Biomod/2013/Harvard/introduction: Difference between revisions
(→Input) |
(→Input) |
||
| Line 3: | Line 3: | ||
==Input== | ==Input== | ||
The goal of the input team is to improve the binding and switching activity of the BlaCaM protein with respect to a previously non-functional analyte. A successful evolution would demonstrate the ability of the BlaCaM switch to sense different molecules, highlighting its potential as a biosensor component. BlaCaM is a fusion of two proteins, a calmodulin center with two halves of β-lactamase attached to the N- and C-termini. Calmodulin displays large conformational changes when it binds to both calcium and varying peptides. These conformational changes adjust the position of the two β-lactamase halves relative to each other, greatly affecting the activity of the enzyme. The ability to turn on or off the activity of the attached enzyme depending on the presence of an analyte gives the BlaCaM protein the ability to act as a sensor. By evolving BlaCaM to bind to different peptides or small molecules, the protein can be made into a sensor for a wide array of compounds. Adapting the BlaCaM switch is performed via directed evolution, where random mutations of the switch are screened and selected for increased effectiveness, and this process is iterated until a satisfactory new switch has been created. | The goal of the input team is to improve the binding and switching activity of the BlaCaM protein with respect to a previously non-functional analyte. A successful evolution would demonstrate the ability of the BlaCaM switch to sense different molecules, highlighting its potential as a biosensor component. BlaCaM is a fusion of two proteins, a calmodulin center with two halves of β-lactamase attached to the N- and C-termini. Calmodulin displays large conformational changes when it binds to both calcium and varying peptides. These conformational changes adjust the position of the two β-lactamase halves relative to each other, greatly affecting the activity of the enzyme. The ability to turn on or off the activity of the attached enzyme depending on the presence of an analyte gives the BlaCaM protein the ability to act as a sensor. By evolving BlaCaM to bind to different peptides or small molecules, the protein can be made into a sensor for a wide array of compounds. Adapting the BlaCaM switch is performed via directed evolution, where random mutations of the switch are screened and selected for increased effectiveness, and this process is iterated until a satisfactory new switch has been created. | ||
The main method driving our directed evolution is bacterial display. In bacterial display, the mutated BlaCaM proteins are displayed upon the surface of bacterial, allowing the proteins to interact with compounds outside of the bacterial. To move the proteins to the outside of the cell, their genes are cloned into bacteria fused to a transporter protein that facilitates transport from the cytoplasm to the surface of the tell. These displaying bacteria are then washed over a media displaying anchored versions of our analyte. Displayed proteins that have been successfully mutated to bind to the analyte will remain fixed to the media, while unsuccessful mutations will be washed away. The bound bacteria are then concentrated, isolated, and analyzed to determine the sequence of the evolved proteins displayed upon their surfaces. | |||
 |  | ||
==Output== | ==Output== | ||
</div> | </div> | ||
Revision as of 07:55, 19 June 2013
<html>
<head>
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'>
</head>
<style>
body {
font-family: 'Open Sans', sans-serif; overflow-y: scroll;
}
.container {
background-color: #ffffff; margin-top:0px
} .OWWNBcpCurrentDateFilled { display: none; }
h1 {
font-size: 36px; line-height: 36px; padding-top: 5px; border-bottom-width: 0;
}
h3 {
font-size: 18px;
}
h5
{
font-family: 'Open Sans', sans-serif; font-size: 11px; font-style: normal; text-align: center; margin:0px; padding:0px;
}
- column-content
{
/* Uncomment to Dewikify width: 0px; float: left; */ margin: 0 0 0 0; padding: 0;
} .firstHeading {
display:none; width:0px;
}
- column-one
{
display:none; width:0px; padding-top: 35px; background-color: #ffffff;
}
- globalWrapper
{
width: 900px; background-color: #ffffff; margin-left: auto; margin-right: auto
}
- content
{
margin-left: 0px; margin-top: 0px; padding-top: 0px; align: center; /*padding: 12px 12px 12px 12px; width: 30%; background-color: #ffffff; border: 0; */
}
- bodyContent
{
width: 850px; align: center; background-color: #fffffff;
}
- column-content
{
width: 900px; background-color: #ffffff;
}
- footer
{
position: center; width: 900px;
} @media screen {
body { background: #000000 0 0 no-repeat; /* changed default background */ }
}
- menu
{
align: left; width: 10em; padding: 0px 10px 10px 10px; background-color: #FFFFFF; float: left;
}
- pagecontent
{
width: 620px; min-height: 400px; float: left; margin-left: 0px;
}
.group:after {
content: ""; display: table; clear: both;
}
.editsection {
/*display: none*/
}
a:link {color:#FF6060;}
a:active {color:#B24343; }
a:hover {color:#B24343; text-decoration: none}
a:visited {color:#FF6060;} /* visited link */
/*Expanding list*/
- exp { list-style: none; }
- exp li {
height: 1.8em; border-top: 1px solid #dedede; margin: 0 0 0 0; padding-top: .2em
}
- exp li:hover { background-color: #F8F8F8}
- exp li a:hover { display: block }
</style> </html>
HARVARD
BIODESIGN
BIOMOD 2013
-
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard"><li>HOME</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/introduction"><li>INTRODUCTION</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/design"><li>DESIGN</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/methods"><li>METHODS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/protocols"><li>PROTOCOLS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/team"><li>TEAM</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/lab"><li>LAB NOTEBOOK</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/results"><li>RESULTS</li></a></html>
<html><a href="http://openwetware.org/wiki/Biomod/2013/Harvard/references"><li>REFERENCES</li></a></html>
Introduction
Input
The goal of the input team is to improve the binding and switching activity of the BlaCaM protein with respect to a previously non-functional analyte. A successful evolution would demonstrate the ability of the BlaCaM switch to sense different molecules, highlighting its potential as a biosensor component. BlaCaM is a fusion of two proteins, a calmodulin center with two halves of β-lactamase attached to the N- and C-termini. Calmodulin displays large conformational changes when it binds to both calcium and varying peptides. These conformational changes adjust the position of the two β-lactamase halves relative to each other, greatly affecting the activity of the enzyme. The ability to turn on or off the activity of the attached enzyme depending on the presence of an analyte gives the BlaCaM protein the ability to act as a sensor. By evolving BlaCaM to bind to different peptides or small molecules, the protein can be made into a sensor for a wide array of compounds. Adapting the BlaCaM switch is performed via directed evolution, where random mutations of the switch are screened and selected for increased effectiveness, and this process is iterated until a satisfactory new switch has been created.
The main method driving our directed evolution is bacterial display. In bacterial display, the mutated BlaCaM proteins are displayed upon the surface of bacterial, allowing the proteins to interact with compounds outside of the bacterial. To move the proteins to the outside of the cell, their genes are cloned into bacteria fused to a transporter protein that facilitates transport from the cytoplasm to the surface of the tell. These displaying bacteria are then washed over a media displaying anchored versions of our analyte. Displayed proteins that have been successfully mutated to bind to the analyte will remain fixed to the media, while unsuccessful mutations will be washed away. The bound bacteria are then concentrated, isolated, and analyzed to determine the sequence of the evolved proteins displayed upon their surfaces. 
