Template:SBB12Project stdt1216

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Melissa Milder

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:     sbb1232
Description:  ToxR-CI'
Genename:     cI
Source:       Lambda phage

We already have a construct with ToxR-linker-cI(wt) in it, pBca9525-Bjc0005. You are going to construct the truncation mutant of this construct. You want the version of CI in which just the N-terminal DNA binding domain remains. This mutation is described in PMID 10411275.

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.

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

PCR ca998/osbb1232R on pBca9525-Bjc0005	  (1586bp, pcrpdt)
Digest pcrpdt                     	  (NheI/BamHI, 1083+492+11, M, pcrdig)
Digest pBca9525-Bca1834                   (NheI/BamHI, L, vectdig)
Ligate pcrdig and vectdig, product is pBca9525-sbb1232
----
>ca998	Forward external annealing for purification of P_R part
gtatcacgaggcagaatttcag
>osbb1232R
CTGTAggatccGCTTGGCTTGGAGCCTGTTGG
>pcrpdt
GTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCATGAGATCTCTCATTAATAAACTGGCAATGACCAAGACCAATGACGATTTCTTCGAAATGATGAAACGCTCATAAATTTGTCTTATGCCAAAAACGCCACGTGTTTACGTGGCGTTTTGCTTTTATATCTGTAATCTTAATGCCGCGCTGGCGATGTTAGGAAAATTCCTGGAATTTGCTGGCATGTTATGCAATTTGCATATCAAATGGTTAATTTTTGCACAGGACTGGTGGGTTTGGAACGGACTTTCCCTTCTGAATAAAGGTCTTCGTGGTTATACTTCTGCTAATAATTTTCTCTGAGAGCATGCATTGTGAATTTACTGACAGTGAGTACTGATCTCATCAGGGATCTGCCACGCACAAAACTACGGTAGCAAATGTTCGGATTAGGACACAACTCAAAAGAAATCTCGATGAGTCATATTGGTACTAAATTCATTCTTGCTGAAAAATTTACCTTCGATCCCCTAAGCAATACTCTGATTGACAAAGAAGATAGTGAAGAGATCATTCGATTAGGCAGCAACGAAAGCCGTATCCTTTGGCTGCTGGCCCAACGTCCAAACGAGGTAATTTCTCGCAATGATTTGCATGACTTTGTTTGGCGAGAGCAAGGTTTTGAAGTCGATGATTCCAGCTTAACCCAAGCCATTTCGACTCTGCGCAAAATGCTCAAAGATTCGACAAAGTCCCCACAATACGTCAAAACGGTTCCGAAGCGCGGTTACCAATTGATCGCCCGAGTGGAAACGGTTGAAGAAGAGATGGCTCGCGAAAACGAAGCTGCTCATGACATCTCTCAGCCGGAGTCCGTCAATGAATACGCAGAATCAAGCAGTGTGCCTTCATCTGCTACCGTAGTGAACACACCGCAGCCAGCCAATGTCGTGGCGAATAAATCGGCTCCAAACTTGGGGAATCGACTGTTTATTCTGATAGCGGTCTTACTTCCCCTCGCAGTATTACTGCTCACTAACCCAAGCCAATCCAGCTTTAAACCCCTAACGGGATCTGCTAGCGGAAGCGGATCGACCAAGAAAAAGCCATTAACACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAAATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTGAGCTTAGAACCTTTACCAAAGGTGATGCGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAAGCCAAGCGGATCCTACAG

Partname:     sbb1215
Featurename:  lz_AAAA-4
Genename:     leucine zipper variant
Source:       Synthetic, see PMID:12459719 

Encode a mutant of the AAAA leucine zipper with the terminal cysteine truncated off. VKEAEDKAEEALSAAYHAANEVARLKKAAGERGG*

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 o16,o17,o18       (pca1)
PCA2 with o16/o12 on pca1 (142 bp, pca2)
Digest pca2               (NheI/BamHI, L, 1215dig)
Digest pBca9525-Bca1834   (NheI/BamHI, L, vectdig)
Ligate 1215dig + vectdig, product is pBca9525-sbb1215
----
>o16	
CCATAgctagcGGCAGTGGATCTGTTAAAGAAGCGGAAGACAAAGCAGAAGAAGCGCTGAGT
>o17
CAAAGCAGAAGAAGCGCTGAGTATCATCTACCACCTGAAAAACGAAGTTGCTCGTCTGA
>o18
cagtaGGATCCTTAGCAGCCGCCACGTTCGCCCGCTGCTTTTTTCAGACGAGCAACTTCGTT
>pca2
CCATAgctagcGGCAGTGGATCTGTTAAAGAAGCGGAAGACAAAGCAGAAGAAGCGCTGAGTATCATCTACCACCTGAAAAACGAAGTTGCTCGTCTGAAAAAAGCAGCGGGCGAACGTGGCGGCTGCTAAGGATCCtactg
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