IGEM:IMPERIAL/2008/New/Chassis 1: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
mNo edit summary
 
(13 intermediate revisions by 2 users not shown)
Line 2: Line 2:
<br>
<br>
<br>  
<br>  
 
&nbsp; <font size=6px color=#E5EBFF><b>Why ''B. subtilis''?</b></font>
{{Imperial/Box2|Why ''B. subtilis''?|This page offers a brief overview of how ''B. subtilis'' meets our main project specifications - to a much higher degree than ''E. coli''! You will find information on the main mechanisms behind our biofabricator and some very interesting biology too. If you would like even ''more'' in-depth information, please click on ''Details'' under each section.  
<br><br>
{{Imperial/Box2||This page offers a brief overview of how ''B. subtilis'' meets our main project specifications - to a much higher degree than ''E. coli''! You will find information on the main mechanisms behind our biofabricator and some very interesting biology too. If you would like even ''more'' in-depth information, please click on ''Details'' under each section.  
|}}
|}}


Line 10: Line 11:
Light is the most obvious candidate, as holography allows us to generate complex patterns with well defined edges in 3D. After examining a number of light sensing pathways, we decided to utilise a native pathway involving YtvA, which is a protein used by ''B. subtilis'' to detect blue light. YtvA triggers a cascade of interactions, but some way down the chain, a molecule called sigma B (σ<sub>B</sub>) is produced. This, in turn, boosts the synthesis of YtvA.
Light is the most obvious candidate, as holography allows us to generate complex patterns with well defined edges in 3D. After examining a number of light sensing pathways, we decided to utilise a native pathway involving YtvA, which is a protein used by ''B. subtilis'' to detect blue light. YtvA triggers a cascade of interactions, but some way down the chain, a molecule called sigma B (σ<sub>B</sub>) is produced. This, in turn, boosts the synthesis of YtvA.


We plan to over-express YtvA and use σ<sub>B</sub> as a promoter for genes which stop movement and produce biomaterial. Therefore, when the bacteria detect blue light, those genes will turn on, the bacteria will stop and biomaterial synthesis will begin.|[[Image:Imperial_2008_Holgram_Art.jpg |200px | 3D blue holographic image by sculptor Eileen Borgeson[http://www.eileenborgeson.com/default.htm]]]|}}
We plan to over-express YtvA and use σ<sub>B</sub> as a promoter for genes which stop movement and produce biomaterial. Therefore, when the bacteria detect blue light, those genes will turn on, the bacteria will stop and biomaterial synthesis will begin. <br><br>[[IGEM:IMPERIAL/2008/Prototype/Light| '''>>> Details >>>''']]|[[Image:Imperial_2008_Holgram_Art.jpg |200px | 3D blue holographic image by sculptor Eileen Borgeson[http://www.eileenborgeson.com/default.htm]]}}




{{Imperial/Box2|Motility|To achieve accurate distribution of our biofabricators, we must exert fine control over their motility. Bacteria's primary method of getting about is via flagellar locomotion. A protein ring on the cell membrane is attached to the flagellum and rotates during locomotion, acting like a propeller to push the cell through its environment.
{{Imperial/Box1|Motility|To achieve accurate distribution of our biofabricators, we must exert fine control over their motility. Bacteria's primary method of getting about is via flagellar locomotion. A protein ring on the cell membrane is attached to the flagellum and rotates during locomotion, acting like a propeller to push the cell through its environment.


The precise mechanism of how this works in ''B. subtilis'' has recently been elucidated. The flagella can be detached from the rotor by expression of a clutch molecule that interacts with the flagella and distorts it, so it is disengaged from the rotor protein. Control over the expression of this clutch should allow us to control the bacteria very quickly. When we want the bacteria to stop, we trigger expression of the clutch, which halts movement.
The precise mechanism of how this works in ''B. subtilis'' has recently been elucidated. The flagella can be detached from the rotor by expression of a clutch molecule that interacts with the flagella and distorts it, so it is disengaged from the rotor protein. Control over the expression of this clutch should allow us to control the bacteria very quickly. When we want the bacteria to stop, we trigger expression of the clutch, which halts movement.
Line 19: Line 20:
To draw a parallel with a car, currently available synthetic methods of stopping bacteria are akin to destroying the engine. Our method is analogous to putting the car into neutral - disengaging the engine from the driveshaft. It is an elegant solution that offers us quick control and also the opportunity for quick reversal (putting the car back into "drive").
To draw a parallel with a car, currently available synthetic methods of stopping bacteria are akin to destroying the engine. Our method is analogous to putting the car into neutral - disengaging the engine from the driveshaft. It is an elegant solution that offers us quick control and also the opportunity for quick reversal (putting the car back into "drive").
<br><br>
<br><br>
[[IGEM:IMPERIAL/2008/Prototype/Motility | '''>>> Details >>>''']]|[[Image:B_subtilis_Clutch_Mechanism.png|200px|Motile ''B. subtilis'' cells are powered by interactions between protein complexes, generating torque for locomotion. The protein EpsE acts as a molecular clutch to disengage the flagellar motor, leaving the flagellum intact but unpowered. This quickly halts locomotion[http://www.sciencemag.org/cgi/reprint/320/5883/1599.pdf]]]|}}
[[IGEM:IMPERIAL/2008/Prototype/Motility | '''>>> Details >>>''']]
|[[Image:B_subtilis_Clutch_Mechanism.png|center|400px|Motile ''B. subtilis'' cells are powered by interactions between protein complexes, generating torque for locomotion. The protein EpsE acts as a molecular clutch to disengage the flagellar motor, leaving the flagellum intact but unpowered. This quickly halts locomotion[http://www.sciencemag.org/cgi/reprint/320/5883/1599.pdf]]]}}




Line 29: Line 31:
3D bio-scaffolds are very useful for tissue culture and regenerative medicine, as they offer a suitable 3D enviroment for implanted cells to grow and proliferate. A good analogy would be scaffolding used in the construction industry. Our blue-sky aim is to construct a genetically-engineered machine that can fabricate bio-scaffolds with precise 3D shapes, directed by 3D holography.  
3D bio-scaffolds are very useful for tissue culture and regenerative medicine, as they offer a suitable 3D enviroment for implanted cells to grow and proliferate. A good analogy would be scaffolding used in the construction industry. Our blue-sky aim is to construct a genetically-engineered machine that can fabricate bio-scaffolds with precise 3D shapes, directed by 3D holography.  
<br><br>
<br><br>
[[IGEM:IMPERIAL/2008/Prototype/Biomaterials | '''>>> Details >>>''']]|[[Image:Imperial_2008_Scaffold.jpg|right|200px|3D Scaffold]]}}
[[IGEM:IMPERIAL/2008/Prototype/Biomaterials | '''>>> Details >>>''']]|[[Image:Imperial_2008_Scaffold.jpg|right|300px|3D Scaffold]]}}
 
<br clear="all"><hr>
<br clear="all">
{{Imperial/Box1||So ''B. subtilis'' fulfils our main specifications perfectly, and can be made to meet our minor specifications with relatively ease. On top of that, it does have other benefits, along with some challenges. These are listed on the next page, together with an overview of our development of ''B. subtilis'' as a chassis.
 
 
<hr>
 
 
{{Imperial/Box2||So ''B. subtilis'' fulfils our main specifications perfectly, and can be made to meet our minor specifications with relatively ease. On top of that, it does have other benefits, along with some challenges. These are listed on the next page, together with an overview of our development of ''B. subtilis'' as a chassis.
<br><br>
<br><br>
[[IGEM:IMPERIAL/2008/New/Chassis_2 | '''>>> Benefits ''vs'' Challenges >>>''']]}}
[[IGEM:IMPERIAL/2008/New/Chassis_2 | '''>>> Benefits ''vs'' Challenges >>>''']]}}


{{Imperial/EndPage|Project|Chassis_2}}
{{Imperial/EndPage|Project|Chassis_2}}

Latest revision as of 07:17, 9 October 2008

<html> <style type="text/css"> .firstHeading {display: none;} </style> </html> <html> <style type="text/css"> 

   table.calendar          { margin:0; padding:2px; }

table.calendar td { margin:0; padding:1px; vertical-align:top; } table.month .heading td { padding:1px; background-color:#FFFFFF; text-align:center; font-size:120%; font-weight:bold; } table.month .dow td { text-align:center; font-size:110%; } table.month td.today { background-color:#3366FF } table.month td { 

   border:2px;
   margin:0;
   padding:0pt 1.5pt;
   font-size:8pt;
   text-align:right;
   background-color:#FFFFFF;
   }
  1. bodyContent table.month a { background:none; padding:0 }

.day-active { font-weight:bold; } .day-empty { color:black; } </style> </html>

<html><script language="JavaScript">

var timeout = 250; var closetimer = 0; var ddmenuitem = 0;

// open hidden layer function mopen(id) { // cancel close timer mcancelclosetime(); // close old layer if(ddmenuitem) ddmenuitem.style.visibility = 'hidden'; // get new layer and show it ddmenuitem = document.getElementById(id); ddmenuitem.style.visibility = 'visible'; } // close showed layer function mclose() { if(ddmenuitem) ddmenuitem.style.visibility = 'hidden'; } // go close timer function mclosetime() { closetimer = window.setTimeout(mclose, timeout); } // cancel close timer function mcancelclosetime() { if(closetimer) { window.clearTimeout(closetimer); closetimer = null; } } // close layer when click-out //document.onclick = mclose; </script> <table background="http://i59.photobucket.com/albums/g305/Timpski/ToolbarBackground.png" style="color:#ffffff;" link="#ffffff" cellpadding="0" cellspacing="1" border="0" bordercolor="#ffffff" align="center" width="100%"><tr><td colspan="6" class="wetlab"><br><br><br></td></tr> <tr><td align="center" width="10%" valign="bottom"><ul id="sddm"><a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Home"> Home </a></ul> </td><td align="center" width="20%" valign="bottom"><ul id="sddm"><a href="#" 

       onclick="mopen('m1')" 
       onmouseover="mopen('m1')" 
       onmouseout="mclosetime()">Biofabricator Subtilis</a>
       <div id="m1" 
           onmouseover="mcancelclosetime()" 
           onmouseout="mclosetime()">
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Project">Project Specifications</a>
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Chassis_1">Why B. subtilis?</a>
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Chassis_2">B. subtilis: Benefits vs Challenges</a>
       </div></ul>

</td><td align="center" width="18%" valign="bottom"><ul id="sddm"><a href="#" 

       onclick="mopen('m2')" 
       onmouseover="mopen('m2')" 
       onmouseout="mclosetime()">Wet Lab</a>
       <div id="m2" 
           onmouseover="mcancelclosetime()" 
           onmouseout="mclosetime()">
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Wet_Lab">Wet Lab Hub</a>
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Cloning_Strategy">Cloning Strategy</a>
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Protocols">Experiments & Protocols</a>
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Major_Results">Experimental Results</a>
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/BioBricks">BioBricks & Characterisation</a>
       </div></ul>

</td><td align="center" width="18%" valign="bottom"><ul id="sddm"><a href="#" 

       onclick="mopen('m3')" 
       onmouseover="mopen('m3')" 
       onmouseout="mclosetime()">Dry Lab</a>
       <div id="m3" 
           onmouseover="mcancelclosetime()" 
           onmouseout="mclosetime()">
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Dry_Lab">Dry Lab Hub</a>
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Growth_Curve">Growth Curves</a>
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Genetic_Circuit">Genetic Circuits</a>
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Motility">Motility Analysis</a>
       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Appendices">Appendices - Code etc.</a>
       </div></ul>

</td><td align="center" width="17%" valign="bottom"><ul id="sddm"><a href="http://2008.igem.org/Team:Imperial_College/Notebook"> Notebook </a></ul> </td><td align="center" width="17%" valign="bottom"><ul id="sddm"><a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Team"> Our Team </a></ul> </td></tr></table></html>

<html><style type="text/css">

div.Section { font:11pt/16pt Calibri, Verdana, Arial, Geneva, sans-serif; background-image: url(http://openwetware.org/images/a/a0/Background.PNG); background-size: 100%; background-origin: content; }

/* Text (paragraphs) */ div.Section p { font:11pt/16pt Calibri, Verdana, Arial, Geneva, sans-serif; text-align:justify; margin-top:0px; margin-left:30px; margin-right:30px; }

/* Headings */ div.Section h1 { font:22pt Calibri, Verdana, Arial, Geneva, sans-serif; text-align:left; color:#3366FF; font-weight:bold; }

/* Subheadings */ div.Section h2 { font:18pt Calibri, Verdana, Arial, Geneva, sans-serif; color:#3366FF; margin-left:5px; font-weight:bold; }

/* Subsubheadings */ div.Section h3 { font:22pt Calibri, Verdana, Arial, sans-serif; color:#E5EBFF; margin-left:10px; font-weight:bold; }

/* Subsubsubheadings */ div.Section h4 { font:22pt Calibri, Verdana, Arial, sans-serif; color:#2B48B3; margin-left:10px; font-weight:bold; }

/* Subsubsubsubheadings */ div.Section h5 { font:12pt Calibri, Verdana, Arial, sans-serif; color:#3366FF; margin-left:20px; }

/* References */ div.Section h6 { font:12pt Calibri, Verdana, Arial, sans-serif; font-weight:bold; font-style:italic; color:#3366FF; margin-left:25px; }

/* Hyperlinks */ div.Section a { }

div.Section a:hover { }

/* Tables */ div.Section td { font:11pt/16pt Calibri, Verdana, Arial, Geneva, sans-serif; text-align:justify; vertical-align:top; padding:2px 4px 2px 4px; }

/* Lists */ div.Section li { font:11pt/16pt Calibri, Verdana, Arial, Geneva, sans-serif; text-align:left; margin-top:0px; margin-left:30px; margin-right:0px; }

/* TOC stuff */ table.toc { margin-left:10px; }

table.toc li { font: 11pt/16pt Calibri, Verdana, Arial, Geneva, sans-serif; text-align: justify; margin-top: 0px; margin-left:2px; margin-right:2px; }

/* [edit] links */ span.editsection { color:#BBBBBB; font-size:10pt; font-weight:normal; font-style:normal; vertical-align:bottom; }

span.editsection a { color:#BBBBBB; font-size:10pt; font-weight:normal; font-style:normal; vertical-align:bottom; }

span.editsection a:hover { color:#3366FF; font-size:10pt; font-weight:normal; font-style:normal; vertical-align:bottom; }

/* Drop-down Menu */

  1. sddm {

margin: 0; padding: 0; z-index: 30 margin: 0; padding: 0; float: center; font: bold 12pt Calibri, Verdana, Arial, Geneva, sans-serif; border: 0px; list-style: none; }

  1. sddm a {

display: block; margin: 0px 0px 0px 0px; padding: 0 0 12px 0; color: #FFFFFF; text-align: center; text-decoration: none; }

  1. sddm a:hover {

border: 0px }

  1. sddm div {

position: absolute; visibility: hidden; margin: 0; padding: 0; background: #66aadd; border: 1px solid #66aadd } #sddm div a { position: relative; left: 0; display: block; margin: 0; padding: 5px 10px; width: auto; white-space: nowrap; text-align: left; text-decoration: none; background: #FFFFFF; color: #2875DE; font: 11pt Calibri, Verdana, Arial, Geneva, sans-serif } #sddm div a:hover { background: #66aadd; color: #FFFFFF } </style></html>



  Why B. subtilis?

This page offers a brief overview of how B. subtilis meets our main project specifications - to a much higher degree than E. coli! You will find information on the main mechanisms behind our biofabricator and some very interesting biology too. If you would like even more in-depth information, please click on Details under each section.


Light Sensing

We need a trigger - a stimulus that our biofabricators can detect and respond to quickly - such that we can control the synthesis of our biomaterial in a set pattern in 3D.

Light is the most obvious candidate, as holography allows us to generate complex patterns with well defined edges in 3D. After examining a number of light sensing pathways, we decided to utilise a native pathway involving YtvA, which is a protein used by B. subtilis to detect blue light. YtvA triggers a cascade of interactions, but some way down the chain, a molecule called sigma B (σB) is produced. This, in turn, boosts the synthesis of YtvA.

We plan to over-express YtvA and use σB as a promoter for genes which stop movement and produce biomaterial. Therefore, when the bacteria detect blue light, those genes will turn on, the bacteria will stop and biomaterial synthesis will begin.

>>> Details >>>

3D blue holographic image by sculptor Eileen Borgeson[http://www.eileenborgeson.com/default.htm



Motility

To achieve accurate distribution of our biofabricators, we must exert fine control over their motility. Bacteria's primary method of getting about is via flagellar locomotion. A protein ring on the cell membrane is attached to the flagellum and rotates during locomotion, acting like a propeller to push the cell through its environment.

The precise mechanism of how this works in B. subtilis has recently been elucidated. The flagella can be detached from the rotor by expression of a clutch molecule that interacts with the flagella and distorts it, so it is disengaged from the rotor protein. Control over the expression of this clutch should allow us to control the bacteria very quickly. When we want the bacteria to stop, we trigger expression of the clutch, which halts movement.

To draw a parallel with a car, currently available synthetic methods of stopping bacteria are akin to destroying the engine. Our method is analogous to putting the car into neutral - disengaging the engine from the driveshaft. It is an elegant solution that offers us quick control and also the opportunity for quick reversal (putting the car back into "drive").

>>> Details >>>

Motile B. subtilis cells are powered by interactions between protein complexes, generating torque for locomotion. The protein EpsE acts as a molecular clutch to disengage the flagellar motor, leaving the flagellum intact but unpowered. This quickly halts locomotion[1]
Motile B. subtilis cells are powered by interactions between protein complexes, generating torque for locomotion. The protein EpsE acts as a molecular clutch to disengage the flagellar motor, leaving the flagellum intact but unpowered. This quickly halts locomotion[1]


Biomaterial Synthesis

After our bacteria are positioned correctly, they need to express a biomaterial. B. subtilis is Gram-positive, meaning it has only a single membrane as opposed to a double membrane in Gram-negative bacteria like E. coli. This means that the expression of a biomaterial is a lot more tractable; biomaterial can be produced and secreted more efficiently. With a double membrane, material may accumulate inside the cell and the efficiency of biomaterial production can be significantly lower.

Another important aspect of our biomaterial specifications is what we want to secrete. We did a lot of research on this and decided to express elastin peptides and EAK16-II. Both are small peptides and their molecular structures favour their self-assembly outside the B. subtilis cells to form 3D bio-scaffolds.

3D bio-scaffolds are very useful for tissue culture and regenerative medicine, as they offer a suitable 3D enviroment for implanted cells to grow and proliferate. A good analogy would be scaffolding used in the construction industry. Our blue-sky aim is to construct a genetically-engineered machine that can fabricate bio-scaffolds with precise 3D shapes, directed by 3D holography.

>>> Details >>>

3D Scaffold
3D Scaffold


So B. subtilis fulfils our main specifications perfectly, and can be made to meet our minor specifications with relatively ease. On top of that, it does have other benefits, along with some challenges. These are listed on the next page, together with an overview of our development of B. subtilis as a chassis.

>>> Benefits vs Challenges >>>



<html><center><table style="color:#ffffff;background-color:#66aadd;" cellpadding="3" cellspacing="1" border="0" bordercolor="#ffffff" align="center"> <tr><td><ul id="sddm"></html>< Previous<html></ul> </td><td><ul id="sddm"><a href="#">Back to top</a></ul> </td><td><ul id="sddm"></html>Next ><html></ul> </td></tr></table> </center></html>

Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:IMPERIAL/2008/New/Chassis_1&oldid=249900"