20.20/Biocomputing/Parts

Tasks remaining:

• Add CLB1 promoter as a BioBrick
• Compose new BioBricks (aka stamp collecting)
  • Get protein coding regions
  • Add RBS & Term if not included
  • Reconcile degradation tags -- remove LVA on things that don't need it, add appropriate deg tags
  • Put nuclear localization tag on everything

IRES

• URE2 IRES
  • Somewhere in the area of nucleotides 98 through 309 on the URE2 mRNA in yeast.

Invertase Systems

• Invertase Hin
  • Protein generator for Hin
    • RBS
    • Hin coding region w/ LVA tag --
    • nuclear tag
    • LVA degradation tag already added
    • Terminator
  • Recognition sites hixC sites
• Invertase Cre
  • Protein generator for Cre
    • RBS
    • Cre coding region w/o LVA deg tag
    • nuclear tag
    • LVA degradation tag
    • Terminator
  • Recognition sites Lox sites for Cre

Inducible Promoters & Activators

• LuxR system
  • Activator protein generator
    • RBS
    • LuxR protein coding region -LVA
    • nuclear tag
    • slightly slower degradation tag AANDENYNYADAS tag rated "fast"
    • Terminator
  • LuxR inducible promoter
• ogr / PF
  • Activator protein generator
    • ogr activator protein coding region -LVA -- pag and delta should also work "to varying degrees" -- tune which one at experiment.
    • nuclear tag
    • slightly slower degradation tag AANDENYNYADAS tag rated "fast"
    • Terminator
  • PF promoter (ogr inducible) from phage

Repressible Promoters & Repressors

• LacI
  • Repressor protein generator
    • LacI protein generator w/ LVA deg tag
    • nuclear tag
    • Deg tag -- LVA already added
  • LacI repressible promoter

Repressors & Binding Sites

• Lambda cI system
  • Repressor protein generator
    • RBS
    • lambda cI protein coding region +LVA
    • nuclear tag
    • slightly faster degradation tag AANDENYALAA tag rated "very fast"
    • Remove LVA!
    • Terminator
  • Lambda cI repressible promoter to cut up
• Lambda cII system
  • Repressor protein generator
    • RBS
    • lambda cII protein coding region +LVA
    • nuclear tag
    • slightly faster degradation tag AANDENYALAA tag rated "very fast"
    • Remove LVA
  • Lambda cII repressible promoter to cut up
• TetR
  • Repressor protein generator
    • RBS
    • TetR protein coding region +LVA
    • nuclear tag
    • slightly faster degradation tag AANDENYALAA tag rated "very fast"
    • Remove LVA
    • Terminator
  • TetR binding site

Other

• Reporter: eCFP for yeast
  • RBS
  • eCFP protein coding region
  • Terminator
• Constitutively active promoter for reporter bit Arbitrarily picked one
• Cyclin clock
  • CLB1 promoter <== our featured part!
  • Sequence is here.

General

• Recipe for protein generator:
  • RBS This one
  • Protein coding region
  • Terminator(s) this one is in common use
• Nuclear localization tag, for yeast, from SV40
• Degradation tags: we're going to use any of the following three: AANDENYALAA "very fast", AANDENYNYADAS "fast", AANDENYADAS "moderately fast" -- because they're rated for relative speeds. Exactly which ones to use will be tuned at experiment -- specific protein-tag pairings given above are provisional. We can use bacterial deg tags because we're going to use that yeast strain that has the bacterial degradation system. In the case of invertases and the repressors that aren't in the pulsers, we're just going to use LVA because it's in common use and it has like ten family members whose relative speeds aren't well characterized (on BioBricks at least...).