Template:SBB12 part sbb1229: Difference between revisions
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Source: Lambda phage / Agrobacterium | Source: Lambda phage / Agrobacterium | ||
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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 ToxR, a homodimer, which will only homodimerize in response to a homoserine lactone compound. 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 | 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 ToxR, a homodimer, which will only homodimerize in response to a homoserine lactone compound. 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: | 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: | ||
Revision as of 20:43, 6 February 2012
Partname: sbb1229 Featurename: cI-TraR-B Genename: cI, traR Source: Lambda phage / Agrobacterium
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 ToxR, a homodimer, which will only homodimerize in response to a homoserine lactone compound. 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-traR!-BamHI
The cI construct can be amplified from pBca9145-jtk2768. The traR sequence is available from pBca9525-bgl1031.
In designing the junction between cI and TraR, you should make the spacer and frame match those of the similar construct described in PMID: 12569101. We want to encode the same protein as they describe for the cI'/traR(wt) fusion.
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.
