Coding
MUC-AB-B

Part:BBa_K4574063

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


AR301-HC

pcDNA3.4 vector containing anti-toxin A (hemolsyin) heavy chain of AR-301 (tosatoxumab) monoclonal IgG1 antibody. This part is commonly referred to as construct B (MUC-AB-B) 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
  • hVH-Leader (Part:BBa_K4574002): Human heavy chain leader sequence for antibody secretion
  • AR-301-VH (Part:BBa_K4574007): Variable heavy chain locus targeting toxin A (hemolysin) of Staphylococcus aureus
  • hIG-CH (Part:BBa_K4574019): Human immunoglobulin heavy chain constant gamma 1 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_K4574007, and the backbone, comprising Part:BBa_K4574061, PCR fragments were amplified using the following oligonucleotides:

  • B1_F_IgG1-CH_AR301-VH: 5'-GGAGCTGACGGTCACCATGCTTCTACCAAGGGACCCAGC-3'
  • B2_R_Leader(H)_AR301-VH: 5'-GAAGTTCAGAGCACAAACGCCCTGCAG-3'
  • B3_F_AR301-VH_Leader(H): 5'-AGCACAAACGCGAAGTTCAGATGGTGCAGAGCG-3'
  • B4_R_AR301-VH_IgG1-CH: 5'-CGGAGCTGACGGTCACCATAGTT-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 light chain Part:BBa_K4574062) 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 7427
    Illegal SpeI site found at 6768
    Illegal PstI site found at 52
    Illegal PstI site found at 3399
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 6768
    Illegal PstI site found at 52
    Illegal PstI site found at 3399
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2349
    Illegal BglII site found at 6531
    Illegal XhoI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 7427
    Illegal SpeI site found at 6768
    Illegal PstI site found at 52
    Illegal PstI site found at 3399
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 7427
    Illegal SpeI site found at 6768
    Illegal PstI site found at 52
    Illegal PstI site found at 3399
    Illegal NgoMIV site found at 1992
    Illegal NgoMIV site found at 2509
    Illegal NgoMIV site found at 3850
    Illegal NgoMIV site found at 4135
    Illegal AgeI site found at 1450
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 238
    Illegal BsaI.rc site found at 2232
    Illegal BsaI.rc site found at 5663
    Illegal SapI site found at 4580
    Illegal SapI.rc site found at 3699
    Illegal SapI.rc site found at 3909


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