Composite

Part:BBa_K2243031:Design

Designed by: Chen Hong   Group: iGEM17_Peking   (2017-10-23)


Bxb1 attB_322F_Bxb1 attP


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 NgoMIV site found at 4
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 24
    Illegal BsaI site found at 103
    Illegal BsaI.rc site found at 147


Design Notes

A set of desirable unidirectional terminators were selected based on Nasri H, et. al Nature Methods 2013 and they were then constructed on the pSB4C5 vector. Terminator strength was tested using flow cytometry and corresponding Ts value was calculated.


Peking simple terminators.png


Then attB/P sites were then introduced.


Peking sandwiched terminators.png


Two systems- the system containing sandwiched terminators and the system expressing corresponding recombinases were co-transformed into DH5alpha cells. Ts values of terminators sandwiched with attB/P sites were measured with flow cytometry.

Source

Streptomyces bacteria Phage phiC31

Terminators were commercially synthesized.

References

Dyakonov T, Muir A, Nasri H, et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints[J]. Nature Methods, 2013, 10(7):659-664.

Yang L, Voigt CA, et al. Permanent genetic memory with >1-byte capacity. Nat Methods. 2014 Dec; 11(12):1261-6.