Device

Part:BBa_K2082151:Design

Designed by: Cassandra Königs   Group: iGEM16_Bielefeld-CeBiTec   (2016-10-13)


Stop Beta-Lactamase (K30*)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 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 777

Picture with not growing on ampicillin and growing on chloramphenicol
Figure 1: Possible mutants plated on ampicillin containing agar (right) and chloramphenicol containing agar (left). One of the candidates was used for sequencing and part amplification(red square).

Design Notes

In the first step the beta-lactamase cds of pSB1AK3 was cloned in pSB1C3 containing the other parts of the device by gibson cloning. After verification by plating on ampicillin and chloramphenicol containing agar, one clone was picked and amplified. The plasmid DNA was mutated by using primers containing a mismatch leading to a single point mutation after the first amplification round and a 20 bp overlap similar to the other end of the linear DNA. The DNA has been closed by a one part gibson cloning and transformed to screen for a loss of function (Fig. 1). Single point mutation was verified by sanger sequening.


Source

Coming soon

References