SBB11Ntbk-ChrisAnderson

Stress Promoter Features
Toxic Protein Features

Joanna Chen
Template:SBB10_ProjectGeneralNotes

Overview

Your two parts are very similar and they require gene synthesis. In designing your oligos, you should codon optimize the gene sequence for E. coli and try to maximize re-use of your oligos in making this. You might need to write a small program to help you generate this, and JCA can provide you some starter files to help you out.

sbb17: Zinc Finger Domain (zf+)

 Source:  Synthetic
 Target Sequence:    MAPKKKRKVGIHGVPAAMAERPFQCRICMRNFSRSDTLSEHIRTHTGEKPFACDICGRKFAARSTRTTHTKIHTGSQKPFQCRICMRNFSRSDSLSKHIRTHTGEKPFACDICGRKFAQRSNLKVHTKIHLR  
 Vector:  pBjk2741
 Short desciprion:  {<zf+>} 
 Flavor:  {<part>} 
 Family:  Zinc Finger

This part encodes a DNA binding domain

DNA binding domains are proteins that bind to a specific DNA sequences. In the context of this project, they will be fused to other coding domains to generate chimeric enzymes that hopefully perform useful behavior that is somehow the sum of the two activities from which it is composed. Different DNA binding domain parts are needed in different flavors, so take special note of what type this one should be.

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

This part is associated with Zinc Finger Transposase devices

A zinc finger transposase is an engineered protein derived from a zinc finger DNA binding protein and a transposase. A transposase is an enzyme that reacts with DNA at specific sites and then clips that DNA region out of the source DNA forming a "transposome". This DNA/protein complex is highly reactive towards other DNAs. It reacts by inserting the DNA sequence mostly randomly into the target DNA.

Zinc Finger proteins (ZF) are DNA binding proteins. The special thing about them is that 1) the ZF's 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. By fusing ZF's to transposase, you can recruit the transposome to a specific site in the genome and thereby bias the insertion site of a transposome into a cell's genome.

You should read the following papers
References: PMID 11821858, PMID 17344320, PMID 19965773