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