DNA

Part:BBa_K174006:Design

Designed by: The Newcastle 2009 iGEM team   Group: iGEM09_Newcastle   (2009-10-10)
Revision as of 18:59, 10 October 2009 by Goksel (Talk | contribs)

Sac single crossover site for Bacillus subtilis


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]

Construction

366 bp long sacA sequence is selected from 1440 bp long sacA CDS (from base 392 to base 757). This sacA sequence is amplified by PCR with dangling end primers with EcoRI-NotI-XbaI restriction site at 5’ and SpeI at 3’ and inserted into a Biobrick compatible vector. The sequence is taken from B. subtilis strain 168. Genbank accession number is [ http://www.ncbi.nlm.nih.gov/nuccore/AL009126?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum AL00912]


Forward primer used: GATCTG-GAATTCGCGGCCGCTTCTAGAG-TTCTCATTTCTGCGCATGG (Clamp sequence - Standard Biobrick prefix - first 19 base from the Biobrick)

Reverse primer used: TGTGAC-ACTAGTA-AGGATCGGATGTGCTGATTG (Clamp sequence - SpeI site - last 20 base from the Biobrick) Start of biobrick suffix TACTAGT is written as complemantary which is ACTAGTA (speI site is ACTAGT) to have the scar as in Biobrick standards.


  • Plasmid part BBa_J04450 (pSB1AT3) and our construct were treated with EcoRI and SpeI

Newcastle 2009 sac 1.png

  • The backbone and the sac insert were then ligated

Newcastle 2009 sac 2.png


Design Notes

The sequence selected does not contain any restriction site specific to Biobrick standards


Source

The sequence is taken from 'B. subtilis' 168' sacA gene.

References