Difference between revisions of "Part:BBa K4806010"
Line 1: Line 1:
−
 
−
__NOTOC__
 
−
<partinfo>BBa_K4806010 short</partinfo>
 
−
 
−
aaaaaaaaaaaaaaaaaa
 
−
 
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
−
 
−
<!-- -->
 
−
<span class='h3bb'>Sequence and Features</span>
 
−
<partinfo>BBa_K4806010 SequenceAndFeatures</partinfo>
 
−
 
−
 
−
<!-- Uncomment this to enable Functional Parameter display
 
−
===Functional Parameters===
 
−
<partinfo>BBa_K4806010 parameters</partinfo>
 
−
<!-- -->
 
−
 
  
 
__NOTOC__
 
__NOTOC__
Line 49: Line 30:
 
</p>
 
</p>
 
<p>
 
<p>
−
   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 (<a href=" https://parts.igem.org/Part:BBa_K3002010">BBa_K3002010</a>), or the PSAD-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806010">BBa_K4806010</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</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>) and one the mtTP70C transit peptide to the mitochondria (<a href=" https://parts.igem.org/Part:BBa_K4806011">BBa_K4806011</a>)
+
   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the PSAD-promotor the constructs either contain the hygromycin (<a href=" https://parts.igem.org/Part:BBa_K4806100">BBa_K4806100</a>), paromomycin (<a href=" https://parts.igem.org/Part:BBa_K4806101">BBa_K4806101</a>) or the spectinomycin resistance cassette (<a href=" https://parts.igem.org/Part:BBa_K3002000">BBa_K3002000</a>), the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>), either the POR (<a href=" https://parts.igem.org/Part:BBa_K4806003">BBa_K4806003</a>), CYP2D6 (<a href=" https://parts.igem.org/Part:BBa_K4806001">BBa_K4806001</a>), CYPCamC (<a href=" https://parts.igem.org/Part:BBa_K4806002">BBa_K4806002</a>) or the CYP3A4 coding sequence (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a> for detection and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>).
 
</p>
 
</p>
  
 
<h2>Sequence and Features</h2>
 
<h2>Sequence and Features</h2>
 
</html>
 
</html>
−
<partinfo>BBa_K4806002 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K4806010 SequenceAndFeatures</partinfo>
  
−
<partinfo>BBa_K4806002 parameters</partinfo>
+
<partinfo>BBa_K4806010 parameters</partinfo>
  
  
 
<html>
 
<html>
 
<h2>Results</h2>
 
<h2>Results</h2>
−
<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>We tried to detected the expression of the POR, CYP2D6, CYPCamC and CYP3A4 targeted to the chloroplast with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806212">BBa_K4806212</a>,<a href=" https://parts.igem.org/Part:BBa_K4806208">BBa_K4806208</a>,<a href=" https://parts.igem.org/Part:BBa_K4806217">BBa_K4806217</a>,<a href=" https://parts.igem.org/Part:BBa_K4806203">BBa_K4806203</a>) via immunoblotting.</p>
 
<p>
 
<p>
 
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/fig2-cypcamcbba-k4806002.png">
 
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/fig2-cypcamcbba-k4806002.png">

Revision as of 15:39, 13 September 2023


PSAD-promotor for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the PSAD-promotor (A1-B1). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a coding sequence like CYP3A4 (BBa_K4806000) and a terminator like tRPL23 (BBa_K3002006), this level 0 part leads to expression of your target protein (Einhaus et al., 2021). To detect the target protein a tag like HA-tag (BBa_K3002017) is recommended.


Constructs

Fig.1 Construct design
We designed 4 level 2 parts containing the PSAD-promotor using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. The POR gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806212)
  • 2. CYP2D6 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806208)
  • 3. CYPCamC gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806217)
  • 4. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the PSAD-promotor the constructs either contain the hygromycin (BBa_K4806100), paromomycin (BBa_K4806101) or the spectinomycin resistance cassette (BBa_K3002000), the CTPPSAD transit peptide to the chloroplast (BBa_K4806014), either the POR (BBa_K4806003), CYP2D6 (BBa_K4806001), CYPCamC (BBa_K4806002) or the CYP3A4 coding sequence (BBa_K4806000), the HA-tag (BBa_K3002017 for detection and the tRPL23-terminator (BBa_K3002006).

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We tried to detected the expression of the POR, CYP2D6, CYPCamC and CYP3A4 targeted to the chloroplast with HA-tag (BBa_K4806212,BBa_K4806208,BBa_K4806217,BBa_K4806203) 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.