Coding

Part:BBa_K2790004

Designed by: Jatin Khaimani   Group: iGEM18_IISER-Bhopal-India   (2018-10-09)


Three gene construct of smmoC, smmoY and smmoD linked with viral 2A peptide

This part contains the coding sequence, codon optimised for P. pastoris, for the three subunits of soluble methane monooxygenase (sMMO) namely, sMMO C (reductase), sMMO Y (Hydroxylase) and sMMO D (possible regulatory subunit). The coding sequences are linked via viral 2A peptides and are tagged with a hexa-His tag at their C-terminus. These parts will be expressed as a polycistronic mRNA but during translation, ribosome will skip over a peptide bond in the 2A sequence leading to the formation of separate individual protein subunits. This part can be expressed under any promoter (constitutive or inducible) and equimolar quantities of each subunit is expected. This part was synthesized so as to reduce the number of antibiotic selection markers required to clone all 6 subunits of sMMO enzyme complex in a eukaryotic system as this eliminates the need of individual promoter and terminator sequences flanking the genes. The other 3 sMMO subunits (sMMO B, sMMO X and sMMO Z), codon optimised for P. pastoris and linked via viral 2A peptides, can be found at <a href="https://parts.igem.org/Part:BBa_K2790005"> BBa_K2790005 </a>.

<img src="T--IISER-Bhopal-India--P2A1_pGKB.png" alt="" class="team-center" width="100" height="190"/>

  • Figure 1 Cloning of P2A1 in pGKB vector and clone confirmation by restriction digestion with EcoRI and KpnI- g block P2A1 (2733bp) cloned in pGKB Plasmid (3506bp) and the resultant clone P2A1_pGKB (6239bp) was run on 1% Agarose gel for validation

    • <img src="T--IISER-Bhopal-India--p2a1submission.png" class="team-center" width="100" height="190">

  • Figure 2 Cloning of P2A1 in pSB1C3 vector and clone confirmation by restriction digestion with EcoRI and PstI- g block P2A1 (2733bp) cloned in pSB1C3 Plasmid (2Kb) and the resultant clone P2A1_pSB1C3 (~5Kb) was run on 1% Agarose gel for validation

    • Usage and Biology

    Sequence and Features


    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      COMPATIBLE WITH RFC[12]
    • 21
      INCOMPATIBLE WITH RFC[21]
      Illegal BglII site found at 79
      Illegal BglII site found at 490
      Illegal BglII site found at 820
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal NgoMIV site found at 1117
      Illegal NgoMIV site found at 2371
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI.rc site found at 405


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    Categories
    Parameters
    None