Semi-Synthetic DNA Shuffling and Doramectin - Revision history

Hustone: /* Background */

2007-12-06T21:26:50Z

‎Background

Hustone: /* Conclusion */

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‎Conclusion

Hustone: /* Sources */

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‎Sources

Hustone: /* Sources */

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‎Sources

Hustone: /* Background */

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‎Background

Hustone: /* Conclusion */

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‎Conclusion

Hustone: /* Conclusion */

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‎Conclusion

Hustone: /* In the Lab */

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‎In the Lab

Hustone: /* The Experiment */

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‎The Experiment

Hustone: /* The Goal */

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‎The Goal

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 Doramectin is a drug used to treat gastrointestinal roundworms, lungworms, eyeworms, grubs, and sucking lice in cattle. The drug is one of several avermectins, compounds used for the treatment of parasites in animals and river blindness in humans. Over 1 billion dollars is spent each year on avermectin derivatives in the United States.
-Avermectins are produced by the soil-borne bacteria ''Streptomyces avermitilis.'' The subspecies ''Streptomyces'' is the largest genus of antibiotic-producing bacteria; erythromycin, neomycin, streptomycin, and tetracycline were all originally derived from species of Streptomyces.
+Avermectins are produced by the soil-borne bacteria ''Streptomyces avermitilis.'' The subspecies [http://en.wikipedia.org/wiki/Streptomyces ''Streptomyces''] is the largest genus of antibiotic-producing bacteria; erythromycin, neomycin, streptomycin, and tetracycline were all originally derived from species of Streptomyces.
 ==The Experiment==

← Older revision		Revision as of 17:55, 6 December 2007
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	==Conclusion==		==Conclusion==

−	Semi-synthetic shuffling proved successful in quickly and efficiently improving the function of the ''aveC'' gene. The method also appears adept for recombining beneficial mutations when using directed evolution to improve genes ('''Fig.''') and allows for some degree of rational input into directed evolution through the use of oligonucleotides in shuffling.	+	Semi-synthetic shuffling proved successful in quickly and efficiently improving the function of the ''aveC'' gene. The method also appears adept for recombining beneficial mutations when using directed evolution to improve genes ('''Fig. 4''') and allows for some degree of rational input into directed evolution through the use of oligonucleotides in shuffling.

← Older revision		Revision as of 17:55, 6 December 2007
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	===Sources===		===Sources===
		+
		+	[http://www.pnas.org/cgi/reprint/91/22/10747 Stemmer, W.P.C. 1994. DNA shuffling by random fragmentation and reassembly: ''in vitro'' recombination for molecular evolution. ''PNAS'' 91: 10747-10751]

	[http://www.bio.davidson.edu/courses/synthetic/papers/doramectin.pdf Stutzman-Engwall, K., S. Conlon, R. Fedechko, H. McArthur, K. Pekrun, Y. Chen, S. Jenne, C. La, N. Trinh, S. Kim, Y. Zhang, R. Fox, C. Gustafsson & A. Krebber. 2005. Semi-synthetic DNA shuffling of ''ave''C leads to improved industrial scale production of doramectin by ''Streptomyces avermitilis''. ''Metabolic Engineering'' 7: 27-37.]		[http://www.bio.davidson.edu/courses/synthetic/papers/doramectin.pdf Stutzman-Engwall, K., S. Conlon, R. Fedechko, H. McArthur, K. Pekrun, Y. Chen, S. Jenne, C. La, N. Trinh, S. Kim, Y. Zhang, R. Fox, C. Gustafsson & A. Krebber. 2005. Semi-synthetic DNA shuffling of ''ave''C leads to improved industrial scale production of doramectin by ''Streptomyces avermitilis''. ''Metabolic Engineering'' 7: 27-37.]
−
−	~~[http://www.pnas.org/cgi/reprint/91/22/10747 Stemmer, W.P.C. 1994. DNA shuffling by random fragmentation and reassembly: ''in vitro'' recombination for molecular evolution. ''PNAS'' 91: 10747-10751]~~

← Older revision		Revision as of 17:54, 6 December 2007
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	[http://www.bio.davidson.edu/courses/synthetic/papers/doramectin.pdf Stutzman-Engwall, K., S. Conlon, R. Fedechko, H. McArthur, K. Pekrun, Y. Chen, S. Jenne, C. La, N. Trinh, S. Kim, Y. Zhang, R. Fox, C. Gustafsson & A. Krebber. 2005. Semi-synthetic DNA shuffling of ''ave''C leads to improved industrial scale production of doramectin by ''Streptomyces avermitilis''. ''Metabolic Engineering'' 7: 27-37.]		[http://www.bio.davidson.edu/courses/synthetic/papers/doramectin.pdf Stutzman-Engwall, K., S. Conlon, R. Fedechko, H. McArthur, K. Pekrun, Y. Chen, S. Jenne, C. La, N. Trinh, S. Kim, Y. Zhang, R. Fox, C. Gustafsson & A. Krebber. 2005. Semi-synthetic DNA shuffling of ''ave''C leads to improved industrial scale production of doramectin by ''Streptomyces avermitilis''. ''Metabolic Engineering'' 7: 27-37.]
		+
		+	[http://www.pnas.org/cgi/reprint/91/22/10747 Stemmer, W.P.C. 1994. DNA shuffling by random fragmentation and reassembly: ''in vitro'' recombination for molecular evolution. ''PNAS'' 91: 10747-10751]

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 ==Background==
-Doramectin is a drug used to treat gastrointestinal roundworms, lungworms, eyeworms, grubs, and sucking lice in cattle. The drug is one of several avermectins, compounds used for the treatment of parasites in animals and river blindness in humans. Over 1 billion dollars is spent each on avermectin derivatives in the United States.
+Doramectin is a drug used to treat gastrointestinal roundworms, lungworms, eyeworms, grubs, and sucking lice in cattle. The drug is one of several avermectins, compounds used for the treatment of parasites in animals and river blindness in humans. Over 1 billion dollars is spent each year on avermectin derivatives in the United States.
 Avermectins are produced by the soil-borne bacteria ''Streptomyces avermitilis.'' The subspecies ''Streptomyces'' is the largest genus of antibiotic-producing bacteria; erythromycin, neomycin, streptomycin, and tetracycline were all originally derived from species of Streptomyces.

← Older revision		Revision as of 17:53, 6 December 2007
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	Semi-synthetic shuffling proved successful in quickly and efficiently improving the function of the ''aveC'' gene. The method also appears adept for recombining beneficial mutations when using directed evolution to improve genes ('''Fig.''') and allows for some degree of rational input into directed evolution through the use of oligonucleotides in shuffling.		Semi-synthetic shuffling proved successful in quickly and efficiently improving the function of the ''aveC'' gene. The method also appears adept for recombining beneficial mutations when using directed evolution to improve genes ('''Fig.''') and allows for some degree of rational input into directed evolution through the use of oligonucleotides in shuffling.
		+
		+
		+
		+	===Sources===
		+
		+	[http://www.bio.davidson.edu/courses/synthetic/papers/doramectin.pdf Stutzman-Engwall, K., S. Conlon, R. Fedechko, H. McArthur, K. Pekrun, Y. Chen, S. Jenne, C. La, N. Trinh, S. Kim, Y. Zhang, R. Fox, C. Gustafsson & A. Krebber. 2005. Semi-synthetic DNA shuffling of ''ave''C leads to improved industrial scale production of doramectin by ''Streptomyces avermitilis''. ''Metabolic Engineering'' 7: 27-37.]

← Older revision		Revision as of 17:46, 6 December 2007
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	==Conclusion==		==Conclusion==

−	Semi-synthetic shuffling proved successful in quickly and efficiently improving the function of the ''aveC'' gene.	+	Semi-synthetic shuffling proved successful in quickly and efficiently improving the function of the ''aveC'' gene. The method also appears adept for recombining beneficial mutations when using directed evolution to improve genes ('''Fig.''') and allows for some degree of rational input into directed evolution through the use of oligonucleotides in shuffling.

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 The researchers sought to engineer a strain of ''S. avermitilis'' that would produce higher yields of the B1 form of doramectin. The team had already identified that the gene ''aveC'' was responsible for the B2:B1 ratio. This knowledge led the team to conduct directed evolution upon the ''aveC'' gene.
-==In the Lab==
+===In the Lab===
 The researchers conducted three rounds of directed evolution on the ''aveC'' gene. To generate a mutant library of the gene, the researchers used the mutagenic agent, Mutazyme, and semi-synthetic DNA shuffling (see following section). The mutant ''aveC'' genes were inserted into gene replacement vectors, which were then transformed into ''S. avermitilis'' cells. These cells were diluted into 96 well plates for high throughput culturing under conditions suitable for doramectin production. Selection of the best mutants was conducted by testing the doramectin output and B2:B1 ratios of each well (quantifiable with [http://en.wikipedia.org/wiki/MS/MS MS/MS]). DNA isolated was isolated from the best mutants and the ''aveC'' gene was amplified using PCR. The amplified DNA from these mutants was recombined using semi-synthetic shuffling, subjected to random mutagenesis, and resubmitted to selection.

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 Avermectins are produced by the soil-borne bacteria ''Streptomyces avermitilis.'' The subspecies ''Streptomyces'' is the largest genus of antibiotic-producing bacteria; erythromycin, neomycin, streptomycin, and tetracycline were all originally derived from species of Streptomyces.
-==The Goal==
+==The Experiment==
 Researchers Stutzman-Engwall ''et al.'' began their experiment with a strain of ''S. avermitilis'' capable of producing doramectin from supplemented cyclohexancaroxylic acid. This strain produced doramectin in two forms: CHC-B1, the most useful form of doramectin, and CHC-B2, a related compound less effective as an antiparasital than CHC-B1. The ratio of ineffective CHC-B2 to effective CHC-B1 produced by this strain was 1:1.