Difference between revisions of "Part:BBa K4806001"

Revision as of 11:05, 18 September 2023

CYP2D6 gene for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of CYP2D6 (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 FLAG-tag (BBa_K4806012) is recommended.

Constructs

Fig.1 Construct design
We designed 5 level 2 constructs containing CYP2D6 using the modular cloning system (MoClo).

Here are the links to the built constructs:

1. CYP2D6 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806205)
2. CYP2D6 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806207)
3. CYP2D6 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806206)
4. CYP2D6 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806208)
5. CYP2D6 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806215)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the CYP2D6 coding sequence the constructs contain either the AβSAP(i)-promotor (BBa_K4806013) 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). The resistance cassette for hygromycin or 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 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.

Contribution

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

@@ Line 10: / Line 10: @@
-<p>This basic part contains the coding sequence of CYP2D6 (B3-B4). This part is codon-optimized for <i>Chlamydomonas reinhardtii</i> and was built as part of the CYPurify Collection. In combination with a promoter like AβSAP(i) (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>) and a terminator like tRPL23 (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>), this level 0 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015). To detect the target protein a tag like FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806012">BBa_K4806012</a>) is recommended. </p>
+<p>This basic part contains the coding sequence of CYP2D6 (B3-B4). This part is codon-optimized for <i>Chlamydomonas reinhardtii</i> and was built as part of the CYPurify Collection. In combination with a promoter like AβSAP(i) (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>) and a terminator like tRPL23 (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>, this level 0 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015). To detect the target protein a tag like FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806012">BBa_K4806012</a>) is recommended. </p>
 <br>
 <h2>Constructs</h2>
@@ Line 31: / Line 31: @@
 </p>
 <p>
-    These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP2D6 coding sequence the constructs contain 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>). The resistance cassette for hygromycin or 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).
+    These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP2D6 coding sequence the constructs contain either the AβSAP(i)-promotor (<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>)<sup>*</sup> 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>)<sup>*</sup>. Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>). The resistance cassette for hygromycin or 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>
@@ Line 53: / Line 53: @@
 <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>
+<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>