IGEM:MIT/2007/Notebook/2007-6-26

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