Part:BBa_K823040:Design

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

pBAD: BBa_I0500 PCR fragment RyhB: pURyhB [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852835/ Vecerek B., Bläsi U., 2007] uof: pRuof_CGU-lacZ [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852835/ Vecerek B., Bläsi U., 2007] lacZalpha: pRuof_CGU-lacZ [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852835/ Vecerek B., Bläsi U., 2007]

References

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852835/ Vecerek B., Bläsi U., 2007]