IGEM:MIT/2007/Notebook/2007-6-26: Difference between revisions
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(New page: 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 s...) |
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*move passenger peptide before his tag and trypsin so that it will be displayed last | *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 | *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 |
Latest revision as of 14:03, 26 June 2007
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