Composite

Part:BBa_K4806212

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-09-18)
Revision as of 10:37, 19 September 2023 by Lulang (Talk | contribs)


POR gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick)


This composite part contains the PSAD-promotor (BBa_K4806010), the CTPPSAD-transit peptide (BBa_K4806014), the POR coding sequence (BBa_K4806003), the HA-tag (BBa_K3002017)* for detection and the tRPL23-terminator (BBa_K3002006)*. This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. This level 2 part leads to potential detoxification of specific chemicals (Ohkawa & Inui, 2015).


Construct

Fig.1 Construct design
This construct was designed using the modular cloning system (MoClo).

The resistance cassette for hygromycin 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 1280
    Illegal PstI site found at 2636
    Illegal PstI site found at 3069
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1280
    Illegal PstI site found at 2636
    Illegal PstI site found at 3069
    Illegal NotI site found at 1786
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1280
    Illegal PstI site found at 2636
    Illegal PstI site found at 3069
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1280
    Illegal PstI site found at 2636
    Illegal PstI site found at 3069
    Illegal NgoMIV site found at 1923
    Illegal NgoMIV site found at 2272
    Illegal NgoMIV site found at 3613
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We confirmed that this construct is built correctly via agarose gel electrophoresis.

Fig.2 Test digest of the POR level 2 targeted to the chloroplast
We digested this level 2 MoClo part with the restriction enzymes NotI and EcoRI.

The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.


We tried to detected the expression the POR targeted to the chloroplast with HA-tag via immunoblotting.

Fig.3 Expression of the POR with the CTPPSAD-transit peptide
(a)Level 2 MoClo construct for expression of the POR containing the CTPPSAD-tranist peptide with HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. 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 the POR (~ 56 kDa) is not 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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