Composite

Part:BBa_K2350016:Design

Designed by: JO-NING HUNG   Group: iGEM17_NYMU-Taipei   (2017-10-21)


BBa_B0015 and Ampicillin resistance gene

We construct a vector containing ampicillin resistance gene (AmpR) for antibiotic selection. Because our ampicillin resistance gene source, pBR322, is without effective terminator that could be functional in cyanobacteria, we fused AmpR with BBa_B0015 using fusion PCR. BBa_B0015 is double terminator which is proved to be functional in cyanobacteria. Ampicillin resistance gene coding sequence and promoter is retrieved from pBR322. To insert AmpR-B0015 with EcoR1 and Pst1 cutting sites, we use site-directed mutagenesis to remove Pst1 cutting sites in AmpR nucleotide sequence.



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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 950


Design Notes

To insert AmpR-B0015 with EcoR1 and Pst1 cutting sites, we use site-directed mutagenesis to remove Pst1 cutting sites in AmpR nucleotide sequence.


Source

BBa_B0015 is from iGEM distribution kit, and Ampicillin resistance gene is from pBR322 nucleotide sequence.


References

Pei-Hong Chen, Hsien-Lin Liu, Yin-Ju Chen, Yi-Hsiang Cheng, Wei-Ling Lin, Chien-Hung Yeh and Chuan-Hsiung Chang (2012). Enhancing CO2 bio-mitigation by genetic engineering of cyanobacteria