Template:SBB12 part sbb1218: Difference between revisions
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{{SBB12_TemplateDNAAvailable}} | {{SBB12_TemplateDNAAvailable}} | ||
<pre> | |||
PCR ca998F/mh0501R on pBca9145-jtk2768 (643 bp, cIpcrpdt) | |||
PCR mh0501F/mh0502R on pBca9525-Bca1835 (548 bp, oBPpcrpdt) | |||
---------------------------------------------------------------------------------------- | |||
PCR ca998F/mh0502R on cIpcrpdt+oBPpcrpdt (1145 bp, EcoRI/BamHI, pcrdig) | |||
Digest PBca9525-Bca1835 (EcoRI/BamHI, 2411+1520+61, L, vectdig) | |||
Ligate pcrdig and vectdig (PBca9525-Bmh0001) | |||
---------------------------------------------------------------------------------------- | |||
>ca998F Forward cloning of cI construct | |||
Gtatcacgaggcagaatttcag | |||
>mh0502R Reverse cloning of oBP reading frame | |||
cgatcgttaGGATCCttaTTCCGGGTGCGGGTGGTTTTC | |||
>mh0501F Forward removal of HindIII site | |||
caacaggctccaagccaagCtttGGgAGcGGATCagctagcGGCAG | |||
>mh0501R Reverse removal of HindIII site | |||
CTGCCgctagctGATCCgCTcCCaaaGcttggcttggagcctgttg | |||
>PBca9525-Bmh0001 | |||
gtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctggaattcatgAGATCTGTTAATGCTAGCTCAGTCCTAGGGACTCTGCTAGCGGATCTAGACATAAAAACGGCAAAGTatgagcacaaaaaagaaaccattaacacaagagcagcttgaggacgcacgtcgccttaaagcaatttatgaaaaaaagaaaaatgaacttggcttatcccaggaatctgtcgcagacaagatggggatggggcagtcaggcgttggtgctttatttaatggcatcaatgcattaaatgcttataacgccgcattgcttgcaaaaattctcaaagttagcgttgaagaatttagcccttcaatcgccagagaaatctacgagatgtatgaagcggttagtatgcagccgtcacttagaagtgagtatgagtaccctgttttttctcatgttcaggcagggatgttctcacctgagcttagaacctttaccaaaggtgatgcggagagatgggtaagcacaaccaaaaaagccagtgattctgcattctggcttgaggttgaaggtaattccatgaccgcaccaacaggctccaagccaagCtttGGgAGcGGATCagctagcGGCAGTGGATCTGCTCAGGAAGAAGAAGCTGAACAGAACCTGTCTGAACTGTCTGGTCCGTGGCGTACCGTTTACATCGGTTCTACCAACCCGGAAAAAATCCAGGAAAACGGTCCGTTCCGTACCTACTTCCGTGAACTGGTTTTCGACGACGAAAAAGGTACCGTTGACTTCTACTTCTCTGTTAAACGTGACGGTAAATGGAAAAACGTTCACGTTAAAGCTACCAAACAGGACGACGGTACCTACGTTGCTGACTACGAAGGTCAGAACGTTTTCAAAATCGTTTCTCTGTCTCGTACCCACCTGGTTGCTCACAACATCAACGTTGACAAACACGGTCAGACCACCGAACTGACCGAACTGTTCGTTAAACTGAACGTTGAAGACGAAGACCTGGAAAAATTCTGGAAACTGACCGAAGACAAAGGTATCGACAAAAAAAACGTTGTTAACTTCCTGGAAAACGAAAACCACCCGCACCCGGAAtaaGGATCCtaaCTCGAcgtgcaggcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatCAATTCGACCCAGCTTTCTTGTACAAAGTTGGCATTATAAAAAATAATTGCTCATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATTTGCCATCCAGCTGATATCCCCTATAGTGAGTCGTATTACATGGTCATAGCTGTTTCCTGGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGCCTCATCTAGACCAGCCAGGACAGAAATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAGTGGACATAAGCCTGTTCGGTTCGTAAGCTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAACCCTCGAGCCACCCATGACCAAAATCCCTTAACGTGAGTTACGCGTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTcctaggTGTAAAACGACGGCCAGTCTTAAGCTCGGGCCCCAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAATTGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCAGGCTCCGAATTG | |||
</pre> |
Revision as of 18:13, 12 February 2012
Partname: sbb1218 Featurename: cI-N-bOBP-B Genename: cI, bOBP Source: Lambda phage / cow
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 bOBP, a homodimer, which hopefully will do something in response to a chemical ligand. 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-bOBP!-BamHI
The cI construct can be amplified from pBca9145-jtk2768. The NheI-spacer-bOBP!-BamHI cassette is available from pBca9525-Bca1835.
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.
PCR ca998F/mh0501R on pBca9145-jtk2768 (643 bp, cIpcrpdt) PCR mh0501F/mh0502R on pBca9525-Bca1835 (548 bp, oBPpcrpdt) ---------------------------------------------------------------------------------------- PCR ca998F/mh0502R on cIpcrpdt+oBPpcrpdt (1145 bp, EcoRI/BamHI, pcrdig) Digest PBca9525-Bca1835 (EcoRI/BamHI, 2411+1520+61, L, vectdig) Ligate pcrdig and vectdig (PBca9525-Bmh0001) ---------------------------------------------------------------------------------------- >ca998F Forward cloning of cI construct Gtatcacgaggcagaatttcag >mh0502R Reverse cloning of oBP reading frame cgatcgttaGGATCCttaTTCCGGGTGCGGGTGGTTTTC >mh0501F Forward removal of HindIII site caacaggctccaagccaagCtttGGgAGcGGATCagctagcGGCAG >mh0501R Reverse removal of HindIII site CTGCCgctagctGATCCgCTcCCaaaGcttggcttggagcctgttg >PBca9525-Bmh0001 gtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctggaattcatgAGATCTGTTAATGCTAGCTCAGTCCTAGGGACTCTGCTAGCGGATCTAGACATAAAAACGGCAAAGTatgagcacaaaaaagaaaccattaacacaagagcagcttgaggacgcacgtcgccttaaagcaatttatgaaaaaaagaaaaatgaacttggcttatcccaggaatctgtcgcagacaagatggggatggggcagtcaggcgttggtgctttatttaatggcatcaatgcattaaatgcttataacgccgcattgcttgcaaaaattctcaaagttagcgttgaagaatttagcccttcaatcgccagagaaatctacgagatgtatgaagcggttagtatgcagccgtcacttagaagtgagtatgagtaccctgttttttctcatgttcaggcagggatgttctcacctgagcttagaacctttaccaaaggtgatgcggagagatgggtaagcacaaccaaaaaagccagtgattctgcattctggcttgaggttgaaggtaattccatgaccgcaccaacaggctccaagccaagCtttGGgAGcGGATCagctagcGGCAGTGGATCTGCTCAGGAAGAAGAAGCTGAACAGAACCTGTCTGAACTGTCTGGTCCGTGGCGTACCGTTTACATCGGTTCTACCAACCCGGAAAAAATCCAGGAAAACGGTCCGTTCCGTACCTACTTCCGTGAACTGGTTTTCGACGACGAAAAAGGTACCGTTGACTTCTACTTCTCTGTTAAACGTGACGGTAAATGGAAAAACGTTCACGTTAAAGCTACCAAACAGGACGACGGTACCTACGTTGCTGACTACGAAGGTCAGAACGTTTTCAAAATCGTTTCTCTGTCTCGTACCCACCTGGTTGCTCACAACATCAACGTTGACAAACACGGTCAGACCACCGAACTGACCGAACTGTTCGTTAAACTGAACGTTGAAGACGAAGACCTGGAAAAATTCTGGAAACTGACCGAAGACAAAGGTATCGACAAAAAAAACGTTGTTAACTTCCTGGAAAACGAAAACCACCCGCACCCGGAAtaaGGATCCtaaCTCGAcgtgcaggcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatCAATTCGACCCAGCTTTCTTGTACAAAGTTGGCATTATAAAAAATAATTGCTCATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATTTGCCATCCAGCTGATATCCCCTATAGTGAGTCGTATTACATGGTCATAGCTGTTTCCTGGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGCCTCATCTAGACCAGCCAGGACAGAAATGCCTCGACTTCGCTGCTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAGTGGACATAAGCCTGTTCGGTTCGTAAGCTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAACCCTCGAGCCACCCATGACCAAAATCCCTTAACGTGAGTTACGCGTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTcctaggTGTAAAACGACGGCCAGTCTTAAGCTCGGGCCCCAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAATTGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCAGGCTCCGAATTG