i)-promotor (AβSAP(i) (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>), the PAR-promotor (), or the PSAD-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806010">BBa_K4806010</a>), either the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>) or the FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806012">BBa_K4806012</a>) for detection and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>). Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>)
 </p>