Composite

Part:BBa_K4806218

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-09-18)
Revision as of 08:59, 21 September 2023 by Lulang (Talk | contribs)


CYPCamC gene for expression in the mitochondria for Chlamydomonas reinhardtii (Phytobrick)


This composite part contains the PAR-promotor (BBa_K3002010), the mtTP70C-transit peptide (BBa_K4806011), the CYPCamC coding sequence (BBa_K4806002), 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 228
    Illegal PstI site found at 409
    Illegal PstI site found at 918
    Illegal PstI site found at 1190
    Illegal PstI site found at 1914
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 228
    Illegal PstI site found at 409
    Illegal PstI site found at 918
    Illegal PstI site found at 1190
    Illegal PstI site found at 1914
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 424
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 228
    Illegal PstI site found at 409
    Illegal PstI site found at 918
    Illegal PstI site found at 1190
    Illegal PstI site found at 1914
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 228
    Illegal PstI site found at 409
    Illegal PstI site found at 918
    Illegal PstI site found at 1190
    Illegal PstI site found at 1914
    Illegal NgoMIV site found at 2935
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

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

Fig.2 Test digest of CYPCamC level 2 targeted to the mitochondria
We digested this level 2 MoClo part with the restriction enzymes NheI and EcoRV.

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.

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Categories
Parameters
None