Difference between revisions of "Part:BBa K3767004:Design"

 
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__NOTOC__
 
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<partinfo>BBa_K3767004 short</partinfo>
 
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<partinfo>BBa_K3767004 SequenceAndFeatures</partinfo>
 
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===Design Notes===
 
===Design Notes===
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===Source===
 
===Source===
  
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Since it is difficult to prepare OspA biochemically, an alternative is to recombinantly&#8239;express and purify&#8239;OspA&#8239;from&#8239;E.Coli&#8239;by cloning the&#8239;ospA&#8239;gene (2). By closely following the procedure outlined in a paper written from Cornell University (2), we were able to recombinantly&#8239;express and purify&#8239;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&#8239;and vectors&#8239;pREST-A and B.&#8239;&#8239;The BamHI-SalI&#8239;insert fragment of pYFC99 was subcloned into pREST-A and B resulting in pYFC101 and 104.  pYFC99 was then subcloned onto the BamHI-SalI&#8239;site of pTTQ18&#8239;resulting in pYFC103. The&#8239;PstI-SalI&#8239;fragment&#8239;from pYFC87&#8239;containing&#8239;apXIIA&#8239;was then inserted into the&#8239;pYFC99&#8239;PstI-SalI&#8239;site to create pYFC105 clone. The E.Coli&#8239;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).
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__NOTOC__
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<partinfo>BBa_K3767004 SequenceAndFeatures</partinfo>
  
 
===References===
 
===References===
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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. 
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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. 
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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).
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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.

Latest revision as of 19:05, 3 October 2021

Design Notes

...


Source


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.