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Amy Kristofferson


Welcome to your project page!

For each part listed, you should:

  • Design oligos to make your part
  • Write up proper construction files and put it on the Construction Files page
  • Put your oligos on the Oligo Log page
  • Put your parts into Clotho

You should design your construction strategy to put your part into plasmid vectorName-Bca1144 (Where vectorName is indicated for each part) using EcoRI and BamHI or just BamHI or BglII for EIPCR. The maps for all plasmids involved in our project can be downloaded here.

Several references have been provided to give you some background on the biology of your part.

Also, use the link above left to upload a picture of yourself, your name, and anything else you'd like.

Finally, you should create a notebook on the main page of the wiki

sbb22: FokI Cleavage Domain (fok-)

 Source:  Registry part BBa_K243001  in vector BBa_K157000
 Target Sequence:  CAGCTGGTTaaatctgaactggaggagaaaaaatccgagctgcgccacaaactgaaatatgtgcctcacgagtatatcgaactgatcgagatcgcccgtaatagtacccaagaccgtatcctggaaatgaaagtgatggagttcttcatgaaagtctatggctatcgtggcaaacatctgggtggtagccgtaaaccagACggtgccatttataccgttggttccccgatcgattatggcgttatcgtggAcacaaaagcgtattctggcggttataatctgccgattggtcaggctgatgagatggaacgttatgtggaagagaatcagacccgtaacaaacatctgaacccgaacgaatggtggaaagtgtatccgtcaagtgtcaccgagttcaaatttctgttcgtgagcggccactttaaaggcaactataaagcccagctgactcgtctgaaccatatcaccaatTgcaatggggcagtgctgagtgttgaggaactgctgatcggtggagaaatgatcaaagcaggcaccctgactctggaagaagttcgccgtaaattcaacaatggcgagatcaatttt
 Vector:  pBjk2741
 Short description: <FokI-> 
 Flavor:  {<part>} 
 Family:  Restriction Endonuclease

Your part exists in the Registry of standard biological parts, but this template sequence differs from what you want by 3 mutations and 3 extra AA at N-term. Take careful note in designing your oligos that you generate the correct product.

This part encodes a DNA modifying enzyme

Different DNA modification enzymes are needed in different flavors, so take special note of what type this one should be. DNA modification enzymes are often toxic to E. coli, so you may have some difficulty making this part. Make careful note in your notebook about the size distribution of colonies you get on your plates and the ratio of red to white colonies.

This part is associated with Zinc Finger Nuclease devices

A zinc finger nuclease is an engineered protein derived from two zinc finger DNA binding proteins and cutting domains usually from a type IIS restriciton enzyme. The special thing about them is that 1) they bind long sequences of DNA, long enough that it might be unique in a eukaryotic genome, and 2) zinc finger proteins that bind to most sequences can be generated somewhat easily. So, you can use a zinc finger nuclease to introduce a ds break at a specific user-defined site in a eukaryotic genome if the protein is delivered to the nucleus of the cell. Eukaryotic ds breaks stimulate homologous recombination. So, if you deliver both the ZFN and a DNA homologous to the target site, you get insertion of your DNA into that site of the genome.

You should read the following papers
References: PMID 18545224, PMID 17603475, PMID 11821858

sbb36: P9 origin of replication

 Source:  pBca9145-jtk2642
 Vector:  pBca9523
 Short description: {ColE2 ori med}     
 Family:  Replication protein

This part exists but requires an EcoRI/BamHI transfer

This part has already been made, but it's with the wrong vector for our assembly program. We need to move the part into a different plasmid. We'll do this with an EcoRI/BamHI transfer. The protocol describing this is here.