Template:SBB12Project stdt1209
Your name here
Your part uses pBgl00001-Bjk2828 as its vector.... You need a special antibiotic.
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: sbb1220 Description: P_R-CmR Genename: P_R, CmR Source: Lambda phage / synthetic
You are going to make a CmR reporter of the P_R promoter from lambda phage. This promoter shuts down when bound to CI protein. CmR confers chloramphenicol resistance when expressed, and it can also be assayed biochemically. The sequence of the P_R part, which is present in pBjk2741-jtk2801 is:
GATCTtaaatctatcaccgcaagggataaatatctaacaccgtgcgtgttgactattttacctctggcggtgataatggttgcG
You can get the rbs.CmR part from plasmid pKGC438. What you want to make is a BglBrick part that would result from joining the P_R part with the rbs.CmR part. In designing this, you'll need to make arbitrary decisions about where to 'cut' the non-BioBrick pKGC438 sequence. I would recommend starting from the CCAC... after the T7 promoter and then ending after the stop codon. Though the final construct should be a BglBrick part, you'll be making the construct by SOEing. In designing your construction file for this, note that the P_R part is really short, and things will go better if you use oligo ca998 as the forward external oligo instead of some annealing region that binds within the part. Ask JCA if that doesn't make sense.
The vector for your final product should be pBgl00001, and you can use plasmid pBgl00001-Bjk2828. Note that this plasmid has a p15A origin of replication, and as such is much lower copy than most other parts you'll work with. As a result, you won't get much material in your minipreps, and you should adjust things accordingly.
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 ca998/gfRevPR on pBjk2741-jtk2801 (160bp, A) PCR gfForRbsCmR/gfRevRbsCmR on pKGC438 (920bp, B) PCR ca998/gfRevRbsCmR on A+B (1059bp, pcrpdt) Digest pcrpdt (EcoRI/BamHI, L, pcrdig) Digest pBgl00001-Bjk2828 (EcoRI/BamHI, L, plasdig) Ligate pcrdig and plasdig, product is pBgl00001-sbb1220 ---- >ca998 Forward external annealing for purification of P_R part gtatcacgaggcagaatttcag >gfForRbsCmR ggtgataatggttgcGGATCTCCACAACGGTTTCCCTC >gfRevRbsCmR gctagGGATCCTTACGCCCCGCCCTGCCAC >gfRevPR AGATCCgcaaccattatcacc >pcrpdt GTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATCTGAAGTGGAATTCATGAGATCTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGCGGATCTCCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGAGCGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACGATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAGGATCCCTAGC
Partname: sbb1210 Featurename: lz_IILK-1 Genename: leucine zipper variant Source: Synthetic, see PMID:12459719
Encode the IILK leucine zipper, but with the terminal cysteine truncated off. VKELEDKNEELLSIIYHLKNEVARLKKLVGERGG*
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,o10,o12 (pca1) PCA2 with o1/o12 on pca1 (139 bp, pca2) Digest pca2 (NheI/BamHI, L, 1210dig) Digest pBca9525-Bca1834 (NheI/BamHI, L, vectdig) Ligate 1210dig + vectdig, product is pBca9525-sbb1210 ---- >o1 CCATAgctagcGGCAGTGGATCTGTTAAAGAACTGGAAGACAAAAACGAAGAACTGCTGAGT >o10 CAAAAACGAAGAACTGCTGAGTATCATCTACCACCTGAAAAACGAAGTTGCTCGTCTGA >o12 CAGTAGGATCCTTAGCCGCCACGTTCGCCAACCAGTTTTTTCAGACGAGCAACTTCGTT >pca2 CCATAgctagcGGCAGTGGATCTGTTAAAGAACTGGAAGACAAAAACGAAGAACTGCTGAGTATCATCTACCACCTGAAAAACGAAGTTGCTCGTCTGAAAAAACTGGTTGGCGAACGTGGCGGCTAAGGATCCTACTG
