CH391L/S12/MAGE lycopene production, CAGE "Amberless" E. coli - Revision history

Jeffrey E. Barrick: /* Specificity of MAGE */

2012-04-29T20:20:30Z

Specificity of MAGE

←Older revision		Revision as of 20:20, 29 April 2012
Line 26:		Line 26:

	====Specificity of MAGE====		====Specificity of MAGE====
-	Through the lycopene production pathway, the Church group showed that MAGE could be extremely specific if well-defined oligos are introduced. From the DXP pathway, translation optimization of lycopene production genes such as idi alone (EcHW2a) increased lycopene production 40%, while optimizing dxs and idi increased production by 390% (ExHW2e). <cite>Wang2009</cite> It was also shown that in the secondary pathway, inactivation of gdhA increases lycopene production but lowers growth rate in EcHW2b ~~by32~~%. The specificity possible by MAGE use was expanded by the Church group to other projects.	+	Through the lycopene production pathway, the Church group showed that MAGE could be extremely specific if well-defined oligos are introduced. From the DXP pathway, translation optimization of lycopene production genes such as idi alone (EcHW2a) increased lycopene production 40%, while optimizing dxs and idi increased production by 390% (ExHW2e). <cite>Wang2009</cite> It was also shown that in the secondary pathway, inactivation of gdhA increases lycopene production but lowers growth rate in EcHW2b by 32%. The specificity possible by MAGE use was expanded by the Church group to other projects.

	==CAGE "Amberless" ''E. Coli''==		==CAGE "Amberless" ''E. Coli''==

James L. Bachman: /* Whole-genome Synthesis */

2012-04-16T22:19:43Z

Whole-genome Synthesis

←Older revision		Revision as of 22:19, 16 April 2012
Line 46:		Line 46:

	===Whole-genome Synthesis===		===Whole-genome Synthesis===
-	In 2010 the Venter group of JCVI synthesized a 1.08–mega–base pair genome and transplanted it to M. capricolum to create the new M. mycoides. <cite>Venter</cite> This synthetic biology achievement took 400 scientists year to create, along with a whopping price tag of $40 million. So unless you are Craig Venter, this approach to genome synthesis is likely unrealistic, but this is where MAGE ~~and CAGE may~~ shine. Due to the relatively inexpensive nature of MAGE and the ability to modify much of the genome simultaneously many think that it could provide an alternative to the venter de novo synthesis.<cite>Wang2009</cite>However, with the very low frequency of both oligo transformation and recombination, and the fact that the Amberless ''E. coli'' remains unfinished, it does not seem that the Church groups' techniques provide a definite alternative to whole-genome synthesis in its current form.	+	In 2010 the Venter group of JCVI synthesized a 1.08–mega–base pair genome and transplanted it to M. capricolum to create the new M. mycoides. <cite>Venter</cite> This synthetic biology achievement took 400 scientists year to create, along with a whopping price tag of $40 million. Meaning that, unless you are Craig Venter, this approach to whole-genome synthesis is likely unrealistic, but this is where MAGE has the potential to shine. Due to the relatively inexpensive nature of MAGE and the ability to modify much of the genome simultaneously many think that it could provide an alternative to the venter de novo synthesis.<cite>Wang2009</cite>However, with the very low frequency of both oligo transformation and recombination, and the fact that the Amberless ''E. coli'' remains unfinished, it does not seem that the Church groups' techniques provide a definite alternative to whole-genome synthesis in its current form.

	[[Image:MAGE_Machine.jpg\|thumb\|200px\|right\|Machine for the automation of MAGE.<cite>techreview</cite>]]		[[Image:MAGE_Machine.jpg\|thumb\|200px\|right\|Machine for the automation of MAGE.<cite>techreview</cite>]]
		+
	===MAGE Automation===		===MAGE Automation===
	Due to the cyclical and scalable features of the MAGE system, the Church group constructed a device to automate the MAGE process. It has chambers that maintain the growth of healthy cells and a device to electroporate the cells, dramatically speeding up the technique.<cite>Wang2009</cite> http://nextbigfuture.com/2011/07/accelerated-evolution-machine-being.html		Due to the cyclical and scalable features of the MAGE system, the Church group constructed a device to automate the MAGE process. It has chambers that maintain the growth of healthy cells and a device to electroporate the cells, dramatically speeding up the technique.<cite>Wang2009</cite> http://nextbigfuture.com/2011/07/accelerated-evolution-machine-being.html

James L. Bachman: /* Lamba-Red Bacteriophage */

2012-04-16T14:54:52Z

Lamba-Red Bacteriophage

←Older revision		Revision as of 14:54, 16 April 2012
Line 6:		Line 6:

	====Lamba-Red Bacteriophage====		====Lamba-Red Bacteriophage====
-	Modification in MAGE is done through oligo-mediated allelic replacement, which is controlled by the λ-Red single-stranded DNA binding protein β. This protein works by binding the single-stranded oligo and helping it to displace the ~~okazaki~~ fragment on the lagging strand. Normally, the cell's repair proteins would spot the mismatch, but one of the key genes for the repair mechanism has been knocked out in the strain used, EcNR2. Upon the next round of DNA replication, the introduced oligo is copied and becomes part of genome. It is also possible that the fragments can be replaced by other near matching oligos in succeeding rounds, generating even more diversity. The Church group found using 90-mer oligos were the most efficient for replacement. This is due to the λ-Red protein needing at least 30 bps to complex DNA, and that 90bp presents a good chance of homology to the target, while oligos larger than 90-mer have a higher chance of forming secondary structure, greatly reducing replacement effieciency.	+	Modification in MAGE is done through oligo-mediated allelic replacement, which is controlled by the λ-Red single-stranded DNA binding protein β. This protein works by binding the single-stranded oligo and helping it to displace the Okazaki fragment on the lagging strand. Normally, the cell's repair proteins would spot the mismatch, but one of the key genes for the repair mechanism has been knocked out in the strain used, EcNR2. Upon the next round of DNA replication, the introduced oligo is copied and becomes part of genome. It is also possible that the fragments can be replaced by other near matching oligos in succeeding rounds, generating even more diversity. The Church group found using 90-mer oligos were the most efficient for replacement. This is due to the λ-Red protein needing at least 30 bps to complex DNA, and that 90bp presents a good chance of homology to the target, while oligos larger than 90-mer have a higher chance of forming secondary structure, greatly reducing replacement effieciency.

	[[Image:Figurer_2.jpg\|thumb\|300px\|left\|Efficiency of allelic replacement (a) mismatch, (b) insertion, (c) deletion.l<cite>Wang2009</cite>]]		[[Image:Figurer_2.jpg\|thumb\|300px\|left\|Efficiency of allelic replacement (a) mismatch, (b) insertion, (c) deletion.l<cite>Wang2009</cite>]]

James L. Bachman at 14:54, 16 April 2012

2012-04-16T14:54:06Z

←Older revision		Revision as of 14:54, 16 April 2012
Line 39:		Line 39:

	===The "amberless" ''E. Coli''===		===The "amberless" ''E. Coli''===
-	The original 32 recoded strains were turned into 8 strains, each with 1/8 of the genome recoded. Two of these strains had a dysfunctional tolC phenotype, meaning that it passed the positive-negative control selections. MAGE was used to reconstruct these strains from the ancestral strain (also had mutation). From this, 4 strains each with 1/4 of the genome and 80 codon modifications were created and conjugation into one strain was attempted. In the end, 28 of the 31 conjugations were accomplished, still falling short of providing entire-genome modification. If all TAG stop codons can be successfully replaced with TAA stop codons then it may be possible to use the TAA codon for other uses such as incorporation of unnatural amino acids.	+	The original 32 recoded strains were turned into 8 strains, each with 1/8 of the genome recoded. Two of these strains had a dysfunctional tolC phenotype, meaning that it passed the positive-negative control selections. MAGE was used to reconstruct these strains from the ancestral strain (also had mutation). From this, 4 strains each with 1/4 of the genome and 80 codon modifications were created and conjugation into one strain was attempted. In the end, 28 of the 31 conjugations were accomplished, still falling short of providing entire-genome modification. If all TAG stop codons can be successfully replaced with TAA stop codons then it may be possible to use the TAA codon for other uses such as incorporation of unnatural amino acids.<cite>Uaa</cite>

	==Other MAGE Applications==		==Other MAGE Applications==
Line 46:		Line 46:

	===Whole-genome Synthesis===		===Whole-genome Synthesis===
-	In 2010 the Venter group of JCVI synthesized a 1.08–mega–base pair genome and transplanted it to M. capricolum to create the new M. mycoides. <cite>Venter</cite> This synthetic biology achievement took 400 scientists year to create, along with a whopping price tag of $40 million. So unless you are Craig Venter, this approach to genome synthesis is likely unrealistic, but this is where MAGE and CAGE may shine. Due to the relatively inexpensive nature of MAGE and the ability to modify much of the genome simultaneously many think that it could provide an alternative to the venter de novo synthesis. However, with the very low frequency of both oligo transformation and recombination, and the fact that the Amberless ''E. coli'' remains unfinished, it does not seem that the Church groups' techniques provide a definite alternative to whole-genome synthesis in its current form.	+	In 2010 the Venter group of JCVI synthesized a 1.08–mega–base pair genome and transplanted it to M. capricolum to create the new M. mycoides. <cite>Venter</cite> This synthetic biology achievement took 400 scientists year to create, along with a whopping price tag of $40 million. So unless you are Craig Venter, this approach to genome synthesis is likely unrealistic, but this is where MAGE and CAGE may shine. Due to the relatively inexpensive nature of MAGE and the ability to modify much of the genome simultaneously many think that it could provide an alternative to the venter de novo synthesis.<cite>Wang2009</cite>However, with the very low frequency of both oligo transformation and recombination, and the fact that the Amberless ''E. coli'' remains unfinished, it does not seem that the Church groups' techniques provide a definite alternative to whole-genome synthesis in its current form.

	[[Image:MAGE_Machine.jpg\|thumb\|200px\|right\|Machine for the automation of MAGE.<cite>techreview</cite>]]		[[Image:MAGE_Machine.jpg\|thumb\|200px\|right\|Machine for the automation of MAGE.<cite>techreview</cite>]]
Line 68:		Line 68:
	#techreview http://www.technologyreview.com/biomedicine/22299/		#techreview http://www.technologyreview.com/biomedicine/22299/
	//MAGE Machine		//MAGE Machine
		+	#Uaa pmid=20147747
		+	//Unnatural amino acid incorporation

James L. Bachman at 22:02, 15 April 2012

2012-04-15T22:02:48Z

←Older revision		Revision as of 22:02, 15 April 2012
Line 28:		Line 28:
	Through the lycopene production pathway, the Church group showed that MAGE could be extremely specific if well-defined oligos are introduced. From the DXP pathway, translation optimization of lycopene production genes such as idi alone (EcHW2a) increased lycopene production 40%, while optimizing dxs and idi increased production by 390% (ExHW2e). <cite>Wang2009</cite> It was also shown that in the secondary pathway, inactivation of gdhA increases lycopene production but lowers growth rate in EcHW2b by32%. The specificity possible by MAGE use was expanded by the Church group to other projects.		Through the lycopene production pathway, the Church group showed that MAGE could be extremely specific if well-defined oligos are introduced. From the DXP pathway, translation optimization of lycopene production genes such as idi alone (EcHW2a) increased lycopene production 40%, while optimizing dxs and idi increased production by 390% (ExHW2e). <cite>Wang2009</cite> It was also shown that in the secondary pathway, inactivation of gdhA increases lycopene production but lowers growth rate in EcHW2b by32%. The specificity possible by MAGE use was expanded by the Church group to other projects.

-	==CAGE "Amberless" E. Coli==	+	==CAGE "Amberless" ''E. Coli''==
-	Hierarchical Conjugative Assembly (CAGE) was developed in George Church's lab as a means to merge sets of codon modifications from MAGE into genomes (each 1/4 size of E. coli genome) with 80 precise codon modifications. It was demonstrated that synonymous codon changes can be combined into strains without lethal effects on the cell population. E. coli has three stop codons and two release factors. Release factor 1 (RF1) recognizes UAA and UAG, while RF2 recognizes UAA and UGA. The hypothesis was that replacing all TAG codons with TAA codons, the genetic dependence on RF1 would be abolished and the newly introduced TAA codons would be recognized by RF2. The group sought to test if E. coli that had replaced all 314 TAG stop codons with TAA codons would be viable. (MG1655 genome) <cite>Isaacs2011</cite>	+	Hierarchical Conjugative Assembly (CAGE) was developed in George Church's lab as a means to merge sets of codon modifications from MAGE into genomes (each 1/4 size of ''E. coli'' genome) with 80 precise codon modifications. It was demonstrated that synonymous codon changes can be combined into strains without lethal effects on the cell population. ''E. coli'' has three stop codons and two release factors. Release factor 1 (RF1) recognizes UAA and UAG, while RF2 recognizes UAA and UGA. The hypothesis was that replacing all TAG codons with TAA codons, the genetic dependence on RF1 would be abolished and the newly introduced TAA codons would be recognized by RF2. The group sought to test if ''E. coli'' that had replaced all 314 TAG stop codons with TAA codons would be viable. (MG1655 genome) <cite>Isaacs2011</cite>

	===Codon Modification Strategy===		===Codon Modification Strategy===
Line 36:		Line 36:

	===Assembling Stop Codon Modifications===		===Assembling Stop Codon Modifications===
-	Through the use of Hierarchical Conjugative Assembly (CAGE), merging the modifications from MAGE was accomplished. This technique is dependent upon bacterial conjugation to transfer the modified segments. The oriT sequence typically used for conjugation is fused with kanR for integration into E. coli genome by the λ-Red mediated dsDNA recombination. This makes for precise control of conjugation initiation location and use of a ~2-kb casette in place of the 30-kb Hfr fragment(conjugation factors are maintained on recipients as well). <cite>Isaacs2011</cite> [[Image:Figure_4a.jpg\|thumb\|300px\|Right\|The site specific conjugation of donor and plasmid. <cite>Isaacs2009</cite>]] The 32 strains were converted to 16 pairs for conjugation, with a donor strain transferring its genomic region to a recipient. Selectable markers control placement of transfered DNA, in the donor strain the recoded region was flanked upstream by the oriT-Kan cassete and downstream by a positive selectable marker (ie: antibiotic resistance). The recipient strain contained a different positive selectable marker and a positive-negative selectable marker (tolC), about the recoded region. Through the use of multiplex allele-specific colony PCR (MASC-PCR) 81 integration sites were tested, 12 gave no recombination, 23 sites had a recombination frequency of ~10^-7, 38 sites with ~10^-6, and 8 sites with ~10^-5 recombination frequencies.	+	Through the use of Hierarchical Conjugative Assembly (CAGE), merging the modifications from MAGE was accomplished. This technique is dependent upon bacterial conjugation to transfer the modified segments. The oriT sequence typically used for conjugation is fused with kanR for integration into ''E. coli'' genome by the λ-Red mediated dsDNA recombination. This makes for precise control of conjugation initiation location and use of a ~2-kb casette in place of the 30-kb Hfr fragment(conjugation factors are maintained on recipients as well). <cite>Isaacs2011</cite> [[Image:Figure_4a.jpg\|thumb\|300px\|Right\|The site specific conjugation of donor and plasmid. <cite>Isaacs2009</cite>]] The 32 strains were converted to 16 pairs for conjugation, with a donor strain transferring its genomic region to a recipient. Selectable markers control placement of transfered DNA, in the donor strain the recoded region was flanked upstream by the oriT-Kan cassete and downstream by a positive selectable marker (ie: antibiotic resistance). The recipient strain contained a different positive selectable marker and a positive-negative selectable marker (tolC), about the recoded region. Through the use of multiplex allele-specific colony PCR (MASC-PCR) 81 integration sites were tested, 12 gave no recombination, 23 sites had a recombination frequency of ~10^-7, 38 sites with ~10^-6, and 8 sites with ~10^-5 recombination frequencies.

-	===The "amberless" E. Coli===	+	===The "amberless" ''E. Coli''===
	The original 32 recoded strains were turned into 8 strains, each with 1/8 of the genome recoded. Two of these strains had a dysfunctional tolC phenotype, meaning that it passed the positive-negative control selections. MAGE was used to reconstruct these strains from the ancestral strain (also had mutation). From this, 4 strains each with 1/4 of the genome and 80 codon modifications were created and conjugation into one strain was attempted. In the end, 28 of the 31 conjugations were accomplished, still falling short of providing entire-genome modification. If all TAG stop codons can be successfully replaced with TAA stop codons then it may be possible to use the TAA codon for other uses such as incorporation of unnatural amino acids.		The original 32 recoded strains were turned into 8 strains, each with 1/8 of the genome recoded. Two of these strains had a dysfunctional tolC phenotype, meaning that it passed the positive-negative control selections. MAGE was used to reconstruct these strains from the ancestral strain (also had mutation). From this, 4 strains each with 1/4 of the genome and 80 codon modifications were created and conjugation into one strain was attempted. In the end, 28 of the 31 conjugations were accomplished, still falling short of providing entire-genome modification. If all TAG stop codons can be successfully replaced with TAA stop codons then it may be possible to use the TAA codon for other uses such as incorporation of unnatural amino acids.

	==Other MAGE Applications==		==Other MAGE Applications==
	===His-Tagging with MAGE===		===His-Tagging with MAGE===
-	MAGE was used to simultaneously modify all of the translational machinery of E. coli, while maintaining them functionally. Through 110 MAGE cycles, hexa-histidine tags were successfully added to the genes that code for all 38 translational proteins. The incorporation of His-tags into up to 8 different translation factors in a single strain does not dramatically affect the fitness of the cell. Additionally, three ribosomal subunit genes 50s, 30s, and 70s were his-tagged for purification of ribosomes using Ni-NTA. <cite>Wang2012</cite>	+	MAGE was used to simultaneously modify all of the translational machinery of ''E. coli'', while maintaining them functionally. Through 110 MAGE cycles, hexa-histidine tags were successfully added to the genes that code for all 38 translational proteins. The incorporation of His-tags into up to 8 different translation factors in a single strain does not dramatically affect the fitness of the cell. Additionally, three ribosomal subunit genes 50s, 30s, and 70s were his-tagged for purification of ribosomes using Ni-NTA. <cite>Wang2012</cite>

	===Whole-genome Synthesis===		===Whole-genome Synthesis===
-	In 2010 the Venter group of JCVI synthesized a 1.08–mega–base pair genome and transplanted it to M. capricolum to create the new M. mycoides. <cite>Venter</cite> This synthetic biology achievement took 400 scientists year to create, along with a whopping price tag of $40 million. So unless you are Craig Venter, this approach to genome synthesis is likely unrealistic, but this is where MAGE and CAGE may shine. Due to the relatively inexpensive nature of MAGE and the ability to modify much of the genome simultaneously many think that it could provide an alternative to the venter de novo synthesis. However, with the very low frequency of both oligo transformation and recombination, and the fact that the Amberless E. coli remains unfinished, it does not seem that the Church groups' techniques provide a definite alternative to whole-genome synthesis in its current form.	+	In 2010 the Venter group of JCVI synthesized a 1.08–mega–base pair genome and transplanted it to M. capricolum to create the new M. mycoides. <cite>Venter</cite> This synthetic biology achievement took 400 scientists year to create, along with a whopping price tag of $40 million. So unless you are Craig Venter, this approach to genome synthesis is likely unrealistic, but this is where MAGE and CAGE may shine. Due to the relatively inexpensive nature of MAGE and the ability to modify much of the genome simultaneously many think that it could provide an alternative to the venter de novo synthesis. However, with the very low frequency of both oligo transformation and recombination, and the fact that the Amberless ''E. coli'' remains unfinished, it does not seem that the Church groups' techniques provide a definite alternative to whole-genome synthesis in its current form.

	[[Image:MAGE_Machine.jpg\|thumb\|200px\|right\|Machine for the automation of MAGE.<cite>techreview</cite>]]		[[Image:MAGE_Machine.jpg\|thumb\|200px\|right\|Machine for the automation of MAGE.<cite>techreview</cite>]]

Jeffrey E. Barrick: /* References */

2012-04-14T17:02:09Z

References

←Older revision		Revision as of 17:02, 14 April 2012
Line 58:		Line 58:
	#Isaacs2011 pmid=21764749		#Isaacs2011 pmid=21764749
	//CAGE article		//CAGE article
-	#Nature pmid= 20395970	+	#Nature pmid=20395970
	//Synthetic biology review		//Synthetic biology review
	http://en.wikipedia.org/wiki/Lycopene		http://en.wikipedia.org/wiki/Lycopene

James L. Bachman: /* The "amberless" E. Coli */

2012-04-09T18:12:49Z

The "amberless" E. Coli

←Older revision		Revision as of 18:12, 9 April 2012
Line 39:		Line 39:

	===The "amberless" E. Coli===		===The "amberless" E. Coli===
-	The original 32 recoded strains were turned into 8 strains, each with 1/8 of the genome recoded. Two of these strains had a dysfunctional tolC phenotype, meaning that it passed the positive-negative control selections. MAGE was used to reconstruct these strains from the ancestral strain (also had mutation). In the end, 28 of the 31 conjugations were accomplished, still falling short of providing entire-genome modification. If all TAG stop codons can be successfully replaced with TAA stop codons then it may be possible to use the TAA codon for other uses such as incorporation of unnatural amino acids.	+	The original 32 recoded strains were turned into 8 strains, each with 1/8 of the genome recoded. Two of these strains had a dysfunctional tolC phenotype, meaning that it passed the positive-negative control selections. MAGE was used to reconstruct these strains from the ancestral strain (also had mutation). From this, 4 strains each with 1/4 of the genome and 80 codon modifications were created and conjugation into one strain was attempted. In the end, 28 of the 31 conjugations were accomplished, still falling short of providing entire-genome modification. If all TAG stop codons can be successfully replaced with TAA stop codons then it may be possible to use the TAA codon for other uses such as incorporation of unnatural amino acids.

	==Other MAGE Applications==		==Other MAGE Applications==

James L. Bachman: /* Assembling Stop Codon Modifications */

2012-04-09T18:11:04Z

Assembling Stop Codon Modifications

←Older revision		Revision as of 18:11, 9 April 2012
Line 36:		Line 36:

	===Assembling Stop Codon Modifications===		===Assembling Stop Codon Modifications===
-	Through the use of Hierarchical Conjugative Assembly (CAGE), merging the modifications from MAGE was accomplished. This technique is dependent upon bacterial conjugation to transfer the modified segments. The oriT sequence typically used for conjugation is fused with kanR for integration into E. coli genome by the λ-Red mediated dsDNA recombination. This makes for precise control of conjugation initiation location and use of a ~2-kb casette in place of the 30-kb Hfr fragment(conjugation factors are maintained on recipients as well). <cite>Isaacs2011</cite> [[Image:Figure_4a.jpg\|thumb\|300px\|Right\|The site specific conjugation of donor and plasmid. <cite>Isaacs2009</cite>]] The 32 strains were converted to 16 pairs for conjugation, with a donor strain transferring its genomic region to a recipient. Selectable ~~makers~~ control placement of transfered DNA, in the donor strain the recoded region was flanked upstream by the oriT-Kan cassete and downstream by a positive selectable marker (ie: antibiotic resistance). The recipient strain contained a different positive selectable marker and a positive-negative selectable marker (tolC), about the recoded region. Through the use of multiplex allele-specific colony PCR (MASC-PCR) 81 integration sites were tested, 12 gave no recombination, 23 sites had a recombination frequency of ~10^-7, 38 sites with ~10^-6, and 8 sites with ~10^-5 recombination frequencies.	+	Through the use of Hierarchical Conjugative Assembly (CAGE), merging the modifications from MAGE was accomplished. This technique is dependent upon bacterial conjugation to transfer the modified segments. The oriT sequence typically used for conjugation is fused with kanR for integration into E. coli genome by the λ-Red mediated dsDNA recombination. This makes for precise control of conjugation initiation location and use of a ~2-kb casette in place of the 30-kb Hfr fragment(conjugation factors are maintained on recipients as well). <cite>Isaacs2011</cite> [[Image:Figure_4a.jpg\|thumb\|300px\|Right\|The site specific conjugation of donor and plasmid. <cite>Isaacs2009</cite>]] The 32 strains were converted to 16 pairs for conjugation, with a donor strain transferring its genomic region to a recipient. Selectable markers control placement of transfered DNA, in the donor strain the recoded region was flanked upstream by the oriT-Kan cassete and downstream by a positive selectable marker (ie: antibiotic resistance). The recipient strain contained a different positive selectable marker and a positive-negative selectable marker (tolC), about the recoded region. Through the use of multiplex allele-specific colony PCR (MASC-PCR) 81 integration sites were tested, 12 gave no recombination, 23 sites had a recombination frequency of ~10^-7, 38 sites with ~10^-6, and 8 sites with ~10^-5 recombination frequencies.

	===The "amberless" E. Coli===		===The "amberless" E. Coli===

James L. Bachman: /* Codon Modification Strategy */

2012-04-09T18:09:33Z

Codon Modification Strategy

←Older revision		Revision as of 18:09, 9 April 2012
Line 33:		Line 33:
	===Codon Modification Strategy===		===Codon Modification Strategy===
	[[Image:CAGE_Method.jpg\|thumb\|300px\|Right\|Strategy for splitting up genome and combining codon modifications.<cite>Isaacs2009</cite>]]		[[Image:CAGE_Method.jpg\|thumb\|300px\|Right\|Strategy for splitting up genome and combining codon modifications.<cite>Isaacs2009</cite>]]
-	The genome of MG1655 (a mismatch repair-deficient strain) with 314 TAG stop codons (at least 43 essential genes and 39 TAG codon overlaps of ORF of other genes) was split up into 32 regions, 31 of which had 10 TAG stop codons and one with four. This strategy was selected because pools of at least 10 oligos have been shown to have high replacement efficiency and that the total number of cell divisons to achieve replacement. MAGE was used to introduce all 10 TAG=>TAA codon modifications. The 314 oligos encoding these specific mutations were designed computationally on the basis of previous MAGE experiments. After 18 cycles of MAGE allelic replacement frequencies were analyzed in 1504 clones (47 clones for each 32 segments). The average replacement frequency was 37 +/- 19% after 18 cycles, with 42% of the population unconverted. Of the remaning population, replacements from 1-10 alleles were observed. It was apparent two types of cells were shown to have been evolving: one that ready permits replacement and one largely resistant. It was also shown that no TAG stop codons were ~~essentil~~ for survival or robust growth.<cite>Isaacs2011</cite>	+	The genome of MG1655 (a mismatch repair-deficient strain) with 314 TAG stop codons (at least 43 essential genes and 39 TAG codon overlaps of ORF of other genes) was split up into 32 regions, 31 of which had 10 TAG stop codons and one with four. This strategy was selected because pools of at least 10 oligos have been shown to have high replacement efficiency and that the total number of cell divisons to achieve replacement. MAGE was used to introduce all 10 TAG=>TAA codon modifications. The 314 oligos encoding these specific mutations were designed computationally on the basis of previous MAGE experiments. After 18 cycles of MAGE allelic replacement frequencies were analyzed in 1504 clones (47 clones for each 32 segments). The average replacement frequency was 37 +/- 19% after 18 cycles, with 42% of the population unconverted. Of the remaning population, replacements from 1-10 alleles were observed. It was apparent two types of cells were shown to have been evolving: one that ready permits replacement and one largely resistant. It was also shown that no TAG stop codons were essential for survival or robust growth.<cite>Isaacs2011</cite>

	===Assembling Stop Codon Modifications===		===Assembling Stop Codon Modifications===

James L. Bachman: /* CAGE "Amberless" E. Coli */

2012-04-09T18:06:58Z

CAGE "Amberless" E. Coli

←Older revision		Revision as of 18:06, 9 April 2012
Line 29:		Line 29:

	==CAGE "Amberless" E. Coli==		==CAGE "Amberless" E. Coli==
-	Hierarchical Conjugative Assembly (CAGE) was developed in George Church's lab as a means to merge sets of codon modifications from MAGE into genomes (1/4 size of E. coli genome) with 80 precise ~~changes~~. It was demonstrated that synonymous codon changes can be combined into strains without lethal effects on the cell population. E. coli has three stop codons and two release factors. Release factor 1 (RF1) recognizes UAA and UAG, while RF2 recognizes UAA and UGA. The hypothesis was that replacing all TAG codons with TAA codons, the genetic dependence on RF1 would be abolished and the newly introduced TAA codons would be recognized by RF2. The group sought to test if E. coli that had replaced all 314 TAG stop codons with TAA codons would be viable. (MG1655 genome) <cite>Isaacs2011</cite>	+	Hierarchical Conjugative Assembly (CAGE) was developed in George Church's lab as a means to merge sets of codon modifications from MAGE into genomes (each 1/4 size of E. coli genome) with 80 precise codon modifications. It was demonstrated that synonymous codon changes can be combined into strains without lethal effects on the cell population. E. coli has three stop codons and two release factors. Release factor 1 (RF1) recognizes UAA and UAG, while RF2 recognizes UAA and UGA. The hypothesis was that replacing all TAG codons with TAA codons, the genetic dependence on RF1 would be abolished and the newly introduced TAA codons would be recognized by RF2. The group sought to test if E. coli that had replaced all 314 TAG stop codons with TAA codons would be viable. (MG1655 genome) <cite>Isaacs2011</cite>

	===Codon Modification Strategy===		===Codon Modification Strategy===