Composite

Part:BBa_K823040:Design

Designed by: Julia Bartels   Group: iGEM12_LMU-Munich   (2012-09-10)
Revision as of 08:00, 26 September 2012 by JuliaB (Talk | contribs) (→‎Design Notes)

Inverter: pBad-RyhB-pLac(R0011)-uof-lacZalpha


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
    Illegal BamHI site found at 495
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Fusion of the small RNA directly to promotor to ensure exact +1 site and length. Translational fusion of uof to lacZalpha. These had to be done by fusionPCRs


1. Primer design:

  • Promoter (which you would like to invert): BB-suffix + Promoter primer forward (a), GTCTTCCTGATCGCGAGACA + Promoter reverse primer from -10 (b)
  • RyhB: TGTCTCGCGATCAGGAAGAC (RyhB fwd) (c), BB-suffix + AAAGCCAGCACCCGGCTGGCTAAG (RyhB rev) (d)
  • uof: BB-prefix + GTGGTTTTCATTTAGGCGTG (Uof fwd) (e), Output/Reporter forward primer + GTTATCAGTCATGCGGAATC (uof rev) (f)
  • Output/Reporter: Forward primer without BB-prefix (g), Reverse primer with BB-suffix (h)

2. PCRs and Fusion PCRs:

  • basic PCRs: Promoter (a+b), RyhB (c+d), uof (e+f), Output/Reporter (g+h)
  • Fusion PCRs: ~ 200 ng equimolar with forward primer of the front and reverse primer of the back fusion part: to be inversed promoter to RyhB (a+d) and uof to Output/Reporter (e+h)

3. Bring these into BioBrick vectors to facilitate 3a assemblies

4. 3a assemblies

  • 3a assembly of Output/Reporter with a constitutive/inducible promoter like BBa_R0011
  • 3a assembly promoter + RyhB with product of step above

Source

E.coli, Plasmids

References