Device

Part:BBa_K598002:Design

Designed by: Yan GONG, Shuobin CHEN, Yunpeng LIU, Xiaowei YAN   Group: iGEM11_Peking_R   (2011-09-27)

Bistable Switch Mutant 68


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 10
    Illegal AgeI site found at 122
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2699


Design Notes

This part (BBa_K598002) is one of the mutation libraries which has a nearly 3:1 ratiometric of the green colonies to the red colonies (G/R ratio) of bistable switch.The library mutating the ribosome binding site (RBS) of cI434 gene is constructed using site-directed mutagenesis method, as a demonstration of RBS calculation of the bistable switch.(Figure 1)

Figure 1 Construction of bistable switch library via site-directed mutagenesis. Forward and reverse primers binding sites are schematized.

Primers are designed pairing the template (BBa_K228003) but replacing the RBS of cI434 gene with NNNNNNN. After PCR amplification, the PCR product was treated with DpnI and column-purified. The linear DNA was phosphoryated by T4 polynucleotide kinase and column-purified. The DNA was then self-ligated and transformed into DH5α cells. Thus, the library is established.

Source

The template (BBa_K228003) for site-directed mutagenesis is inherited from 2009 Peking iGEM team.

References