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

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