Difference between revisions of "Part:BBa K4806100"
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<p> This level 1 composite part contains the PSAD-promoter (<a href="https://parts.igem.org/Part:BBa_K3002001">BBa_K3002001</a>), the coding sequence of the hygromycin resistance cassette (<a href="https://parts.igem.org/Part:BBa_K4806015">BBa_K4806015</a>)and the PSAD-Terminator (<a href="https://parts.igem.org/Part:BBa_K3002002">BBa_K3002002</a>). This part mediates resistance to spectinomycin. </p>
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<p> This level 1 composite part contains the PSAD-promoter (<a href="https://parts.igem.org/Part:BBa_K3002001">BBa_K3002001</a>), the coding sequence of the hygromycin resistance cassette (<a href="https://parts.igem.org/Part:BBa_K4806015">BBa_K4806015</a>)and the PSAD-terminator (<a href="https://parts.igem.org/Part:BBa_K3002002">BBa_K3002002</a>). This part mediates resistance to hygromycin. </p>
 
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<h2>Constructs</h2>
 
<h2>Constructs</h2>
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   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the POR 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 FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806012">BBa_K4806012</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)<sup>*</sup> or mNeonGreen (<a href=" https://parts.igem.org/Part:BBa_K4806006">BBa_K4806006</a>) for detection or mStop (<a href=" https://parts.igem.org/Part:BBa_K4806009">BBa_K4806009</a>) 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 spectinomycin or hygromycin is already built in the level 2 vector pMBS807 we are using (exept for the tandem construct). The usage of this vector allows the direct assembly of level 0 parts to level 2 constructs, facilitating the cloning time (Niemeyer & Schroda, 2022).
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Revision as of 11:24, 2 October 2023


Hygromycin Resistance for Chlamydomonas reinhardtii (Phytobrick)

This level 1 composite part contains the PSAD-promoter (BBa_K3002001), the coding sequence of the hygromycin resistance cassette (BBa_K4806015)and the PSAD-terminator (BBa_K3002002). This 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)

part beschreibung fehlt

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 1270
    Illegal NgoMIV site found at 1452
  • 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.