Difference between revisions of "Part:BBa K4197008"

Revision as of 15:05, 6 October 2022

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

Ara h 2 gene ordered on IDT was amplified by PCR using the high fidelity Phusion polymerase with primers IF3_allergen F and IF4_Ara h 2. Expected size of the amplicon was 567 bp.

To merge Ara h 2 to OmpA, the gene was inserted in a pET-21 b (+) plasmid containing OmpA merged to Gal d 2, another allergen. The plasmid was linearized, excluding the Gal d 2 fragment by PCR. The primers used were IF1_allergen and IF2_plasmid. Expected size of the amplicon was 5924 bp.

Amplification product sizes were checked on EtBr stained agarose gel (Figure 6).

Figure 6: pET-21 b (+)_OmpA linearized with Gal d 2 exclusion (A) and Ara h 2 amplified fragment (B). Expected sizes of the amplicons were 5924 bp (A) and 567 bp (B). PCR amplicon sizes of pET-21 b (+)_OmpA (A) and Ara h 2 (B) were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).

Amplification products matched expected sizes, they were further purified from the gel.

The Ara h 2 fragment was then inserted into pET-21 b (+)_OmpA by In-Fusion. The In-Fusion assemby reaction was transformed into Stellar competent cells. Transformants were selected on LB-ampicillin plates. 15 transformants were then screened by colony PCR with primer pairs flanking the insertion zone (primers used: screening_inserts-F and screening_inserts-R, expected size of the amplicons: 1450 bp). 13 positive transformants were detected (Figure 7).

Figure 7: Identifying strains that bear pET21 b (+)_OmpA_Ara h 2 by colony PCR. The expected size of the amplicons was 1450 bp. 13 positive transformants were detected. PCR amplicon sizes of colonies with Ara h 2 plasmid were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).

Figure 7: Identifying strains that bear pET21 b (+)_OmpA_Ara h 2 by colony PCR. The expected size of the amplicons was 1450 bp. 13 positive transformants were detected. PCR amplicon sizes of colonies with Ara h 2 plasmid were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).

Four of these transformants (colonies 21, 22, 23, 24) had their plasmid extracted by Miniprep. Sequences were all validated by Sanger sequencing. The plasmid was named pET-21 b (+)_OmpA_Ara h 2.

The plasmid was finally used to transform E. coli Tuner cells to express OmpA_Ara h 2 at the cell membrane.

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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

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