Template:SBB12 part sbb1216

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In designing the junction between cI and the NheI site, you should make the spacer and frame match those of the ToxR constructs (see [[Media:sbb2012_homodimerParts.pdf | this illustration]]).
In designing the junction between cI and the NheI site, you should make the spacer and frame match those of the ToxR constructs (see [[Media:sbb2012_homodimerParts.pdf | this illustration]]).
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{{SBB12_TemplateDNAAvailable}}
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<blockquote style="background: white; border: 1px solid rgb(117, 174, 255); font: typewriter; padding: 2em;">
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{{SBB12_TemplateDNAAvailable}}
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<blockquote style="background: white; border: 1px solid rgb(117, 174, 255); font: typewriter; padding: 2em;"><tt>
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===Construction File===
===Construction File===
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tba
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<pre>
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</tt></blockquote>
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PCR aj1000F/aj1000R on pBca9145-jtk2768         (387 bp, A)
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PCR aj2000F/aj2000R on pBjk2741-jtk3346 (868 bp, B)
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PCR aj1000F/aj2000R on A+B (1231 bp, pcrpdt)
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Digest pcrpdt (EcoRI/BamHI, L, pcrdig)
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Digest pBca9525-Bca1839 (EcoRI/BamHI, 627+3494, plasdig)
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Ligate pcrdig and plasdig, product is  pBca9525-sbb1216 (3689bp)
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------------
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>aj1000F
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ccaaaGAATTCatgAGATCTgttaatgctagctcagtcct
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>aj1000R
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TCCACTGCCgctagcTCCTGACCCGCTTCCaaccgcttcatacatctcgt
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>aj2000F
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GGGTCAGGAgctagcGGCAGTGGATCTGGTgaaagtgaaacgtgatttca
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>aj2000R
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ttaGGATCCttattaggcgaccacaggtttgc
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</pre></blockquote>
__NOTOC__
__NOTOC__

Revision as of 21:47, 13 February 2012

Partname:     sbb1216
Featurename:  cI-N-AmilCP-B
Genename:     cI, AmilCP
Source:       Lambda phage / via the Registry

This one is a little different. You aren't putting a homodimer onto ToxR; you're building out a different homodimerization system. You're replacing ToxR with a different dimerization reporter, and then fusing it with amilCP, a very blue protein, which will facilitate future cloning. This one is the cI transcription repressor from lambda phage, which has been shown to do similar reporting of homodimerization as is reported for ToxR (see PMID 10411275). You'll need to carefully read this paper to determine how much of cI to include in your system -- you probably don't want 100% of the ORF for this part.

Your final construct should be a normal BglBricks part in plasmid pBca9525. You most likely will want to piece the part together from two pcr products using SOEing to get something like:

 EcoRI-BglII-Pcon.rbs.cI-spacer-NheI-spacer-PglpT.rbs.AmilCP!-BamHI

Note that this construct has 2 promoters and no terminator in between them. It's a little unusual design-wise, but usually works out ok. You can get the PglpT-AmilCP fragment from plasmid pBjk2741-jtk3346. The cI construct can be amplified from Media:pBca9145-jtk2768.str

In designing the junction between cI and the NheI site, you should make the spacer and frame match those of the ToxR constructs (see this illustration).

Genomic or plasmid DNA with this feature is available

Either genomic DNA or plasmid DNA is available for this sequence. Use your usual considerations of size and number of internal restriction sites to decide whether you should use that template for construction. You'll also need to think through which polymerase to use in making the construct.


Construction File

PCR aj1000F/aj1000R on pBca9145-jtk2768	        (387 bp, A)
PCR aj2000F/aj2000R on pBjk2741-jtk3346		(868 bp, B)
PCR aj1000F/aj2000R on A+B			(1231 bp, pcrpdt)
Digest pcrpdt				(EcoRI/BamHI, L, pcrdig)
Digest pBca9525-Bca1839			(EcoRI/BamHI, 627+3494, plasdig)
Ligate pcrdig and plasdig, product is  pBca9525-sbb1216 (3689bp)
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>aj1000F
ccaaaGAATTCatgAGATCTgttaatgctagctcagtcct
>aj1000R
TCCACTGCCgctagcTCCTGACCCGCTTCCaaccgcttcatacatctcgt
>aj2000F
GGGTCAGGAgctagcGGCAGTGGATCTGGTgaaagtgaaacgtgatttca
>aj2000R
ttaGGATCCttattaggcgaccacaggtttgc
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