Difference between revisions of "Part:BBa K4806015"

Revision as of 11:43, 6 October 2023

Hygromycin resistance for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the hygromycin resistance cassette (B3-B5). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promotor like PSAD (BBa_K3002001), and a terminator like PSAD (BBa_K3002002) this level 0 part mediates resistance to hygromycin.

Constructs

Fig.1 Construct design
We designed 2 level 2 constructs containing the hygromycin resistance cassette using the modular cloning system (MoClo).

Here are the links to the built constructs:

1. CYP3A4 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806214)
2. CYP2D6 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806215)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the hygromycin resistance cassette the constructs contains the AβSAP(i)-promotor (BBa_K4806013), either the POR (BBa_K4806003), CYP3A4 (BBa_K4806000) or the CYP2D6 coding sequence (BBa_K4806001), the HA-tag (BBa_K3002017)^* for detection and and the tRPL23-terminator (BBa_K3002006)^*. Further a dummy for F3 (BBa_K4806017) and an endlinker(BBa_K4806016) were used.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 455
Illegal NgoMIV site found at 637
1000
COMPATIBLE WITH RFC[1000]

Results

We detected the expression of CYP3A4 tandem together with the POR with HA-tag (BBa_K4806214) via immunoblotting.

Fig.2 Expression of CYP3A4 tandem together with the POR with HA-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 tandem together with the POR containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP3A4/POR 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 CYP3A4 (~ 57 kDa) and the POR (~77 kDa) is visible.

Contribution

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

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-    These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the hygromycin resistance cassette the constructs contains the AβSAP(i)-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>), either the POR (<a href=" https://parts.igem.org/Part:BBa_K4806003">BBa_K4806003</a>), CYP3A4 (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>) or CYP2D6 coding sequence (<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 and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>. Further a dummy for F3 (<a href=" https://parts.igem.org/Part:BBa_K4806017">BBa_K4806017</a>) and an endlinker(<a href=" https://parts.igem.org/Part:BBa_K4806016">BBa_K4806016</a>) were used.
+    These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the hygromycin resistance cassette the constructs contains the AβSAP(i)-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>), either the POR (<a href=" https://parts.igem.org/Part:BBa_K4806003">BBa_K4806003</a>), CYP3A4 (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>) or the CYP2D6 coding sequence (<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 and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>. Further a dummy for F3 (<a href=" https://parts.igem.org/Part:BBa_K4806017">BBa_K4806017</a>) and an endlinker(<a href=" https://parts.igem.org/Part:BBa_K4806016">BBa_K4806016</a>) were used.
 </p>