Part:BBa_K660201:Design

Latherin (Surfactant Protein) + 6xHIS Tag

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 691
  Illegal PstI site found at 922
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 691
  Illegal PstI site found at 922
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 462
  Illegal BglII site found at 1005
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 691
  Illegal PstI site found at 922
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 691
  Illegal PstI site found at 922
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

We are aware of two illegal restriction sites that exist in our latherin biobrick. Due to time constraints, we were unable to complete site directed mutagenesis. The primers we designed for this are below, for any future team.

1st PstI site:
5': GCG GAA ATT AAA CTG CAA GAT ACC CGT CTG CAA CTG

    Forward Primer: 5' CAG TTG CAG ACG GGT ATC TTG CAG TTT AAT TTC CGC

    Reverse Primer: 5' GCG GAA ATT AAA CTG CAA GAT ACC CGT CTG CAA CTG

MTP= 79.33

2nd PstI site:
5': GCG ATT GAG CTG CAA AGC GGC AAT CCG C

    Forward Primer: 5' G CGG ATT GCC GCT TTG CAG CTC AAT CGC

    Reverse Primer: 5' GCG ATT GAG CTG CAA AGC GGC AAT CCG C

MTP= 78.71

Source

Latherin was originally isolated from horse sweat. It was expressed in E.coli by researchers at University of Glasgow. We received it from them in a plasmid and amplified it up by PCR to add a biobrick prefix and suffix and a 6xHIS tag.

References

Beegley J, et al., 1986. Isolation and characterization of latherin, a surface-active protein from horse sweat
http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=3753435>.

Kennedy M., 2011., Latherin and other biocompatible surfactant proteins
http://www.biochemsoctrans.org/bst/039/bst0391017.htm

Information on "Affinity Purification"
http://www.brunschwig-ch.com/pdf/downloads/ABT_RapidRun_Procedure.pdf?PHPSESSID=cb79f5862da62a654bb1a6aefe87c62b