Team:Harvard/Technology

From 2011.igem.org

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__NOTOC__
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Our project integrates 4 main technologies: 
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*chip-based synthesis of DNA[[#References|[1]]]
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*multiplex automated genome engineering (MAGE)[[#References|[2]]][[#References|[3]]]
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*lamba red recombination[[#References|[4]]][[#References|[5]]]
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*bioinformatics
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==Chip-Based DNA Synthesis==
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Team members combined these technologies - 3 recently developed, one already widely used - in ways that no one else has tried: we successfully utilized all four over the course of our 10 week project. Considering that two of these methods were originally published less than 2 years ago (MAGE and chip synthesis), Harvard iGEM has reduced new ideas into practice: see our [https://2011.igem.org/Team:Harvard/Protocols Protocols] page and use our methods for other applications.
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We are creating 55,000 zinc fingers using microchip synthesis (Kosuri et al). These fingers will then be tried against the DNA sequences we wish to bind.  
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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[[#References|[2]]]. Chip-based DNA synthesis method was developed by Sri Kosuri et al[[#References|[1]]], and the actual oligo synthesis was generously provided by Agilent Technologies, a sponsor of iGEM. Lambda red was originally developed by Yu et al[[#References|[4]]].  
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===Summary (Adapted from Kosuri et al)<sup>[[#References|1]]</sup>===
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'''By submitting the necessary [[Team:Harvard/Results/Biobricks|strains and parts to the registry]], and publishing these [[Team:Harvard/Protocols|easy-to-follow protocols]] along with our [[Team:Harvard/Results/Tools|source code]], it is our hope that future iGEM teams will also use these techniques in their own synthetic biology projects. '''
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=Bioinformatics=
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See our [https://2011.igem.org/Team:Harvard/Project/Design Design] page for details on the computational aspects of our project technology.
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The synthesis of DNA encoding regulatory elements, genes, pathways and entire genomes provides powerful ways to both test biological hypotheses and harness biology for our use. For example, from the use of oligonucleotides in deciphering the genetic code to the recent complete synthesis of a viable bacterial genome, DNA synthesis has engendered tremendous progress in biology. Currently, almost all DNA synthesis relies on the use of phosphoramidite chemistry on controlled-pore glass (CPG) substrates. The synthesis of gene-sized fragments (500–5,000 base pairs (bp)) relies on assembling many CPG oligonucleotides together using a variety of gene synthesis techniques. Technologies to assemble verified gene-sized fragments into much larger synthetic constructs are now fairly mature.
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Our bioinformatics source code for our zinc finger sequence generator and zinc finger binding site finder is available [https://2011.igem.org/Team:Harvard/Results/Tools here].
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[[File:HARVChip_synthesis.png|thumb|Getting zinc finger DNA off of a chip using OLS pools (Kosuri et al)]]
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=Chip-Based Synthesis=
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See [https://2011.igem.org/Team:Harvard/Technology/Chip_Synthesis Chip Synthesis] for details about the technology, and read our [https://2011.igem.org/Team:Harvard/Project/Synthesize Synthesize] page for details on how we applied chip synthesis to zinc finger proteins.
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The price of gene synthesis has fallen drastically over the last decade. However, the current commercial price of gene synthesis, ~$0.40–1.00/bp, has begun to approach the relatively stable cost of the CPG oligonucleotide precursors (~$0.10–0.20/bp)1, suggesting that oligonucleotide cost is limiting. At these prices, the construction of large gene libraries and synthetic genomes is out of reach to most.  
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Using microchip synthesis (provided by Agilent Technologies), we have 55,000 potential zinc fingers (whose sequences were generated by Team Harvard's [[Team:Harvard/Project/Design|bioinformatics]]) to test. These fingers will then be tried against the DNA sequences we wish to bind.  
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*Original Paper: http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html
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*Our protocols for getting DNA pools off of the chip: https://2011.igem.org/Team:Harvard/Protocols#Chip_DNA_Extraction
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A promising route is to harness existing DNA microchips, which can produce up to a million different oligonucleotides on a single chip, as a source of DNA. Recently, the quality of microchip-synthesized oligonucleotides was improved by controlling depurination during the synthesis process. These arrays produce up to 55,000 200-mer oligonucleotides on a single chip and are sold as a ~1–10 picomole pools of oligonucleotides, termed OLS pools (oligo library synthesis). Estimations of the frequency of transitions, transversions, insertions and deletions in OLS pools found the overall error rate to be ~1/500 bp both before and after PCR amplification, suggesting that OLS pools can be used for accurate large-scale gene synthesis.
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=MAGE=
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See [https://2011.igem.org/Team:Harvard/Technology/MAGE MAGE] for details about the technology, and read our [https://2011.igem.org/Team:Harvard/Project/Test#Building_the_selection_strain:_MAGE Test] page for details on how we used MAGE to build our selection strain.  
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==MAGE==
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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.
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*Original Paper: http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html
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*Our Protocols: https://2011.igem.org/Team:Harvard/Protocols#MAGE
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===Summary (Adapted from Wang et al)<sup>[[#References|2]],[[#References|3]]</sup>===
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=Lambda Red Mediated Recombineering=
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The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes.
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See [https://2011.igem.org/Team:Harvard/Technology/Lambda_Red Lambda Red] for details about the technology, and read our [https://2011.igem.org/Team:Harvard/Project/Test#Building_the_selection_strain:_Lambda_Red_Recombineering Test] page for details on how we used lambda red to build our selection strain.  
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[[File:HARVmage.jpg|thumb|Each cell contains a different set of mutations, producing a heterogeneous population of rich diversity (denoted by distinct chromosomes in different cells). Degenerate oligo pools that target specific genomic positions enable the generation of a diverse set of sequences at each chromosomal location. (Wang et al)]]
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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.
-
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. Because the process is cyclical and scalable, MAGE facilitates rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). This multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.
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*Gene Knockouts and Exchanges by Linear Transformation: http://rothlab.ucdavis.edu/protocols/Lin.Transform.html
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*Open Wet Ware Protocol: http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement
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*Our Protocol: https://2011.igem.org/Team:Harvard/Protocols#Lambda_Red
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MAGE provides a highly efficient, inexpensive and automated solution to simultaneously modify many genomic locations (for example, genes, regulatory regions) across different length scales, from the nucleotide to the genome level.
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=Gibson (Isothermal) Assembly=
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See our [https://2011.igem.org/Team:Harvard/Project/Synthesize#Zinc_Finger_Expression_Plasmids Synthesize] page for how we used Gibson Assemble to create our zinc finger expression plasmids.
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==Gibson (Isothermal) Assembly==
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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.
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===Summary (Adapted from Gibson et al)<sup>[[#References|4]],[[#References|5]]</sup>===
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*Original Paper: http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html
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*Source: http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html
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*Protocol: http://www.nature.com/protocolexchange/protocols/554#/main
*Protocol: http://www.nature.com/protocolexchange/protocols/554#/main
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*Our Protocol: https://2011.igem.org/Team:Harvard/Protocols#Isothermal_assembly
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</div>
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==References==
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<div class="whitebox">
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=References=
'''1.''' Sriram Kosuri, Nikolai Eroshenko, Emily M LeProust, Michael Super, Jeffrey Way, Jin Billy Li, George M Church. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. ''Nature Biotechnology'', 28(12):1295-9. [http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html]
'''1.''' Sriram Kosuri, Nikolai Eroshenko, Emily M LeProust, Michael Super, Jeffrey Way, Jin Billy Li, George M Church. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. ''Nature Biotechnology'', 28(12):1295-9. [http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html]
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'''2.''' Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest, George M. Church. Programming cells by multiplex genome engineering and accelerated evolution. (2009). ''Nature'', 460(7257):894-8. [http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html]
'''2.''' Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest, George M. Church. Programming cells by multiplex genome engineering and accelerated evolution. (2009). ''Nature'', 460(7257):894-8. [http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html]
 +
'''3.''' Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. ''Science'', 333(6040):348-53. [http://www.sciencemag.org/content/333/6040/348.full]
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'''3.''' Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. ''Science'', 333(6040):348-53. [http://www.sciencemag.org/content/333/6040/348.short]
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'''4.''' Yu D., H. M. Ellis, et al. (2000). An efficient recombination system for chromosome engineering in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 97(11): 5978-5983.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165854/]
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+
-
 
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'''4.''' Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. ''Nature Methods,''6(5):343-5. Epub 2009 Apr 12.
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[http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html]
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-
 
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'''5.''' Gibson D. (2009). One-step enzymatic assembly of DNA molecules up to several hundred kilobases in size. ''Nature Protocols,''Published online 16 April 2009, doi:10.1038/nprot.2009.77.
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'''5.''' Mosberg JA, Lajoie MJ, Church GM. Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. ''Genetics'' 2010;186:791-799.[http://www.genetics.org/content/186/3/791]
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[http://dx.doi.org/10.1038/nprot.2009.77]
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Latest revision as of 02:24, 29 October 2011

bar

Overview | MAGE | Chip-Based Synthesis | Lambda Red | Protocols

Our project integrates 4 main technologies:

  • chip-based synthesis of DNA[1]
  • multiplex automated genome engineering (MAGE)[2][3]
  • lamba red recombination[4][5]
  • bioinformatics

Team members combined these technologies - 3 recently developed, one already widely used - in ways that no one else has tried: we successfully utilized all four over the course of our 10 week project. Considering that two of these methods were originally published less than 2 years ago (MAGE and chip synthesis), Harvard iGEM has reduced new ideas into practice: see our Protocols page and use our methods for other applications.

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[2]. Chip-based DNA synthesis method was developed by Sri Kosuri et al[1], and the actual oligo synthesis was generously provided by Agilent Technologies, a sponsor of iGEM. Lambda red was originally developed by Yu et al[4].

By submitting the necessary strains and parts to the registry, and publishing these easy-to-follow protocols along with our source code, it is our hope that future iGEM teams will also use these techniques in their own synthetic biology projects.

Bioinformatics

See our Design page for details on the computational aspects of our project technology.

Our bioinformatics source code for our zinc finger sequence generator and zinc finger binding site finder is available here.

Chip-Based Synthesis

See Chip Synthesis for details about the technology, and read our Synthesize page for details on how we applied chip synthesis to zinc finger proteins.

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.

MAGE

See MAGE for details about the technology, and read our Test page for details on how we used MAGE to build our selection strain.

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.

Lambda Red Mediated Recombineering

See Lambda Red for details about the technology, and read our Test page for details on how we used lambda red to build our selection strain.

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

See our Synthesize page for how we used Gibson Assemble to create our zinc finger expression plasmids.

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.

References

1. Sriram Kosuri, Nikolai Eroshenko, Emily M LeProust, Michael Super, Jeffrey Way, Jin Billy Li, George M Church. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature Biotechnology, 28(12):1295-9. [http://www.nature.com/nbt/journal/v28/n12/full/nbt.1716.html]

2. Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest, George M. Church. Programming cells by multiplex genome engineering and accelerated evolution. (2009). Nature, 460(7257):894-8. [http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html]

3. Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science, 333(6040):348-53. [http://www.sciencemag.org/content/333/6040/348.full]

4. Yu D., H. M. Ellis, et al. (2000). An efficient recombination system for chromosome engineering in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 97(11): 5978-5983.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165854/]

5. Mosberg JA, Lajoie MJ, Church GM. Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 2010;186:791-799.[http://www.genetics.org/content/186/3/791]
Retrieved from "http://2011.igem.org/Team:Harvard/Technology"