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Morning: Edit CPX insert(w/ Brian)

  • move passenger peptide before his tag and trypsin so that it will be displayed last
  • get rid of HindIII and SalI restriction sites and redesign insert so pp/his/tryp mini insert can be switched using SfiI embedded in linkers

Afternoon Meeting Notes

Editing our insertion sequences

Restriction sites

  • Our insert design used Sfi, HindIII and SalI
  • Switched the passenger order on CPX: His6, Tryp, PP-> PP, His6, Tryp
  • The BioBricks assembly restriction sites are: EcoRI, XbaI, SpeI, PstI
    • SpeI and XbaI have matching overhangs (but when you ligate them together, the restriction site is destroyed)
  • Getting a grad from Rice lab to give us eCPX instead of the PI
    • try calling Jeffrey Rice
  • Repeating PP sequence, does that help? Yes, you get better affinity (put a really small glycine/alganine in between)
  • New insert: EcoRI SS Sfi HindIII Peptide SalI Epitope(T7) SfiI CPX Double-stop XbaI
    • T7 is a peptide displayed by T7 phage, we will use its matching antibody to basically stain show that peptide has been displayed
  • What order do we want EcoRI and XbaI? Biobricks has EcoRI first
  • Reduce codon repetition (don't want to deplete tRNA pool)
  • How are we going to clip OmpC?

Other Stuff

  • SP3k3, biobrick part has same origin of replication as plasmid in Rice paper (low copy)
  • GeneArc will run the sequence you send through optimization programs
    • if we e-mail, she will insert restriction sites where you want
    • Send protein you want sequenced (in AA), highlight areas where you want to specify exact sequence of DNA (eg the restriction sites)
  • Checked Lpp-OmpA display protein biobrick part from Harvard, junk.
  • In the meantime while we wait for our inserts to be ordred, work on:
    • vector, RBS (-10 -35), metal promoters, team name
    • Check out vector in Rice paper (pBAD33, has arabinose). We want one with medium copy, p15A origin, arabinose inducible promoter
    • email biostuff@mit.edu (if you need something)
  • BioBricks has plasmids
  • BioBrick has a prefix and suffix standard