Composite
RFC 37

Part:BBa_K245099:Design

Designed by: Špela Miklavič   Group: iGEM09_Slovenia   (2009-10-16)

APH-p53


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]


Design Notes

Part BBa_K245099 is intended to be cloned into the BioBrick vector under the strong promotor, such as T7. It has been experimentally in vector BBa_K245005

The gene for APH is of synthetic origin, optimized for expression in E. coli and based on aminoacid sequence of a designed homodimeric antiparallel coiled-coil from the reference (Gurnon et al., 2003). The gene for p53 tetramerization domain was also of synthetic origin, based on aminoacid sequence of human p53 tetramerization domain, optimized for expression in E. coli.

Assembly of part BBa_K245099 First, each of the coding sequences was cloned into the BioBrick vector (BBa_K245005). The vector already contains T7 promoter, RBS and ATG at 5' of multiple-cloning site, T7 terminator and STOP codon at 3' of multiple-cloning site. His-tag coding sequence is also incorporated between ATG and multiple-cloning site. Resulting basic parts were BBa_K245133 (APH inserted in functionalized vector BBa_K245005) and BBa_K245128 (p53 inserted in functionalized vector BBa_K245005).

The part with p53 was cut with NgoMIV and XbaI restriction enzymes and the part containing APH was cut with BspeEI and XbaI restriction enzymes. The APH-coding fragment was ligated into the linearized p53 part (BBa_K245128). Ligation resulted in a new composite part (BBa_K245099) where APH gene is located 5’ of p53. A joining of NgoMIV and BspEI restriction sites generates a protein-domain friendly scar tccggc (SG) after the ligation of basic parts BBa_K245133 and BBa_K245128. Resulting composite part BBa_K245099 has also His-tag-coding sequence due to design of our vector.

Source

The gene for APH is of synthetic origin, optimized for expression in E. coli and based on aminoacid sequence of a designed homodimeric antiparallel coiled-coil from the reference (Gurnon et al., 2003). The gene for p53 tetramerization domain was also of synthetic origin, based on aminoacid sequence of human p53 tetramerization domain, optimized for expression in E. coli.

References