Template:SBB12Project stdt1204
Welcome to your project page!
I've given you two parts to make.
- Design oligos to make your part
- Write up a proper construction file on the wiki template associated with your part
- Enter your Features (REMEMBER: It's not a bug, it's a feature. We just don't know what it does yet), Oligos, Parts, and Plasmids into Clotho
Also, use the link above left to upload a picture of yourself, your name, and anything else you'd like, and rename the link from "Your Name Here" to your name.
Finally, you should create a notebook on the on the main class page of the wiki
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 File
PCA1 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
Partname: sbb1204 Featurename: lz_EILK Genename: leucine zipper variant Source: Synthetic, see PMID:12459719
This part encodes a leucine zipper
We will be using a set of leucine zippers as homodimer domains. In total, we'll have 8 of them all with different Kd's for homodimerization. However, the sequences only differ at the same 4 amino acid sites. This will allow us to test whether the activation of ToxR is dependent on the Kd of the homodimer that is attached to it. These sequences are entirely synthetic, but all should encode the following peptide:
VKELEDKNEELLS XX YH XX NEVARLKKLVGERGGC*
Where the x's are the amino acids I tell you. So, if I gave you the lz_IILK zipper to make, the peptide you should encode is: VKELEDKNEELLSIIYHLKNEVARLKKLVGERGGC*
Here is an example of what your trying to make pBca9525-sbb1230.
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 File
PCA1 on o1,o4,o2 (pca1) PCA2 with o1/o2 on pca1 (142 bp, pca2) Digest pca2 (NheI/BamHI, L, 1204dig) Digest pBca9525-Bca1834 (NheI/BamHI, L, vectdig) Ligate 1204dig + vectdig, product is pBca9525-sbb1204 ---- >o1 CCATAgctagcGGCAGTGGATCTGTTAAAGAACTGGAAGACAAAAACGAAGAACTGCTGAGT >o2 CAGTAGGATCCTTAGCAGCCGCCACGTTCGCCAACCAGTTTTTTCAGACGAGCAACTTCGTT >o4 CAAAAACGAAGAACTGCTGAGTGAGATCTACCACCTGAAAAACGAAGTTGCTCGTCTGA >pca2 CCATAgctagcGGCAGTGGATCTGTTAAAGAACTGGAAGACAAAAACGAAGAACTGCTGAGTGAGATCTACCACCTGAAAAACGAAGTTGCTCGTCTGAAAAAACTGGTTGGCGAACGTGGCGGCTGCTAAGGATCCTACTG
