Coding
MUC-AB-A

Part:BBa_K4574062

Designed by: Igor Koop   Group: iGEM23_Munich   (2023-10-06)


AR301-LC

pcDNA3.4 vector containing anti-toxin A (hemolsyin) light chain of AR-301 (tosatoxumab) monoclonal IgG1 antibody. This part is commonly referred to as construct A (MUC-AB-A) in the context of Team Munich's iGEM 2023 project.

Design

This construct is comprised of the following components:

  • XhoI (Part:BBa_K4574029): XhoI restriction site, allows for insert extraction
  • Kozak (Part:BBa_K4574027): Consensus sequence which enhances the initiation of translation
  • hVL-Leader (Part:BBa_K4574001): Human light chain lambda leader sequence for antibody secretion
  • AR-301-VL (Part:BBa_K4574006): Variable light chain locus targeting toxin A (hemolysin) of Staphylococcus aureus
  • hIG-CL (Part:BBa_K4574018): Human immunoglobulin light chain constant lambda region
  • 2xStop (Part:BBa_K4574028): Double stop codon sequence ensuring enhanced translation termination
  • SacI (Part:BBa_K4574030): SacI restriction site, allows for insert extraction
  • pcDNA3.4 (Part:BBa_K4574000): pcDNA3.4-TOPO backbone for constitutive high-level transgene expression in mammalian systems

Cloning

The cloning of this part has been performed using Gibson (scarless) assembly in DH5α E. coli competent cells. The insert, comprising Part:BBa_K4574006, and the backbone, comprising Part:BBa_K4574060, PCR fragments were amplified using the following oligonucleotides:

  • A1_F_IgL2_CL_AR301-VL: 5'-TGACCGTTCTGGGACAACCTAAGGCCGCTCC-3'
  • A2_R_Leader(L)_AR301-VL: 5'-GAGGACGCTTTGGGCCCAAGATCCGGTACAGTG-3'
  • A3_F_AR301-VL_Leader(L): 5'-GGCCCACAAAGCGTCCTCACCCAATCCC-3'
  • A4_R_AR301-VL_IgL2_CL: 5'-GGACAACCCAGAACGGTCACCTTTGTTCCTGTAC-3'

The provided oligonucleotides include a binding region (indicated by an underline) as well as an overhang required by the assembly method.

Usage and Biology

This construct has been used in mammalian cell culture (HEK293T, Exi293, RAMOS). Specifically, it has been co-expressed (together with its respective heavy chain Part:BBa_K4574063) using a high-yield Expi293 expression system (Gibco, a brand of Thermo Fisher Scientific, Braunschweig, Germany; catalog number: A14635) and purified in FPLC using Protein A agarose cartridges (ibidi, Goettingen, Germany; catalog number: 6-2022-001).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 6725
    Illegal SpeI site found at 6066
    Illegal PstI site found at 457
    Illegal PstI site found at 2697
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 6066
    Illegal PstI site found at 457
    Illegal PstI site found at 2697
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1647
    Illegal BglII site found at 5829
    Illegal XhoI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 6725
    Illegal SpeI site found at 6066
    Illegal PstI site found at 457
    Illegal PstI site found at 2697
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 6725
    Illegal SpeI site found at 6066
    Illegal PstI site found at 457
    Illegal PstI site found at 2697
    Illegal NgoMIV site found at 553
    Illegal NgoMIV site found at 1290
    Illegal NgoMIV site found at 1807
    Illegal NgoMIV site found at 3148
    Illegal NgoMIV site found at 3433
    Illegal AgeI site found at 647
    Illegal AgeI site found at 748
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1530
    Illegal BsaI.rc site found at 4961
    Illegal SapI site found at 3878
    Illegal SapI.rc site found at 2997
    Illegal SapI.rc site found at 3207


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