Difference between revisions of "A Simple Method for Highly Evolved Enzymes"
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==The Problem== | ==The Problem== | ||
| − | The team conducted two rounds of directed evolution on the hEcCM gene | + | The team conducted two rounds of directed evolution on the hEcCM gene using error-prone PCR and DNA shuffling to create the mutant library of hEcCM and selected for increased catalytic activity by testing the enzyme ''in vitro'' in an auxotrophic strain of ''E. coli''. However, the resulting form of the evolved hEcCM, tEcCM , lacked a significant increase in enzymatic activity when quantified by their enzyme-specific assays ('''FIGURE X'''). |
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==The Solution in Theory== | ==The Solution in Theory== | ||
Revision as of 04:52, 5 December 2007
Contents
The Goal
Researchers Neunschwander et al. had been working with the enzyme chorismate mutase (EcCM), which is responsible for converting the metabolic intermediate chorismate to prephanate. For an unspecified reason, the researchers desired the insertion of 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. The team hoped to utilize directed evolution to restore enzymatic activity to the new form of the enzyme, hEcCM.
The Problem
The team conducted two rounds of directed evolution on the hEcCM gene using error-prone PCR and DNA shuffling to create the mutant library of hEcCM and selected for increased catalytic activity by testing the enzyme in vitro in an auxotrophic strain of E. coli. However, the resulting form of the evolved hEcCM, tEcCM , lacked a significant increase in enzymatic activity when quantified by their enzyme-specific assays (FIGURE X).
The Solution in Theory
The authors hypothesized that if they were somehow able increase selective pressure for catalytic activity in their selection tests, directed evolution would be much more effective at restoring the enzymatic activity of hEcCM.
The Experiment
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.
The researchers changed the expression of hEcCM in two ways to keep the enzyme at low concentrations. First, they put hEcCM gene behind a Ptet promoter cassette, which allowed the team to control intercellur levels of the enzyme as a function of tetracycline concentrations introduced externally. Secondly, they inserted an ssrA tag behind the enzyme, making the enzyme susceptible to degradation by the protease ClpXP.
To test the effectiveness of this new method, they created a mutated library of the hEcCM 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. As predicted, lower levels of tetracycline resulted in lower levels of complementation in the auxotrophic strain. Of the six clones selected at a complementation frequency of 2 +/- 1%.
The enzymatic activity of the most effective hEcCM hEcCM mutant, EcCM-200/4, can be seen after one round of directed evolution in figure X. The researchers have found EcCM-200/4 to have a satisfactory restoration in catalytic activity for their purposes.
Advantages of the Method
The method Neuenschwander et al. proved useful and restoring enzymating efficiency to their synthetically-engineered catalyst.
Modularity – change the concentration of the protein depending on the protein.
This method appears to be very useful for improving individuals proteins.
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.
Requires knowledge of gene to function
In addition, the researchers reported a high number of false positives – E. coli cells which had received two or more copies of the heEcCM expression plasmid – when concentrations of tetracycline to induce hEcCM gene were too low.
