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hisGFPmek and PDZ domain

hisGFPmek and PDZ domain. His6 tag (purification). G H H H H H H G. Mek2 tag (PDZ binding). PDZ domain. E. coli. Q P G T P T R T A V. hisGFPmek protein. LppOmpA. *not to scale, at all. Initial cell binding assay. Ex 488, Em 507 (published for EGFP clone).

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hisGFPmek and PDZ domain

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  1. hisGFPmek and PDZ domain His6 tag (purification) G H H H H H H G Mek2 tag (PDZ binding) PDZ domain E. coli Q P G T P T R T A V hisGFPmek protein LppOmpA *not to scale, at all

  2. Initial cell binding assay Ex 488, Em 507 (published for EGFP clone)

  3. In vitrohisGFPmek bindingassay with GST-PDZ fusion protein Q P G T P T R T A V --GSH hisGFPmek Glutathione bead GST (glutathione S-transferase) PDZ

  4. In vitro hisGFPmek assay Ex 485, Em 538 (quorum-sensing group protocol)

  5. In vitro hisGFPmek assay Ex 488, Em 507 (published for EGFP clone)

  6. Rebuilding the Voigt construct Voigt et al., 2006

  7. Alternative PCR/digest assembly • Amplification by PCR, digestion/ligation of PCR’d fragments • Pros • Faster, more processive protocol • No overnight liquid cultures or transformations (until the end) • No gel isolation, just PCR purification • No colony PCR • PCR is better amplifier than bacteria • Can reconstitute unavailable subparts • Cons • Can’t save intermediate constructs (except by cloning PCR products into Topo vector) • Requires primer design and synthesis • Problems with identical/homologous BioBrick parts

  8. Alternative PCR/digest assembly BB_F E X S P VR BBa_T9002 BBa_J23039 E X S P E X S P VR BBa_T9002 E X S P VR

  9. BB_R as well as BB_F E E X S P VR BB_R X S P

  10. Plans • Explore AIDA construct • AIDA-streptavidin • AIDA-PDZ • Make a new hisGFPmek? • Longer Mek tag, or longer linker • Finish the Voigt construct by PCR/digest • Forever abandon traditional BioBricks protocol?

  11. Alternative PCR/digest assembly E X S P VR BBa_T9002 E X S P VR

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