Difference between revisions of "Part:BBa K4806012"
Line 29: Line 29:
 
</p>
 
</p>
 
<p>
 
<p>
−
   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the FLAG-tag the constructs either contain the hygromycin (<a href=" https://parts.igem.org/Part:BBa_K4806100">BBa_K4806100</a>), spectinomycin (<a href=" https://parts.igem.org/Part:BBa_K3002102">BBa_K3002102</a>) or the paromocyin resistance cassette (<a href=" https://parts.igem.org/Part:BBa_K4806101">BBa_K4806101</a>), either the POR (<a href=" https://parts.igem.org/Part:BBa_K4806003">BBa_K4806003</a>), the 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>), or the CYP3A4 coding sequence (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>) and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>). The resistance cassette for hygromycin, spectinomycin 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 FLAG-tag the constructs either contain the POR (<a href=" https://parts.igem.org/Part:BBa_K4806003">BBa_K4806003</a>), the 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>), or the CYP3A4 coding sequence (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>) and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>). The resistance cassette for hygromycin, spectinomycin 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>
 
</p>
  

Revision as of 19:06, 14 September 2023


FLAG-tag for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the FLAG-tag (B5). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promotor like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006), this level 0 part leads to detection of your expressed target protein like CYP3A4 (BBa_K4806000).


Constructs

Fig.1 Construct design
We designed 3 level 2 constructs containing the FLAG-tag using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. The POR gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806210)
  • 2. CYP3A4 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806201)
  • 3. CYP2D6 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806206)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the FLAG-tag the constructs either contain the POR (BBa_K4806003), the CYP3A4 (BBa_K4806000) or the CYP2D6 coding sequence(BBa_K4806001), or the CYP3A4 coding sequence (BBa_K4806000) and the tRPL23-terminator (BBa_K3002006). The resistance cassette for hygromycin, spectinomycin 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
    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 1
    Illegal NgoMIV site found at 7
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We tried to detected the expression of the POR, CYP3A4 and CYP2D6 with FLAG-tag (BBa_K4806210, BBa_K4806201, BBa_K4806206) via immunoblotting.

Fig.2 Expression of the POR, CYP3A4, CYP2D6 with FLAG-tag
(1a-3a) Level 2 MoClo constructs for expression of the enzyme POR, CYP3A4, CYP2D6 containing the FLAG-tag were designed (see Fig.1 for part description)
(1b-3b) Picture of resulting western blots. The enzymes are marked by a black arrow, the white arrow marks cross reactions 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 (1a-3a). 30 antibiotic resistant transformants (depending on the construct) 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 the POR (~77 kDa), CYP3A4 (~57 kDa), and CYP2D6 (~56 kDa) is visible.