Template:SBB12 part sbb1223
Partname: sbb1223 Featurename: caff_VHH Genename: n/a Source: synthetic
You will need to synthesize this one using PCA with full E. coli codon usage. What it encodes is a VHH domain of an evolved Camelid antibody. The family of animals including llamas make unusual single-heavy-chain antibodies. When just the business portion of the molecule is employed, it's called a VHH domain. This particular one has been shown to dimerize in response to caffeine (PMID 19572722).
You need to read the paper, its supporting information, and the one it references (PMID 16808459) to figure out the exact peptide sequence you want. This may be easier said than done. If you get stumped, let me know. I do have more sequence info about what we want, but you should try first to figure it out.
This part encodes a homodimer domain
You are going to put the homodimer on the C-terminus of a truncated ToxR. For this part, you're going to go off BioBricks. Kindov. Your final product will be a BioBrick, but you are going to use an ad hoc procedure to create it. You should examine this illustration which refers to two sequences: pBca9525-Bca1834 and pBca9525-Bca1839. The important thing here is that you think through the translation frame of the final product, and make sure that you add some linker between your sequence and the sequence 5' to it. You should also remove the start methionine from you homodimer, unless your project description explicitly says not to. You should include the stop codon.
The product of your construction file should resemble pBca9525-Bca1839 in terms of it encoding a full expression cassette containing a ToxR fusion protein with your designed feature. If you were given part sbb1293, your product would be called pBca9525-sbb1293.
Construction FilePCA1 on caff.01.o01-caff.01.o14 (pca1) PCA2 with caff.01.o01/caff.01.o14 on pca1 (394 bp, pca2) Digest pca2 (NheI/BamHI, L, 1223dig) Digest pBca9525-Bca1834 (NheI/BamHI, L, vectdig) Ligate 1223dig + vectdig, product is pBca9525-sbb1223 ---- >caff.01.o01 CCATAGCTAGCGGCAGTGGATCTCAAGTACAACTGGTAGAAAGCGGTGGTG >caff.01.o02 GGCTACCACCTGCTTGTACCAGACCACCACCGCTTTCTACCAGTTGTA >caff.01.o03 TGGTACAAGCAGGTGGTAGCCTGCGTCTGAGCTGCACCGCTTCCGG >caff.01.o04 ACCAAGCCATACTGTAGATGGTTCCTGTACGACCGGAAGCGGTGCAGCTCA >caff.01.o05 GAACCATCTACAGTATGGCTTGGTTCCGTCAAGCACCAGGTAAAGAACGTGA >caff.01.o06 GACCAACCAACGGTAGCCAGGAACTCACGTTCTTTACCTGGTGCTTGA >caff.01.o07 CTGGCTACCGTTGGTTGGTCCTCCGGTATCACCTACTACATGGACAGCGTAA >caff.01.o08 GTCACGGCTGATGGTGAAACGACCTTTTACGCTGTCCATGTAGTAGGTGA >caff.01.o09 TCGTTTCACCATCAGCCGTGACAAAGGTAAAAACACCGTATACCTCCAGATGG >caff.01.o10 CGGTGTCTTCTGGTTTCAGGCTGTCCATCTGGAGGTATACGGTGTTTTT >caff.01.o11 GCCTGAAACCAGAAGACACCGCAGTTTACTACTGCACCGCTACCCGT >caff.01.o12 GACCCCAGTAGTCGTAACCAACGGAGTAAGCACGGGTAGCGGTGCAGTAG >caff.01.o13 GTTGGTTACGACTACTGGGGTCAAGGCACCCAAGTAACCGTAAGCAGCTAAG >caff.01.o14 CTGGAGGATCCTTAGCTGCTTACGGTTACTTGGG >pca2 CCATAGCTAGCGGCAGTGGATCTCAAGTACAACTGGTAGAAAGCGGTGGTGGTCTGGTACAAGCAGGTGGTAGCCTGCGTCTGAGCTGCACCGCTTCCGGTCGTACAGGAACCATCTACAGTATGGCTTGGTTCCGTCAAGCACCAGGTAAAGAACGTGAGTTCCTGGCTACCGTTGGTTGGTCCTCCGGTATCACCTACTACATGGACAGCGTAAAAGGTCGTTTCACCATCAGCCGTGACAAAGGTAAAAACACCGTATACCTCCAGATGGACAGCCTGAAACCAGAAGACACCGCAGTTTACTACTGCACCGCTACCCGTGCTTACTCCGTTGGTTACGACTACTGGGGTCAAGGCACCCAAGTAACCGTAAGCAGCTAAGGATCCTCCAG