A Simple Method for Highly Evolved Enzymes - Revision history

Hustone: /* Disadvantages of the Method */

2007-12-06T17:56:33Z

‎Disadvantages of the Method

Hustone: /* Advantages of the Method */

2007-12-06T17:50:57Z

‎Advantages of the Method

Hustone: /* Goals */

2007-12-06T17:48:05Z

‎Goals

Hustone: /* The Experiment */

2007-12-06T17:47:55Z

‎The Experiment

Hustone: /* Goals */

2007-12-06T17:47:41Z

‎Goals

Hustone: /* Background */

2007-12-06T17:47:07Z

‎Background

Hustone: /* Disadvantages of the Method */

2007-12-06T07:21:01Z

‎Disadvantages of the Method

Hustone: /* The Experiment */

2007-12-06T07:05:04Z

‎The Experiment

Hustone: /* The Experiment */

2007-12-06T07:03:29Z

‎The Experiment

Hustone: /* Problems with Directed Evolution */

2007-12-06T06:58:12Z

‎Problems with Directed Evolution

← Older revision		Revision as of 17:56, 6 December 2007
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	# The method requires the knowledge of gene function, which often is not the case.		# The method requires the knowledge of gene function, which often is not the case.
	# This method generates a large number false positives due to some ''E. coli'' receiving two or more copies of the expression plasmid.		# This method generates a large number false positives due to some ''E. coli'' receiving two or more copies of the expression plasmid.
		+
		+	==Source==
		+
		+	[http://www.bio.davidson.edu/courses/synthetic/papers/highly_efficient_enzymes.pdf Neuenschwander, M., M. Butz, C. Heintz & D. Hilvert. 2007. A simple selection strategy for evolving highly efficient enzymes. ''Nature Biotechnology'' 25(10): 1145-1147.]

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 ==Advantages of the Method==
-The method Neuenschwander et al. proved useful and restoring enzymating efficiency to their synthetically-engineered catalyst.
+# The method Neuenschwander et al. proved useful and restoring enzymatic activity to their synthetically-engineered catalyst.
+# The "selection vector" used in this experiment is modular: when selective pressure is too low to improve proteins in other experiments with directed evolution, the "selection vector" can be used to increase selective pressure on the protein for phenotype improvement.
 ==Disadvantages of the Method==

← Older revision		Revision as of 17:48, 6 December 2007
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	The team hoped to utilize directed evolution to restore enzymatic activity to the new form of the enzyme, hEcCM.		The team hoped to utilize directed evolution to restore enzymatic activity to the new form of the enzyme, hEcCM.
−

	===Problems with Directed Evolution===		===Problems with Directed Evolution===

← Older revision		Revision as of 17:47, 6 December 2007
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	The team hoped to utilize directed evolution to restore enzymatic activity to the new form of the enzyme, hEcCM.		The team hoped to utilize directed evolution to restore enzymatic activity to the new form of the enzyme, hEcCM.

−	~~==The Experiment==~~

	===Problems with Directed Evolution===		===Problems with Directed Evolution===

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 Researchers Neunschwander ''et al.'' had been working with the enzyme chorismate mutase (EcCM), which is responsible for converting the metabolic intermediate chorismate to prephanate. The researchers wanted to introduce a “five-amino-acid hinge loop” in one of the enzyme’s helixes. However, this insertion drastically affected the enzyme’s capacity to catalyze the chorismate to prephanate reaction ('''Fig. 1''').
-==Goals==
+==The Experiment==
 The team hoped to utilize directed evolution to restore enzymatic activity to the new form of the enzyme, hEcCM.

← Older revision		Revision as of 17:47, 6 December 2007
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		+	__NOTOC__
		+
	==Background==		==Background==

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 ==Disadvantages of the Method==
-The first major disadvantage of this technique is that it requires the use of auxotrophic organisms to create selective pressure for improvement of the protein of interest. While auxotrophic prokaryotes might be easy to engineer and work with, this job would be much more difficult with eukaryotes that contain many enzymes and proteins of interest to synthetic biology, like plants. In addition, the protein of interest itself must be essential to the life of the organism, which may not be the case with completely novel proteins engineered through synthetic biology.
+# The technique requires the use of auxotropihc organisms to produce the selected pressure required to drive the improvement in the protein of interest.
+# The protein of interest must be essential to the life of the organism which may not be the case with completely novel proteins engineered through synthetic biology.
-Requires knowledge of gene to function
+# The method requires the knowledge of gene function, which often is not the case.
+# This method generates a large number false positives due to some ''E. coli'' receiving two or more copies of the expression plasmid.

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 ===Results===
-To test the effectiveness of this new method, they created a mutated library of the tEcCM gene using error-prone PCR and DNA shuffling and inserted these genes into this new expression vector. These plasmids were then transformed into the auxotrophic strain of E. coli and one round of directed evolution was run at a complementation frequency of 2%. The most efficient mutant of this experiment, EcCM-200/4,
+To test the effectiveness of this new method, they created a mutated library of the tEcCM gene using error-prone PCR and DNA shuffling and inserted these genes into this new expression vector. These plasmids were then transformed into the auxotrophic strain of E. coli and the organisms were subjected to a single round of directed evolution. The most efficient mutant of this experiment, EcCM-200/4,
 displayed a level of enzymatic activity comparable to the original wild-type chorismate mutase gene ('''Fig. 1''').

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 ===Problems with Directed Evolution===
-A mutant library of the hEcCM gene was generated using error-prone PCR and DNA shuffling and  selection was run ''in vivo'' in ''E. coli'' auxotrophic for chorismate mutase. However, two rounds of directed evolution of the hEcCM gene (represented as tEcCM) failed to produce significant increases enzymatic activity ('''Fig. 1''').
+A mutant library of the hEcCM gene was generated using error-prone PCR and DNA shuffling and  selection was run ''in vivo'' in ''E. coli'' auxotrophic for chorismate mutase. However, the hEcCM enzyme produced using this protocol, tEcCM, did not have an increase in enzymatic activity ('''Fig. 1''').
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 ===Finding a Solution===
-The authors hypothesized that if they were able increase selective pressure for catalytic activity in their selection tests, directed evolution would be much more useful for restoring the enzymatic activity of hEcCM.
+The authors hypothesized that if they were able increase selective pressure for catalytic activity during the selection process, directed evolution would be much more effective at restoring the enzymatic activity of hEcCM.
-To achieve their goal, the authors came up with the following solution: transform an auxtrophic strain of E. coli with an expression vector containing the hEcCM gene and devise a way to keep the enzyme at very low concentrations. Inefficient catalyst activity would mean lethality for the cell (a common selection scheme) and low levels of the mutated hEcCM would mean only the most efficient enzymes would make it through the selection scheme.
+To achieve their goal, the authors came up with the following solution: transform an auxtrophic strain of E. coli with an expression vector containing the hEcCM gene and devise a way to keep the enzyme at very low concentrations. Inefficient catalyst activity would result in lethality for the cell  (a common selection scheme). Since the levels of the mutated hEcCM would be kept low, only the most efficient enzymes would survive through the selection scheme.
-The researchers changed the expression of hEcCM in two ways to keep the enzyme at low concentrations. First, they inserted the hEcCM gene behind a Ptet promoter cassette, which allowed the team to control intercellur levels of the enzyme as a function of tetracycline concentration ('''Fig. 2'''). Secondly, they inserted an ssrA tag behind the enzyme, making the enzyme susceptible to degradation by the protease ClpXP ('''Fig. 2''').
+The researchers changed the expression of hEcCM in two ways to keep the enzyme at low concentrations. First, they inserted the hEcCM gene behind a Ptet promoter cassette, which allowed the team to control the cellular levels of the enzyme as a function of tetracycline concentration ('''Fig. 2'''). Secondly, they inserted an ssrA tag behind the enzyme, making the enzyme susceptible to degradation by the protease ClpXP ('''Fig. 2''').
 [[Image:EFFICIENTENZYME.jpg]]

← Older revision		Revision as of 06:58, 6 December 2007
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	===Problems with Directed Evolution===		===Problems with Directed Evolution===

−	A mutant library of the hEcCM gene was generated using error-prone PCR and DNA shuffling and selection was run ''in vivo'' in ~~strains of~~ ''E. coli'' auxotrophic for chorismate mutase. However, two rounds of directed evolution of the hEcCM gene (represented as tEcCM) failed to produce significant increases enzymatic activity ('''Fig. 1''').	+	A mutant library of the hEcCM gene was generated using error-prone PCR and DNA shuffling and selection was run ''in vivo'' in ''E. coli'' auxotrophic for chorismate mutase. However, two rounds of directed evolution of the hEcCM gene (represented as tEcCM) failed to produce significant increases enzymatic activity ('''Fig. 1''').