Part:BBa_K3767004:Design

Design Notes

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Source

Since it is difficult to prepare OspA biochemically, an alternative is to recombinantly express and purify OspA from E.Coli by cloning the ospA gene (2). By closely following the procedure outlined in a paper written from Cornell University (2), we were able to recombinantly express and purify OspA in the laboratory. The procedure included an initial PCR to amplify the ospA gene from Borrelia Burgfedori B31 DNA followed by a DNA digestion of the amplified ospA via BamHI and SalI restriction enzymes. The digested DNA was then ligated into the plasmid pHG165 and the plasmid was transformed into isolated E.coli TB1. The recombinant ospA plasmid was then subcloned into the expression vector pTTQ18 which contained the T7 promoter and vectors pREST-A and B.  The BamHI-SalI insert fragment of pYFC99 was subcloned into pREST-A and B resulting in pYFC101 and 104. pYFC99 was then subcloned onto the BamHI-SalI site of pTTQ18 resulting in pYFC103. The PstI-SalI fragment from pYFC87 containing apXIIA was then inserted into the pYFC99 PstI-SalI site to create pYFC105 clone. The E.Coli pYFC101 and pYFC104 was grown in a SOB medium, then was infected with the bacterial microphage M13/T7. pYFC105 was harvested from cultured supernatents from E.Coli harboring pYFC105, then OspA was purified from the M13/TF cells using Immobilized metal ion affinity chromatography (IMIAC).

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Hansson, L., Noppa, L., Nilsson, A., Stromqvist, M., Bergstrom, S. (1994). Expression of Truncated and Full-Length Forms of the Lyme Disease Borrelia Outer Surface Protein A in Escherichia coli, Umed University, Umed, Sweden. 2. Dunn, J., Lade, B., Barbour, A. (1990). Outer Surface Protein A (OspA) from Lyme Disease Spirochete, Borrelia burgdorferi: High Level Expression and Purification of a Soluble Recombinant from of OspA, University of Texas, San Antonio, Texas. 3. Park, J., Prilusky, J., Harel, M and Martz, E. (2015). OspA. Proteopedia [Online].https://proteopedia.org/wiki/index.php/OspA. (Accessed June 19, 2022). 4. Chang, Y-F., Lauderdale, T-L., Lee, W., Shin, J., Jacobson, R., Appel, M and Lein, D. (1993). Expression and secretion of outer surface protein (OSP-A) of Borrelia burgdorferi from Escherichia coli, Cornell University, Ithaca, NY, USA.