CH391L/S13/DnaAssembly - Revision history

Gabriel Wu: /* Restriction Enzyme */

Gabriel Wu — Fri, 03 May 2013 07:06:32 GMT

Restriction Enzyme

←Older revision		Revision as of 07:06, 3 May 2013
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	===Restriction Enzyme===		===Restriction Enzyme===

-	Restriction enzymes recognize a specific nucleotide sequence and then cut the DNA in such a way that results in a double stranded break. If the enzyme cuts within or near the recognition site, it is classified as a Type II restriction enzyme. When a restriction enzyme cuts a piece of DNA, the ~~resulting~~ ends can be straight resulting in a blunt end or with a jagged cut resulting in a sticky end.	+	Restriction enzymes recognize a specific nucleotide sequence and then cut the DNA in such a way that results in a double stranded break. If the enzyme cuts within or near the recognition site, it is classified as a Type II restriction enzyme. When a restriction enzyme cuts a piece of DNA, the ends can be straight resulting in a blunt end or with a jagged cut resulting in a sticky end.

	For example:		For example:

Gabriel Wu: /* Recombination/Homology */

Gabriel Wu — Fri, 03 May 2013 06:51:40 GMT

Recombination/Homology

←Older revision		Revision as of 06:51, 3 May 2013
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	*SLIC sequence and ligation independent cloning T4 DNA polymerase (exonuclease) <cite>Li2007,Li2012</cite><br>		*SLIC sequence and ligation independent cloning T4 DNA polymerase (exonuclease) <cite>Li2007,Li2012</cite><br>

-	[[Image:Infusion.gif\|thumb\| right\|~~500px~~\| [http://openwetware.org/wiki/In-fusion_biobrick_assembly In-Fusion Cloning Diagram]]] <br>	+	[[Image:Infusion.gif\|thumb\| right\|300px\| [http://openwetware.org/wiki/In-fusion_biobrick_assembly In-Fusion Cloning Diagram]]] <br>

	*Variations of the SLIC procedure:		*Variations of the SLIC procedure:

Gabriel Wu: /* Economics */

Gabriel Wu — Fri, 03 May 2013 06:51:15 GMT

Economics

←Older revision		Revision as of 06:51, 3 May 2013
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	Gene synthesis is one of the key enabling technologies of synthetic biology. The increase in efficiency and the decrease in cost is occurring at a staggering rate. Below is a graph comparing DNA synthesis to Moore's Law. In addition, it includes another enabling technology, DNA sequencing. The bottom right graph shows that genome synthesis is technically feasible <cite>Gibson2009</cite>; however, it is not yet a commercial service.		Gene synthesis is one of the key enabling technologies of synthetic biology. The increase in efficiency and the decrease in cost is occurring at a staggering rate. Below is a graph comparing DNA synthesis to Moore's Law. In addition, it includes another enabling technology, DNA sequencing. The bottom right graph shows that genome synthesis is technically feasible <cite>Gibson2009</cite>; however, it is not yet a commercial service.

-	[[Image:Carlson_cost_per_base_june_2011.png\|thumb\|left\|~~350px~~\|Cost per Base of DNA Sequencing and Synthesis.[http://www.synthesis.cc]]]	+	[[Image:Carlson_cost_per_base_june_2011.png\|thumb\|left\|375px\|Cost per Base of DNA Sequencing and Synthesis.[http://www.synthesis.cc]]]
-	[[Image:Cost_per_genome_20130122.jpg\|thumb\|center\|~~400px~~\| Cost per Genome.[http://www.genome.gov/sequencingcosts/]]]	+	[[Image:Cost_per_genome_20130122.jpg\|thumb\|center\|425px\| Cost per Genome.[http://www.genome.gov/sequencingcosts/]]]

	<!-- [http://www.synthesis.cc http://www.synthesis.cc/assets_c/2010/05/carlson_longest_sDNA_2010-thumb-500x457.png]-->		<!-- [http://www.synthesis.cc http://www.synthesis.cc/assets_c/2010/05/carlson_longest_sDNA_2010-thumb-500x457.png]-->

Gabriel Wu: /* Economics */

Gabriel Wu — Fri, 03 May 2013 06:50:31 GMT

Economics

←Older revision		Revision as of 06:50, 3 May 2013
Line 34:		Line 34:
	Gene synthesis is one of the key enabling technologies of synthetic biology. The increase in efficiency and the decrease in cost is occurring at a staggering rate. Below is a graph comparing DNA synthesis to Moore's Law. In addition, it includes another enabling technology, DNA sequencing. The bottom right graph shows that genome synthesis is technically feasible <cite>Gibson2009</cite>; however, it is not yet a commercial service.		Gene synthesis is one of the key enabling technologies of synthetic biology. The increase in efficiency and the decrease in cost is occurring at a staggering rate. Below is a graph comparing DNA synthesis to Moore's Law. In addition, it includes another enabling technology, DNA sequencing. The bottom right graph shows that genome synthesis is technically feasible <cite>Gibson2009</cite>; however, it is not yet a commercial service.

-	[[Image:Carlson_cost_per_base_june_2011.png\|thumb\|left\|~~300px~~\|Cost per Base of DNA Sequencing and Synthesis.[http://www.synthesis.cc]]]	+	[[Image:Carlson_cost_per_base_june_2011.png\|thumb\|left\|350px\|Cost per Base of DNA Sequencing and Synthesis.[http://www.synthesis.cc]]]
	[[Image:Cost_per_genome_20130122.jpg\|thumb\|center\|400px\| Cost per Genome.[http://www.genome.gov/sequencingcosts/]]]		[[Image:Cost_per_genome_20130122.jpg\|thumb\|center\|400px\| Cost per Genome.[http://www.genome.gov/sequencingcosts/]]]

Gabriel Wu: /* Economics */

Gabriel Wu — Fri, 03 May 2013 06:50:13 GMT

Economics

←Older revision		Revision as of 06:50, 3 May 2013
Line 34:		Line 34:
	Gene synthesis is one of the key enabling technologies of synthetic biology. The increase in efficiency and the decrease in cost is occurring at a staggering rate. Below is a graph comparing DNA synthesis to Moore's Law. In addition, it includes another enabling technology, DNA sequencing. The bottom right graph shows that genome synthesis is technically feasible <cite>Gibson2009</cite>; however, it is not yet a commercial service.		Gene synthesis is one of the key enabling technologies of synthetic biology. The increase in efficiency and the decrease in cost is occurring at a staggering rate. Below is a graph comparing DNA synthesis to Moore's Law. In addition, it includes another enabling technology, DNA sequencing. The bottom right graph shows that genome synthesis is technically feasible <cite>Gibson2009</cite>; however, it is not yet a commercial service.

-	[[Image:Carlson_cost_per_base_june_2011.png\|thumb\|left\|~~390px~~\|Cost per Base of DNA Sequencing and Synthesis.[http://www.synthesis.cc]]]	+	[[Image:Carlson_cost_per_base_june_2011.png\|thumb\|left\|300px\|Cost per Base of DNA Sequencing and Synthesis.[http://www.synthesis.cc]]]
-	[[Image:Cost_per_genome_20130122.jpg\|thumb\|center\|~~450px~~\| Cost per Genome.[http://www.genome.gov/sequencingcosts/]]]	+	[[Image:Cost_per_genome_20130122.jpg\|thumb\|center\|400px\| Cost per Genome.[http://www.genome.gov/sequencingcosts/]]]

	<!-- [http://www.synthesis.cc http://www.synthesis.cc/assets_c/2010/05/carlson_longest_sDNA_2010-thumb-500x457.png]-->		<!-- [http://www.synthesis.cc http://www.synthesis.cc/assets_c/2010/05/carlson_longest_sDNA_2010-thumb-500x457.png]-->

Gabriel Wu: /* Genome Synthesis */

Gabriel Wu — Fri, 03 May 2013 06:46:18 GMT

Genome Synthesis

←Older revision		Revision as of 06:46, 3 May 2013
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	===Genome Synthesis===		===Genome Synthesis===
-	J. Craig Venter's team at JCVI ~~have worked to carry~~ gene synthesis ~~to its most advanced level -~~ the creation of a completely synthetic genome. Initially, the JCVI team synthesized a 582,970-base pair "minimized" genome of ''Mycoplasma genitalium'' using a stepwise assembly <cite>Gibson2008</cite>. The genome was divided into 24 cassettes of about 24 kb each which were synthesized by commercial manufacturers. These overlapping cassettes were enzymatically stitched together into bacterial artificial chromosomes, which were attached in a stepwise fashion using a combination of restriction digestion and ''in vitro'' recombination. At the point where the molecules were "quarter genomes", they were transformed into yeast where ''in vivo'' recombination machinery assembled the full ''M. genitalium'' chromosome. This was only proof of synthesis, however, and not of a synthetic genome useful for supporting a living organism.	+	J. Craig Venter's team at JCVI carried out the largest gene synthesis currently reported through the creation of a completely synthetic genome. Initially, the JCVI team synthesized a 582,970-base pair "minimized" genome of ''Mycoplasma genitalium'' using a stepwise assembly <cite>Gibson2008</cite>. The genome was divided into 24 cassettes of about 24 kb each which were synthesized by commercial manufacturers. These overlapping cassettes were enzymatically stitched together into bacterial artificial chromosomes, which were attached in a stepwise fashion using a combination of restriction digestion and ''in vitro'' recombination. At the point where the molecules were "quarter genomes", they were transformed into yeast where ''in vivo'' recombination machinery assembled the full ''M. genitalium'' chromosome. This was only proof of synthesis, however, and not of a synthetic genome useful for supporting a living organism.

	In 2010, the JCVI team reported the creation of a bacterium controlled by a fully synthetic genome <cite>Gibson2010</cite>. The 1.08 mega-base pair genome of ''Mycoplasma mycoides'' was synthesized and transplanted into a ''M. capricolum'' recipient cell to create a synthetic bacterium nicknamed "Synthia". The genome was divided into 1,078 1-kb cassettes, which were synthesized by Blue Heron. These cassettes were assembled in yeast using ''in vivo'' homologous recombination using a three-step hierarchy that built fragments of ~10,000 bp, ~100,000 bp and finally the complete 1.08 mega-bp genome. These were then isolated carefully from contaminating yeast DNA and transplanted into the recipient bacteria. A selectable marker placed in the synthetic genome demonstrated that bacteria that grew after transplantation were controlled by the synthetic genome. The same team demonstrated that a semi-synthetic genome could be assembled and transplanted using a combination of natural and synthetic DNA.		In 2010, the JCVI team reported the creation of a bacterium controlled by a fully synthetic genome <cite>Gibson2010</cite>. The 1.08 mega-base pair genome of ''Mycoplasma mycoides'' was synthesized and transplanted into a ''M. capricolum'' recipient cell to create a synthetic bacterium nicknamed "Synthia". The genome was divided into 1,078 1-kb cassettes, which were synthesized by Blue Heron. These cassettes were assembled in yeast using ''in vivo'' homologous recombination using a three-step hierarchy that built fragments of ~10,000 bp, ~100,000 bp and finally the complete 1.08 mega-bp genome. These were then isolated carefully from contaminating yeast DNA and transplanted into the recipient bacteria. A selectable marker placed in the synthetic genome demonstrated that bacteria that grew after transplantation were controlled by the synthetic genome. The same team demonstrated that a semi-synthetic genome could be assembled and transplanted using a combination of natural and synthetic DNA.

Gabriel Wu: /* Genome Synthesis */

Gabriel Wu — Fri, 03 May 2013 06:42:44 GMT

Genome Synthesis

←Older revision		Revision as of 06:42, 3 May 2013
Line 149:		Line 149:

	In 2010, the JCVI team reported the creation of a bacterium controlled by a fully synthetic genome <cite>Gibson2010</cite>. The 1.08 mega-base pair genome of ''Mycoplasma mycoides'' was synthesized and transplanted into a ''M. capricolum'' recipient cell to create a synthetic bacterium nicknamed "Synthia". The genome was divided into 1,078 1-kb cassettes, which were synthesized by Blue Heron. These cassettes were assembled in yeast using ''in vivo'' homologous recombination using a three-step hierarchy that built fragments of ~10,000 bp, ~100,000 bp and finally the complete 1.08 mega-bp genome. These were then isolated carefully from contaminating yeast DNA and transplanted into the recipient bacteria. A selectable marker placed in the synthetic genome demonstrated that bacteria that grew after transplantation were controlled by the synthetic genome. The same team demonstrated that a semi-synthetic genome could be assembled and transplanted using a combination of natural and synthetic DNA.		In 2010, the JCVI team reported the creation of a bacterium controlled by a fully synthetic genome <cite>Gibson2010</cite>. The 1.08 mega-base pair genome of ''Mycoplasma mycoides'' was synthesized and transplanted into a ''M. capricolum'' recipient cell to create a synthetic bacterium nicknamed "Synthia". The genome was divided into 1,078 1-kb cassettes, which were synthesized by Blue Heron. These cassettes were assembled in yeast using ''in vivo'' homologous recombination using a three-step hierarchy that built fragments of ~10,000 bp, ~100,000 bp and finally the complete 1.08 mega-bp genome. These were then isolated carefully from contaminating yeast DNA and transplanted into the recipient bacteria. A selectable marker placed in the synthetic genome demonstrated that bacteria that grew after transplantation were controlled by the synthetic genome. The same team demonstrated that a semi-synthetic genome could be assembled and transplanted using a combination of natural and synthetic DNA.
-
-	In 2011, a group from Johns Hopkins reported the first partially synthetic eukaryotic genome. They edited approximately 1% of the ''Saccharaomyces cerevisiae'' genome to eliminate introns, repetitive sequence and retrotransposons, replaced all TAG stop codons with TAA, and introduced recombigenic LoxPSym sites to aid in future large-scale manipulations of the genome <cite>Dymond2011</cite>.
-
-	Overall error rates in synthetic genome assembly were controlled by careful cassette synthesis and verification, as well as relying on high-fidelity ''in vivo'' recombination methods for hierarchical construction. Even low error rates can have huge effects, though. One single base deletion in the genome was in an essential gene, ''dnaA'', and delayed progress for many weeks. Isolation, growth and manipulation of mega-base pair DNA molecules either ''in vivo'' or ''in vitro'' is a difficult roadblock as well.
-
-

	Final assembly of large double-stranded DNA products into kilobase and megabase functional molecules, free of errors and with minimal cost remains a process bottleneck for gene and genome synthesis. ''In vitro'' recombination methods have been successfully used to assemble large DNA molecules on the order of tens of kilobases, but this method remains prone to errors and requires considerable homology be engineered into assembly fragments.		Final assembly of large double-stranded DNA products into kilobase and megabase functional molecules, free of errors and with minimal cost remains a process bottleneck for gene and genome synthesis. ''In vitro'' recombination methods have been successfully used to assemble large DNA molecules on the order of tens of kilobases, but this method remains prone to errors and requires considerable homology be engineered into assembly fragments.

Gabriel Wu: /* Genome Synthesis */

Gabriel Wu — Fri, 03 May 2013 06:41:26 GMT

Genome Synthesis

←Older revision		Revision as of 06:41, 3 May 2013
Line 148:		Line 148:
	J. Craig Venter's team at JCVI have worked to carry gene synthesis to its most advanced level - the creation of a completely synthetic genome. Initially, the JCVI team synthesized a 582,970-base pair "minimized" genome of ''Mycoplasma genitalium'' using a stepwise assembly <cite>Gibson2008</cite>. The genome was divided into 24 cassettes of about 24 kb each which were synthesized by commercial manufacturers. These overlapping cassettes were enzymatically stitched together into bacterial artificial chromosomes, which were attached in a stepwise fashion using a combination of restriction digestion and ''in vitro'' recombination. At the point where the molecules were "quarter genomes", they were transformed into yeast where ''in vivo'' recombination machinery assembled the full ''M. genitalium'' chromosome. This was only proof of synthesis, however, and not of a synthetic genome useful for supporting a living organism.		J. Craig Venter's team at JCVI have worked to carry gene synthesis to its most advanced level - the creation of a completely synthetic genome. Initially, the JCVI team synthesized a 582,970-base pair "minimized" genome of ''Mycoplasma genitalium'' using a stepwise assembly <cite>Gibson2008</cite>. The genome was divided into 24 cassettes of about 24 kb each which were synthesized by commercial manufacturers. These overlapping cassettes were enzymatically stitched together into bacterial artificial chromosomes, which were attached in a stepwise fashion using a combination of restriction digestion and ''in vitro'' recombination. At the point where the molecules were "quarter genomes", they were transformed into yeast where ''in vivo'' recombination machinery assembled the full ''M. genitalium'' chromosome. This was only proof of synthesis, however, and not of a synthetic genome useful for supporting a living organism.

-	In 2010, the JCVI team reported the creation of a bacterium controlled by a fully synthetic genome<cite>Gibson2010</cite>. The 1.08 mega-base pair genome of ''Mycoplasma mycoides'' was synthesized and transplanted into a ''M. capricolum'' recipient cell to create a synthetic bacterium nicknamed "Synthia". The genome was divided into 1,078 1-kb cassettes, which were synthesized by Blue Heron. These cassettes were assembled in yeast using ''in vivo'' homologous recombination using a three-step hierarchy that built fragments of ~10,000 bp, ~100,000 bp and finally the complete 1.08 mega-bp genome. These were then isolated carefully from contaminating yeast DNA and transplanted into the recipient bacteria. A selectable marker placed in the synthetic genome demonstrated that bacteria that grew after transplantation were controlled by the synthetic genome. The same team demonstrated that a semi-synthetic genome could be assembled and transplanted using a combination of natural and synthetic DNA.	+	In 2010, the JCVI team reported the creation of a bacterium controlled by a fully synthetic genome <cite>Gibson2010</cite>. The 1.08 mega-base pair genome of ''Mycoplasma mycoides'' was synthesized and transplanted into a ''M. capricolum'' recipient cell to create a synthetic bacterium nicknamed "Synthia". The genome was divided into 1,078 1-kb cassettes, which were synthesized by Blue Heron. These cassettes were assembled in yeast using ''in vivo'' homologous recombination using a three-step hierarchy that built fragments of ~10,000 bp, ~100,000 bp and finally the complete 1.08 mega-bp genome. These were then isolated carefully from contaminating yeast DNA and transplanted into the recipient bacteria. A selectable marker placed in the synthetic genome demonstrated that bacteria that grew after transplantation were controlled by the synthetic genome. The same team demonstrated that a semi-synthetic genome could be assembled and transplanted using a combination of natural and synthetic DNA.

	In 2011, a group from Johns Hopkins reported the first partially synthetic eukaryotic genome. They edited approximately 1% of the ''Saccharaomyces cerevisiae'' genome to eliminate introns, repetitive sequence and retrotransposons, replaced all TAG stop codons with TAA, and introduced recombigenic LoxPSym sites to aid in future large-scale manipulations of the genome <cite>Dymond2011</cite>.		In 2011, a group from Johns Hopkins reported the first partially synthetic eukaryotic genome. They edited approximately 1% of the ''Saccharaomyces cerevisiae'' genome to eliminate introns, repetitive sequence and retrotransposons, replaced all TAG stop codons with TAA, and introduced recombigenic LoxPSym sites to aid in future large-scale manipulations of the genome <cite>Dymond2011</cite>.

Gabriel Wu: /* Genome Synthesis */

Gabriel Wu — Fri, 03 May 2013 06:41:11 GMT

Genome Synthesis

←Older revision		Revision as of 06:41, 3 May 2013
Line 148:		Line 148:
	J. Craig Venter's team at JCVI have worked to carry gene synthesis to its most advanced level - the creation of a completely synthetic genome. Initially, the JCVI team synthesized a 582,970-base pair "minimized" genome of ''Mycoplasma genitalium'' using a stepwise assembly <cite>Gibson2008</cite>. The genome was divided into 24 cassettes of about 24 kb each which were synthesized by commercial manufacturers. These overlapping cassettes were enzymatically stitched together into bacterial artificial chromosomes, which were attached in a stepwise fashion using a combination of restriction digestion and ''in vitro'' recombination. At the point where the molecules were "quarter genomes", they were transformed into yeast where ''in vivo'' recombination machinery assembled the full ''M. genitalium'' chromosome. This was only proof of synthesis, however, and not of a synthetic genome useful for supporting a living organism.		J. Craig Venter's team at JCVI have worked to carry gene synthesis to its most advanced level - the creation of a completely synthetic genome. Initially, the JCVI team synthesized a 582,970-base pair "minimized" genome of ''Mycoplasma genitalium'' using a stepwise assembly <cite>Gibson2008</cite>. The genome was divided into 24 cassettes of about 24 kb each which were synthesized by commercial manufacturers. These overlapping cassettes were enzymatically stitched together into bacterial artificial chromosomes, which were attached in a stepwise fashion using a combination of restriction digestion and ''in vitro'' recombination. At the point where the molecules were "quarter genomes", they were transformed into yeast where ''in vivo'' recombination machinery assembled the full ''M. genitalium'' chromosome. This was only proof of synthesis, however, and not of a synthetic genome useful for supporting a living organism.

-	In 2010, the JCVI team reported the creation of a bacterium controlled by a fully synthetic genome<cite>Gibson2010</cite>. The 1.08 mega-base pair genome of ''Mycoplasma mycoides'' was synthesized and transplanted into a ''M. capricolum'' recipient cell to create synthetic bacterium nicknamed "Synthia". The genome was divided into 1,078 1-kb cassettes, which were synthesized by Blue Heron. These cassettes were assembled in yeast using ''in vivo'' homologous recombination using a three-step hierarchy that built fragments of ~10,000 bp, ~100,000 bp and finally the complete 1.08 mega-bp genome. These were then isolated carefully from contaminating yeast DNA and transplanted into the recipient bacteria. A selectable marker placed in the synthetic genome demonstrated that bacteria that grew after transplantation were controlled by the synthetic genome. The same team demonstrated that a semi-synthetic genome could be assembled and transplanted using a combination of natural and synthetic DNA.	+	In 2010, the JCVI team reported the creation of a bacterium controlled by a fully synthetic genome<cite>Gibson2010</cite>. The 1.08 mega-base pair genome of ''Mycoplasma mycoides'' was synthesized and transplanted into a ''M. capricolum'' recipient cell to create a synthetic bacterium nicknamed "Synthia". The genome was divided into 1,078 1-kb cassettes, which were synthesized by Blue Heron. These cassettes were assembled in yeast using ''in vivo'' homologous recombination using a three-step hierarchy that built fragments of ~10,000 bp, ~100,000 bp and finally the complete 1.08 mega-bp genome. These were then isolated carefully from contaminating yeast DNA and transplanted into the recipient bacteria. A selectable marker placed in the synthetic genome demonstrated that bacteria that grew after transplantation were controlled by the synthetic genome. The same team demonstrated that a semi-synthetic genome could be assembled and transplanted using a combination of natural and synthetic DNA.

	In 2011, a group from Johns Hopkins reported the first partially synthetic eukaryotic genome. They edited approximately 1% of the ''Saccharaomyces cerevisiae'' genome to eliminate introns, repetitive sequence and retrotransposons, replaced all TAG stop codons with TAA, and introduced recombigenic LoxPSym sites to aid in future large-scale manipulations of the genome <cite>Dymond2011</cite>.		In 2011, a group from Johns Hopkins reported the first partially synthetic eukaryotic genome. They edited approximately 1% of the ''Saccharaomyces cerevisiae'' genome to eliminate introns, repetitive sequence and retrotransposons, replaced all TAG stop codons with TAA, and introduced recombigenic LoxPSym sites to aid in future large-scale manipulations of the genome <cite>Dymond2011</cite>.

Gabriel Wu: /* In Vivo */

Gabriel Wu — Fri, 03 May 2013 06:39:48 GMT

In Vivo

←Older revision		Revision as of 06:39, 3 May 2013
Line 122:		Line 122:

	===In Vivo===		===In Vivo===
		+	There is a category of gene assembly tools that operate in live cells:
		+
	*Mating-assisted genetically integrated circuit (MAGIC)-bacterial mating <cite>Li2005</cite><br>		*Mating-assisted genetically integrated circuit (MAGIC)-bacterial mating <cite>Li2005</cite><br>
	*ET recombination <cite>Zhang1998</cite> <br>		*ET recombination <cite>Zhang1998</cite> <br>