https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Directed_protein_evolution&feed=atom&action=historyCH391L/S14/Directed protein evolution - Revision history2024-03-28T23:37:58ZRevision history for this page on the wikiMediaWiki 1.21.1https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Directed_protein_evolution&diff=96390&oldid=prevNathan Shin at 05:15, 3 March 20142014-03-03T05:15:14Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>=== Amplification ===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>=== Amplification ===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Once a variant is selected, in vitro or in vivo methods are used to amplify the desired gene.  </div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Once a variant is selected, in vitro <ins class="diffchange diffchange-inline">methods, such as PCR, </ins>or in vivo methods<ins class="diffchange diffchange-inline">, which simply allow the colonies to propagate, </ins>are used to amplify the desired gene.  </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>== Applications of Directed Protein Evolution ==</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>== Applications of Directed Protein Evolution ==</div></td></tr>
</table>Nathan Shinhttps://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Directed_protein_evolution&diff=96389&oldid=prevNathan Shin at 05:13, 3 March 20142014-03-03T05:13:28Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>== What is directed protein evolution? ==</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>== What is directed protein evolution? ==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Evolution in nature consists of cycles of mutagenesis of an organism's DNA, environmental selection of the most fit mutants, and amplification of those favorable mutations by reproduction. This evolutionary principle has been "successfully exploited by humans over millennia to breed plants and animal." <cite>Jackel2008</cite> However, more recently, researchers have utilized this evolutionary principle to evolve proteins at the molecular level, rather than evolving entire organisms. This <del class="diffchange diffchange-inline">methods </del>is referred to as directed protein evolution. Directed protein evolution is a powerful tool that is able to either optimize preexisting proteins or create novel proteins altogether. By developing a strong selective force, proteins are able to exhibit their plasticity by adapting to the environmental conditions set in a lab. By using directed protein evolution, a researcher does not need to completely understand the underpinnings of protein structures and various folds. Instead, iterative rounds of mutations and artificial selection generates proteins with desirable functions.  </div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Evolution in nature consists of cycles of mutagenesis of an organism's DNA, environmental selection of the most fit mutants, and amplification of those favorable mutations by reproduction. This evolutionary principle has been "successfully exploited by humans over millennia to breed plants and animal." <cite>Jackel2008</cite> However, more recently, researchers have utilized this evolutionary principle to evolve proteins at the molecular level, rather than evolving entire organisms. This <ins class="diffchange diffchange-inline">method </ins>is referred to as directed protein evolution. Directed protein evolution is a powerful tool that is able to either optimize preexisting proteins or create novel proteins altogether. By developing a strong selective force, proteins are able to exhibit their plasticity by adapting to the environmental conditions set in a lab. By using directed protein evolution, a researcher does not need to completely understand the underpinnings of protein structures and various folds. Instead, iterative rounds of mutations and artificial selection generates proteins with desirable functions.  </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Because we don't have nature's advantage of millions of years, we must artificially create large libraries of randomly mutagenated copies of the genes of interest.  Common methods used for library construction include error-prone PCR and DNA shuffling.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Because we don't have nature's advantage of millions of years, we must artificially create large libraries of randomly mutagenated copies of the genes of interest.  Common methods used for library construction include error-prone PCR and DNA shuffling.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>'''Error-Prone PCR''' involves adding agents that increase the error-rate of DNA polymerase in PCR. Error-prone PCR methods generally include a higher concentration of MgCl2, which helps stabilize the non-complementary pairs <cite>Yuan2005</cite>. Other common methods to increase the error-rate of DNA polymerase are adding Mn2+ and varying the ratio of nucleotides in the reaction. The <del class="diffchange diffchange-inline">following </del>methods have been able to increase the error-rate of DNA polymerase from 0.11% to 2% <cite>Yuan2005</cite>. A disadvantage of error-prone PCR, however, comes from the fact that very few mutations are beneficial mutations. As a result, only single beneficial mutants are added during every generation of protein evolution.  </div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>'''Error-Prone PCR''' involves adding agents that increase the error-rate of DNA polymerase in PCR. Error-prone PCR methods generally include a higher concentration of MgCl2, which helps stabilize the non-complementary pairs <cite>Yuan2005</cite>. Other common methods to increase the error-rate of DNA polymerase are adding Mn2+ and varying the ratio of nucleotides in the reaction. The <ins class="diffchange diffchange-inline">previously explained </ins>methods have been able to increase the error-rate of DNA polymerase from 0.11% to 2% <cite>Yuan2005</cite>. A disadvantage of error-prone PCR, however, comes from the fact that very few mutations are beneficial mutations. As a result, only single beneficial mutants are added during every generation of protein evolution.  </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>'''DNA Shuffling''' circumvents the issue presented in error-prone PCR. First, a library of a gene of interest is created by error-prone PCR. Once the desired functions are screened for, the DNA of these clones are shuffled together to amass a large number of beneficial mutations.  As these clones are bred iteratively, the frequency with which dramatic phenotype improvements occur increases dramatically, compared to error-prone PCR alone <cite>Yuan2005</cite>.  </div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>'''DNA Shuffling''' circumvents the issue presented in error-prone PCR. First, a library of a gene of interest is created by error-prone PCR. Once the desired functions are screened for, the DNA of these clones are shuffled together to amass a large number of beneficial mutations.  As these clones are bred iteratively, the frequency with which dramatic phenotype improvements occur increases dramatically, compared to error-prone PCR alone <cite>Yuan2005</cite>.  </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>=== Screening and selection ===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>=== Screening and selection ===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>After a sizable library of DNA is constructed, they must be expressed, either in vivo or in vitro, and evaluated for their ability to perform a particular function.This portion is the greatest challenge in directed protein evolution and lies at the crux of the experiment. To select for these desirable functions, high-throughput assays are used. To develop a high-throughput assay, there are two major hurdles that a protein engineer must navigate. First, the proteins that are being assayed for must be linked to the DNA that codes the polypeptide sequence. This is because DNA is much easier to isolate, sequence, and amplify. Second, a high-throughput assay must be developed that is compatible with the connection between protein and DNA. There are several main methods for tagging the proteins with DNA.  </div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>After a sizable library of DNA is constructed, they must be expressed, either in vivo or in vitro, and evaluated for their ability to perform a particular function.This portion is the greatest challenge in directed protein evolution and lies at the crux of the experiment<ins class="diffchange diffchange-inline">. The protein engineer is able to either select or screen for a desirable trait. The major difference of a selection and a screen is the fact that a selection simply gets rid of all of the variants that do not express a certain protein through the use of selective agents, such as antibiotics. Screens, however, are hand-picked variants that express a desired level of expression or another specific trait</ins>. To select for these desirable functions, high-throughput assays are used. To develop a high-throughput assay, there are two major hurdles that a protein engineer must navigate. First, the proteins that are being assayed for must be linked to the DNA that codes the polypeptide sequence. This is because DNA is much easier to isolate, sequence, and amplify. Second, a high-throughput assay must be developed that is compatible with the connection between protein and DNA. There are several main methods for tagging the proteins with DNA.  </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>'''Physical Linkage Method''' simply creates a physical link between the protein and the DNA that encodes it. Several tactics used to create this physical link include phage display, ribosome display, peptide on plasmid, and cell surface display <cite>Lin2002</cite>.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>'''Physical Linkage Method''' simply creates a physical link between the protein and the DNA that encodes it. Several tactics used to create this physical link include phage display, ribosome display, peptide on plasmid, and cell surface display <cite>Lin2002</cite>.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>=== Amplification ===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>=== Amplification ===</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">Once a variant is selected, in vitro or in vivo methods are used to amplify the desired gene. </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>== Applications of Directed Protein Evolution ==</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>== Applications of Directed Protein Evolution ==</div></td></tr>
</table>Nathan Shinhttps://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Directed_protein_evolution&diff=96308&oldid=prevNathan Shin at 19:59, 17 February 20142014-02-17T19:59:48Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>*study mechanisms of adaptions and protein structures <cite>Kuhlman2003</cite></div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>*study mechanisms of adaptions and protein structures <cite>Kuhlman2003</cite></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>== <del class="diffchange diffchange-inline">PACE (phage-assisted continuous evolution) </del>==</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>== <ins class="diffchange diffchange-inline">Novel Methods for Directed Evolution </ins>==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>[[Image:PACE.jpg | thumb | center | 575 px | Overview of PACE <cite>Esvelt2011</cite>.]]</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>[[Image:PACE.jpg | thumb | center | 575 px | Overview of PACE <cite>Esvelt2011</cite>.]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Although the methods mentioned have proved to be successful, a huge limitation is the amount of time that it takes for each step of the process. The method above is very time-consuming and requires frequent human intervention. As a response, a novel method of continuous protein evolution was developed.  </div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Although the methods mentioned have proved to be successful, a huge limitation is the amount of time that it takes for each step of the process. The method above is very time-consuming and requires frequent human intervention. As a response, a novel method of continuous protein evolution was developed<ins class="diffchange diffchange-inline">, PACE (phage-assisted continuous evolution)</ins>.  </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>A lagoon of bacteriophage are present and E.coli are moved through this "lagoon" at a rate faster than which they can divide, but long enough for the phage to infect them and divide. These phages contain the gene of interest. To preface, phages require the pIII gene in order for it to take over the host cell, replicate, and eventually lyse. Experimentalists deleted this gene from the phages' genome and placed it into an accessory plasmid located in the host cells. Upstream from the pIII gene in the accessory plasmid, there is a selective agent (i.e promoter sequences, protein-protein recognition, etc). Therefore, if the gene product of the phage is able to induce the expression of the pIII gene in the accessory plasmid, the phages replicate, lyse the cell, and reenter the "lagoon". If the phages' gene product does not induce the upstream promoter, however, the phage does not replicate and is instead flushed away with the other bacteria that didn't successfully express the pIII gene. The mutagenesis comes from an arabinose-inducible mutagenesis plasmid; it elevates the error rate by suppressing proofreading and enhancing error-prone lesion bypass <cite>Esvelt2011</cite>.  </div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>A lagoon of bacteriophage are present and E.coli are moved through this "lagoon" at a rate faster than which they can divide, but long enough for the phage to infect them and divide. These phages contain the gene of interest. To preface, phages require the pIII gene in order for it to take over the host cell, replicate, and eventually lyse. Experimentalists deleted this gene from the phages' genome and placed it into an accessory plasmid located in the host cells. Upstream from the pIII gene in the accessory plasmid, there is a selective agent (i.e promoter sequences, protein-protein recognition, etc). Therefore, if the gene product of the phage is able to induce the expression of the pIII gene in the accessory plasmid, the phages replicate, lyse the cell, and reenter the "lagoon". If the phages' gene product does not induce the upstream promoter, however, the phage does not replicate and is instead flushed away with the other bacteria that didn't successfully express the pIII gene. The mutagenesis comes from an arabinose-inducible mutagenesis plasmid; it elevates the error rate by suppressing proofreading and enhancing error-prone lesion bypass <cite>Esvelt2011</cite>.  </div></td></tr>
</table>Nathan Shinhttps://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Directed_protein_evolution&diff=96180&oldid=prevNathan Shin: Created page with "== What is directed protein evolution? == Evolution in nature consists of cycles of mutagenesis of an organism's DNA, environmental selection of the most fit mutants, and amp..."2014-02-10T03:16:37Z<p>Created page with "== What is directed protein evolution? == Evolution in nature consists of cycles of mutagenesis of an organism's DNA, environmental selection of the most fit mutants, and amp..."</p>
<p><b>New page</b></p><div>== What is directed protein evolution? ==<br />
<br />
Evolution in nature consists of cycles of mutagenesis of an organism's DNA, environmental selection of the most fit mutants, and amplification of those favorable mutations by reproduction. This evolutionary principle has been "successfully exploited by humans over millennia to breed plants and animal." <cite>Jackel2008</cite> However, more recently, researchers have utilized this evolutionary principle to evolve proteins at the molecular level, rather than evolving entire organisms. This methods is referred to as directed protein evolution. Directed protein evolution is a powerful tool that is able to either optimize preexisting proteins or create novel proteins altogether. By developing a strong selective force, proteins are able to exhibit their plasticity by adapting to the environmental conditions set in a lab. By using directed protein evolution, a researcher does not need to completely understand the underpinnings of protein structures and various folds. Instead, iterative rounds of mutations and artificial selection generates proteins with desirable functions. <br />
<br />
<br />
== Process ==<br />
[[Image:process_directed_evolution.jpg | thumb | right | 300 px | Overview of Directed Evolution <cite>Esvelt2011</cite>.]]<br />
Each round of directed protein evolution consists of three steps: generating large libraries of randomly mutagenated copies of the gene of interest, appropriately selecting and screening for the desired phenotype or for a particular function, and amplifying the selected genes <cite>Jackel2008</cite>.<br />
<br />
=== Library construction ===<br />
<br />
Because we don't have nature's advantage of millions of years, we must artificially create large libraries of randomly mutagenated copies of the genes of interest. Common methods used for library construction include error-prone PCR and DNA shuffling.<br />
<br />
'''Error-Prone PCR''' involves adding agents that increase the error-rate of DNA polymerase in PCR. Error-prone PCR methods generally include a higher concentration of MgCl2, which helps stabilize the non-complementary pairs <cite>Yuan2005</cite>. Other common methods to increase the error-rate of DNA polymerase are adding Mn2+ and varying the ratio of nucleotides in the reaction. The following methods have been able to increase the error-rate of DNA polymerase from 0.11% to 2% <cite>Yuan2005</cite>. A disadvantage of error-prone PCR, however, comes from the fact that very few mutations are beneficial mutations. As a result, only single beneficial mutants are added during every generation of protein evolution. <br />
<br />
'''DNA Shuffling''' circumvents the issue presented in error-prone PCR. First, a library of a gene of interest is created by error-prone PCR. Once the desired functions are screened for, the DNA of these clones are shuffled together to amass a large number of beneficial mutations. As these clones are bred iteratively, the frequency with which dramatic phenotype improvements occur increases dramatically, compared to error-prone PCR alone <cite>Yuan2005</cite>. <br />
<br />
<br />
=== Screening and selection ===<br />
<br />
After a sizable library of DNA is constructed, they must be expressed, either in vivo or in vitro, and evaluated for their ability to perform a particular function.This portion is the greatest challenge in directed protein evolution and lies at the crux of the experiment. To select for these desirable functions, high-throughput assays are used. To develop a high-throughput assay, there are two major hurdles that a protein engineer must navigate. First, the proteins that are being assayed for must be linked to the DNA that codes the polypeptide sequence. This is because DNA is much easier to isolate, sequence, and amplify. Second, a high-throughput assay must be developed that is compatible with the connection between protein and DNA. There are several main methods for tagging the proteins with DNA. <br />
<br />
'''Physical Linkage Method''' simply creates a physical link between the protein and the DNA that encodes it. Several tactics used to create this physical link include phage display, ribosome display, peptide on plasmid, and cell surface display <cite>Lin2002</cite>.<br />
<br />
'''Compartmentalization Method''' restricts each protein and its associated DNA component into distinct compartments. This method of linking the genotype to the phenotype works especially well with assays that are based on enzyme catalysis. These assays include cell based assays and liposome-based assays <cite>Lin2002</cite>.<br />
<br />
'''Spatially Addressable Methods''' link the identity of a protein to a specific address in space. This way, if a desirable function of a protein is identified, the particular address in space can be linked back to the DNA sequence that encodes it. These assays include Microtiter-plate assays and protein chips <cite>Lin2002</cite>. <br />
<br />
=== Amplification ===<br />
<br />
== Applications of Directed Protein Evolution ==<br />
*Develop novel proteins that can perform functions outside of the context of the cell's survival<br />
*Optimize the function of proteins<br />
**thermostability <cite>Giver1998</cite><br />
**solvent tolerance <cite>Patnaik2002</cite><br />
**pH tolerance<br />
**increase activity/selectivity <cite>Song2002</cite><br />
*study mechanisms of adaptions and protein structures <cite>Kuhlman2003</cite><br />
<br />
== PACE (phage-assisted continuous evolution) ==<br />
<br />
[[Image:PACE.jpg | thumb | center | 575 px | Overview of PACE <cite>Esvelt2011</cite>.]]<br />
<br />
Although the methods mentioned have proved to be successful, a huge limitation is the amount of time that it takes for each step of the process. The method above is very time-consuming and requires frequent human intervention. As a response, a novel method of continuous protein evolution was developed. <br />
<br />
A lagoon of bacteriophage are present and E.coli are moved through this "lagoon" at a rate faster than which they can divide, but long enough for the phage to infect them and divide. These phages contain the gene of interest. To preface, phages require the pIII gene in order for it to take over the host cell, replicate, and eventually lyse. Experimentalists deleted this gene from the phages' genome and placed it into an accessory plasmid located in the host cells. Upstream from the pIII gene in the accessory plasmid, there is a selective agent (i.e promoter sequences, protein-protein recognition, etc). Therefore, if the gene product of the phage is able to induce the expression of the pIII gene in the accessory plasmid, the phages replicate, lyse the cell, and reenter the "lagoon". If the phages' gene product does not induce the upstream promoter, however, the phage does not replicate and is instead flushed away with the other bacteria that didn't successfully express the pIII gene. The mutagenesis comes from an arabinose-inducible mutagenesis plasmid; it elevates the error rate by suppressing proofreading and enhancing error-prone lesion bypass <cite>Esvelt2011</cite>. <br />
<br />
== Future Direction == <br />
<br />
* Further investigation of the evolution of proteins will give insight to protein engineers who seek to rationally design proteins <br />
* Quicker and more accurate screenings <br />
* Engineering networks of interacting proteins<br />
<br />
==References==<br />
<br />
<biblio><br />
#Jackel2008 Christian Jäckel, Peter Kast, and Donald Hilvert. Protein Design by Directed Evolution. Annual Review of Biophysics, 2008.<br />
#Yuan2005 Ling Yuan1, Itzhak Kurek, James English and Robert Keenan. Laboratory-Directed Protein Evolution. Microbiol. Mol. Biol. Rev. September 2005 vol. 69 no. 3 373-392.<br />
#Lin2002 Lin, H. and Cornish, V. W. (2002), Screening and Selection Methods for Large-Scale Analysis of Protein Function. Angew. Chem. Int. Ed., 41: 4402–4425. doi: 10.1002/1521-3773(20021202)41:23<4402::AID-ANIE4402>3.0.CO;2-H<br />
#Giver1998 Giver L, Gershenson A, Freskgard PO, Arnold FH. Directed evolution of a thermostable esterase. Proc Natl Acad Sci USA, 1998.<br />
#Patnaik2002 Patnaik, R., S. Louie, V. Gavrilovic, K. Perry, W. P. Stemmer, C. M. Ryan, and S. del Cardayre. 2002. Genome shuffling of Lactobacillus for improved acid tolerance.<br />
#Peters2003 Matthew W. Peters, Peter Meinhold, Anton Glieder,and Frances H. Arnold†, Regio- and Enantioselective Alkane Hydroxylation with Engineered Cytochromes P450 BM-3. <br />
#Song2002 Song, J. K., B. Chung, Y. H. Oh, and J. S. Rhee. 2002. Construction of DNA-shuffled and incrementally truncated libraries by a mutagenic and unidirectional reassembly method: changing from a substrate specificity of phospholipase to that of lipase. Appl. Environ. Microbiol. 68:6146-6151<br />
#Kuhlman2003 Kuhlman, B., G. Dantas, G. C. Ireton, G. Varani, B. L. Stoddard, and D. Baker. 2003. Design of a novel globular protein fold with atomic-level accuracy. Science 302:1364-1368<br />
#Esvelt2011 Kevin M. Esvelt, Jacob C. Carlson & David R. Liu. A system for the continuous directed evolution of biomolecules. Nature 472, 499–503 (28 April 2011) doi:10.1038/nature09929<br />
#Romero2009 Romero PA and Arnold FH. Exploring protein fitness landscapes by directed evolution. Nat Rev Mol Cell Bio, 2009.<br />
<br />
<br />
</biblio></div>Nathan Shin