Difference between revisions of "Part:BBa K823040:Design"

 
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<partinfo>BBa_K823040 short</partinfo>
 
<partinfo>BBa_K823040 short</partinfo>
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1. Primer design:
 +
 +
* Promoter (which you would like to invert): BB-suffix + Promoter primer forward (a), GTCTTCCTGATCGCGAGACA +  Promoter reverse primer from -10 (b)
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* RyhB: TGTCTCGCGATCAGGAAGAC (RyhB fwd) (c), BB-suffix + AAAGCCAGCACCCGGCTGGCTAAG (RyhB rev) (d)
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* uof:  BB-prefix + GTGGTTTTCATTTAGGCGTG (Uof fwd) (e), Output/Reporter forward primer + GTTATCAGTCATGCGGAATC (uof rev) (f)
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* Output/Reporter: Forward primer without BB-prefix (g), Reverse primer with BB-suffix (h)
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2. PCRs and Fusion PCRs:
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* basic PCRs: Promoter (a+b), RyhB (c+d), uof (e+f), Output/Reporter (g+h)
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* 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)
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3. Bring these into BioBrick vectors to facilitate 3a assemblies
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4. 3a assemblies
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* 3a assembly of Output/Reporter with a constitutive/inducible promoter like [https://parts.igem.org/Part:BBa_R0011 BBa_R0011]
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* 3a assembly promoter + RyhB with product of step above
  
 
===Source===
 
===Source===
  
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E.coli, Plasmids
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pBAD: [https://parts.igem.org/Part:BBa_I0500 BBa_I0500] PCR fragment
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RyhB: pURyhB [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852835/ Vecerek B., Bläsi U., 2007]
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uof: pRuof<sub>CGU</sub>-lacZ [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852835/ Vecerek B., Bläsi U., 2007]
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lacZalpha: pRuof<sub>CGU</sub>-lacZ [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852835/ Vecerek B., Bläsi U., 2007]
  
 
===References===
 
===References===
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 +
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852835/ Vecerek B., Bläsi U., 2007]

Latest revision as of 08:07, 26 September 2012

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

lacZalpha: pRuofCGU-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]