Coding

Part:BBa_K4197008

Designed by: Guillaume Gomez   Group: iGEM22_Toulouse_INSA-UPS   (2022-09-22)
Revision as of 14:26, 6 October 2022 by Laurelamothe (Talk | contribs)


OmpA_Ara h 2 fusion

Gene fusion to express the peanut allergen Ara h 2 on the surface of E. coli .

Introduction

This part is composed of the gene coding for the allergen of peanu Ara h 2 (NCBI: AY158467.1). The peanut allergy prevalence is superior to 5% (lieberman and al. 2018) in developped countries and Ara h 2, among the 17 other peanut allergens, triggers 90% of the patients with peanut allergy (lehmann and al. 2003). Ara h 2 have already been expressed in E. coli and was able to bind the IgE of patient with peanut's allergie (lehmann and al. 2003). Ara h 2 was merged to the membrane protein OmpA of E. coli (BBa_K1694002), to display Ara h on the surface of E. coli . This lippoprotein is the most abundant in E. coli's membrane with 100,000 copies per cell (Ortiz-Suarez and al. 2016) and is often used to display protein on the surface of bacteria (Yang and al. 2016).

Construction

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  • Forward : TAAGAAGGAGATATACCATGGCGGAAGCGGGTATCACC
  • Reverse : CTCGAGTGCGGCCGCAAGCTTCGGATCGTCCTATGATGGAGG

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  • CForward : CGCGGCCGCTTCTAGAGCGGAAGCGGGTATCACC
  • Reverse : AGCGGCCGCTACTAGTCGGATCGTCCTATGATGGAGG

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References

  1. Lieberman, J., Sublett, J., Ali, Y., Haselkorn, T., Damle, V., Chidambaram, A., Rosen, K., & Mahr, T. (2018). INCREASED INCIDENCE AND PREVALENCE OF PEANUT ALLERGY IN CHILDREN AND ADOLESCENTS IN THE UNITED STATES. Annals of Allergy, Asthma & ; Immunology, 121(5), S13. https://doi.org/10.1016/j.anai.2018.09.039
  2. Lehmann, K., Hoffmann, S., Neudecker, P., Suhr, M., Becker, W.-M., & Rösch, P. (2003). High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Ara h 2. Protein Expression and Purification, 31(2), 250–259. https://doi.org/10.1016/s1046-5928(03)00190-6
  3. Ortiz-Suarez, M. L., Samsudin, F., Piggot, T. J., Bond, P. J., & Khalid, S. (2016). Full-Length OmpA : Structure, Function, and Membrane Interactions Predicted by Molecular Dynamics Simulations. Biophysical Journal, 111(8), 1692–1702. https://doi.org/10.1016/j.bpj.2016.09.009
  4. Yang, Chao; Zhao, Qiao; Liu, Zheng; Li, Qiyun; Qiao, Chuanling; Mulchandani, Ashok; et al. (2016): Cell Surface Display of Functional Macromolecule Fusions on Escherichia coli for Development of an Autofluorescent Whole-Cell Biocatalyst. ACS Publications. Journal contribution. https://doi.org/10.1021/es800441t.s001
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Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 130
    Illegal XbaI site found at 47
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 130
    Illegal NheI site found at 92
    Illegal NotI site found at 1188
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 130
    Illegal BamHI site found at 124
    Illegal BamHI site found at 813
    Illegal BamHI site found at 834
    Illegal XhoI site found at 1197
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 130
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 130
    Illegal XbaI site found at 47
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 713


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