Difference between revisions of "Part:BBa K823040:Design"
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1. Primer design: | 1. Primer design: | ||
| − | * Promoter (which you would like to invert): BB-suffix + Promoter primer forward (a), GTCTTCCTGATCGCGAGACA + Promoter reverse primer (b) | + | * 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) | * 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) | * uof: BB-prefix + GTGGTTTTCATTTAGGCGTG (Uof fwd) (e), Output/Reporter forward primer + GTTATCAGTCATGCGGAATC (uof rev) (f) | ||
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* 3a assembly of Output/Reporter with a constitutive/inducible promoter like [https://parts.igem.org/Part:BBa_R0011 BBa_R0011] | * 3a assembly of Output/Reporter with a constitutive/inducible promoter like [https://parts.igem.org/Part:BBa_R0011 BBa_R0011] | ||
| − | * 3a assembly promoter + RyhB with product of step above | + | * 3a assembly promoter + RyhB with product of step above |
| − | + | ||
| − | + | ||
===Source=== | ===Source=== | ||
Revision as of 08:00, 26 September 2012
Inverter: pBad-RyhB-pLac(R0011)-uof-lacZalpha
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
Illegal BamHI site found at 495 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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