Difference between revisions of "Semi-Synthetic DNA Shuffling and Doramectin"

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DNA shuffling begins by using one of these methods to create a mutant library of the gene of interest. However, while normal directed evolution would only resubmit the best mutant from one round of directed evolution to the next, DNA shuffling involves recombining the genetic material of multiple mutants proven effective by the selection scheme. In this way, DNA shuffling aims to recapture other beneficial mutations which would normally be lost through simple selection of the best mutant.
 
DNA shuffling begins by using one of these methods to create a mutant library of the gene of interest. However, while normal directed evolution would only resubmit the best mutant from one round of directed evolution to the next, DNA shuffling involves recombining the genetic material of multiple mutants proven effective by the selection scheme. In this way, DNA shuffling aims to recapture other beneficial mutations which would normally be lost through simple selection of the best mutant.
  
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[[Image:DNAshufflingtheory.jpg]]
  
 
===Semi-synthetic DNA Shuffling===
 
===Semi-synthetic DNA Shuffling===

Revision as of 02:24, 5 December 2007

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. According to the authors, “avermectin derivatives are the most widely used drugs in animal health and agriculture, with current worldwide sales exceeding 1 billion US dollars.”

Avermectins are produced by the soil-borne bacteria Streptomyces avermitilis. The subspecies Streptomyces is the largest genus of antibiotic-producing bacteri; erythromycin, neomycin, streptomycin, and tetracycline were all originally derived from species of Streptomyces.

The Goal

Researchers Stutzman-Engwall et al. began 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 doramecting, and CHC-B2, a related compound that is less effective as an antiparasital than CHC-B1. The ratio of uneffective CHC-B2 to effective CHC-B1 produced by this strain was 1:1.

The researchers were interested in creating a strain of S. avermitilis capable of producing doramectin with a lower ratio of B2 to B1. Previous research had determined that the avecC gene was principally responsible for the ratio of B2 to B1, which led to team to conduct directed evolution upon this gene.

The Problem

S. avermitilis is a strain of bacteria which grows at very slow rates. Because of the slow growth of this bacterium, researchers reported a single round of directed evolution with S. avermitilis took an estimated 2-3 months.

The Solution in Theory

DNA Shuffling

Because of the slow growth rate of S. avermitilis, the researchers desired a method of directed evolution which could compile more beneficial mutations in the evolved mutant in less time. For this task, the team chose to use a method called DNA shuffling in the first process of directed evolution, genetic randomization.

DNA shuffling, sometimes called sexual PCR, is a way of recreating the event of genetic recombination in vitro. This technique, first described in 1994, has already been proven to be much more effective at compiling multiple beneficial mutations in an evolved mutant when compared to genetic randomization using error-prone PCR or mutagenic agents alone.

DNA shuffling begins by using one of these methods to create a mutant library of the gene of interest. However, while normal directed evolution would only resubmit the best mutant from one round of directed evolution to the next, DNA shuffling involves recombining the genetic material of multiple mutants proven effective by the selection scheme. In this way, DNA shuffling aims to recapture other beneficial mutations which would normally be lost through simple selection of the best mutant.

DNAshufflingtheory.jpg

Semi-synthetic DNA Shuffling

As noted previously, DNA shuffling is technique which has already been proven to be effective for directed evolution. However, the authors of this paper have described a variation on DNA shuffling, which they have termed “semi-synthetic DNA shuffling,” which they believe is even more effective at compiling multiple beneficial mutations in the evolved mutant. The key difference in this technique when compared to normal DNA shuffling is that mutations proven to be effective for the selective purpose are stored as oligonucleotides. These beneficial mutations can then be reintroduced at the beginning of each round of directed evolution by keeping these oligonucleotides at high concentration during DNA shuffling. This process means that, theoretically, the total number of beneficial mutations introduced during DNA shuffling increases with each round of directed evolution. Furthermore, this methods ensures proven beneficial mutations are not lost during mutagenesis or error-prone PCR.

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The Experiment

By conducting 5 rounds of semi-synthetic DNA shuffling on the aveC gene, the researchers were able to create a strain of S. avermitilis capable of producing doramectin with a B2:B1 ratio of 0.07:1.

To determine whether semi-synthetic DNA shuffling was effective at assembling multiple beneficial mutations in output strains, the most productive strains of each round of directed evolution were sequenced. While the best mutants from the first round had only five mutations, the most evolved mutations from the fifth round had X mutations. Thus there was a correlation between rounds of directed evolution and beneficial mutations in evolved aveC as well as rounds of directed evolution and decreases in the B2:B1 ratio.

Advantages of the Method

Semi-synthetic DNA shuffling seems to be a very effective method at speeding up the time required for satisfactory results from directed evolution.

Furthermore, semi-synthetic DNA shuffling allows rational input into directed evolution. If previous research has indicated that a certain mutation has a desirable effect on protein function, this mutation can be inserted into the DNA shuffling scheme as an oligonucleotide.

Disadvantages of the Method

The principal disadvantage of semi-synthetic DNA shuffling is that the method requires knowledge of gene to function relationship for the technique to work.


Its applicability to improvements of phenotypes regulated by many genes has not yet to be explored.


RETURN TO PAPER: Directed Evolution and Synthetic Biology - Hunter Stone