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Our project uses 3 new technologies: Multiplex Automated Genome Engineering (MAGE), chip-based synthesis of DNA, and lamba red recombination, along with more traditional bioinformatics. | Our project uses 3 new technologies: Multiplex Automated Genome Engineering (MAGE), chip-based synthesis of DNA, and lamba red recombination, along with more traditional bioinformatics. | ||
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We hope that future iGEM teams will also use these techniques in their own synthetic biology projects. | We hope that future iGEM teams will also use these techniques in their own synthetic biology projects. | ||
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=Bioinformatics= | =Bioinformatics= | ||
See our [https://2011.igem.org/Team:Harvard/Project/Bioinformatics Project:Bioinformatics] page for details on the computational aspects of our project technology. | See our [https://2011.igem.org/Team:Harvard/Project/Bioinformatics Project:Bioinformatics] page for details on the computational aspects of our project technology. | ||
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==Zinc Finger Binding Site Finder== | ==Zinc Finger Binding Site Finder== | ||
Check out our [[Team:Harvard/ZF_Binding_Site_Finder|Zinc Finger Binding Site Finder Tool]]. This tool was designed and used to search the human genome for the six target DNA sequences that we used to design our custom zinc finger arrays. | Check out our [[Team:Harvard/ZF_Binding_Site_Finder|Zinc Finger Binding Site Finder Tool]]. This tool was designed and used to search the human genome for the six target DNA sequences that we used to design our custom zinc finger arrays. | ||
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=Chip-Based Synthesis= | =Chip-Based Synthesis= | ||
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*Original Paper: http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html | *Original Paper: http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html | ||
*Our protocols for getting DNA pools off of the chip: https://2011.igem.org/Team:Harvard/Protocols#Chip_DNA_Extraction | *Our protocols for getting DNA pools off of the chip: https://2011.igem.org/Team:Harvard/Protocols#Chip_DNA_Extraction | ||
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=MAGE= | =MAGE= | ||
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*Original Paper: http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html | *Original Paper: http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html | ||
*Our Protocols: https://2011.igem.org/Team:Harvard/Protocols#MAGE | *Our Protocols: https://2011.igem.org/Team:Harvard/Protocols#MAGE | ||
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=Lambda Red- Mediated Recombineering= | =Lambda Red- Mediated Recombineering= | ||
Genes can be altered by recombination with linear DNA molecules. This requires a high internal DNA concentration, achievable by electroporation. The lambda red system allows efficient recombination between homologous sequences as short as 40 bp, which frees us of the need to provide long tracts of homology for recombination into the chromosome. | Genes can be altered by recombination with linear DNA molecules. This requires a high internal DNA concentration, achievable by electroporation. The lambda red system allows efficient recombination between homologous sequences as short as 40 bp, which frees us of the need to provide long tracts of homology for recombination into the chromosome. | ||
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*Open Wet Ware Protocol: http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement | *Open Wet Ware Protocol: http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement | ||
*Our Protocol: https://2011.igem.org/Team:Harvard/Protocols#Lambda_Red | *Our Protocol: https://2011.igem.org/Team:Harvard/Protocols#Lambda_Red | ||
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=Gibson (Isothermal) Assembly= | =Gibson (Isothermal) Assembly= | ||
An isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase. | An isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase. | ||
Revision as of 16:23, 15 October 2011
Overview | MAGE | Chip-Based Synthesis | Lambda Red | Protocols
Our project uses 3 new technologies: Multiplex Automated Genome Engineering (MAGE), chip-based synthesis of DNA, and lamba red recombination, along with more traditional bioinformatics.
Although the project is the first to utilize several key technologies in novel ways, these technologies were developed outside of Harvard iGEM. The multiplex automated genome engineering (MAGE) method was developed by Harris Wang et al in the Church lab of Harvard University. The chip-based synthesis method that we used to synthesize oligos for our zinc finger pool was developed by Sri Kosuri et al in the Church lab, and the actual oligo synthesis was generously provided by Agilent Technologies,a sponsor of iGEM.
We hope that future iGEM teams will also use these techniques in their own synthetic biology projects.
Contents |
_
Bioinformatics
See our Project:Bioinformatics page for details on the computational aspects of our project technology.
Zinc Finger Binding Site Finder
Check out our Zinc Finger Binding Site Finder Tool. This tool was designed and used to search the human genome for the six target DNA sequences that we used to design our custom zinc finger arrays.
Chip-Based Synthesis
Using microchip synthesis (provided by Agilent Technologies), we have 55,000 potential zinc fingers (whose sequences were generated by Team Harvard's bioinformatics) to test. These fingers will then be tried against the DNA sequences we wish to bind.
- Original Paper: http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html
- Our protocols for getting DNA pools off of the chip: https://2011.igem.org/Team:Harvard/Protocols#Chip_DNA_Extraction
MAGE
Multiplex automated genome engineering (MAGE) is a new method for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome, thus producing combinatorial genomic diversity.
- Original Paper: http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html
- Our Protocols: https://2011.igem.org/Team:Harvard/Protocols#MAGE
Lambda Red- Mediated Recombineering
Genes can be altered by recombination with linear DNA molecules. This requires a high internal DNA concentration, achievable by electroporation. The lambda red system allows efficient recombination between homologous sequences as short as 40 bp, which frees us of the need to provide long tracts of homology for recombination into the chromosome.
- Gene Knockouts and Exchanges by Linear Transformation: http://rothlab.ucdavis.edu/protocols/Lin.Transform.html
- Open Wet Ware Protocol: http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement
- Our Protocol: https://2011.igem.org/Team:Harvard/Protocols#Lambda_Red
Gibson (Isothermal) Assembly
An isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase.
