Difference between revisions of "IGEM:IMPERIAL/2008/New/Cloning Strategy"

From OpenWetWare
Jump to: navigation, search
(Cloning Strategy)
Line 4: Line 4:
 
The Imperial iGEM 2008 team faces the daunting task of working with a chassis that has been rarely used - and never characterised - in the competition to date. While the ''B. subtilis'' chassis offers us many advantages, working from the ground up presents many challenges.
 
The Imperial iGEM 2008 team faces the daunting task of working with a chassis that has been rarely used - and never characterised - in the competition to date. While the ''B. subtilis'' chassis offers us many advantages, working from the ground up presents many challenges.
  
Our cloning strategy is highly complex. In order to build increasingly complicated constructs for our final product, we need to build, test and characterise all the parts and devices leading to the final systems! The diagram below shows the critical pathway for our cloning strategy and as you can see, there are a huge number of closely-linked steps..
+
Our cloning strategy is highly complex. In order to build increasingly complicated constructs for our final product, we need to build, test and characterise all the parts and devices leading to the final systems. The diagram below shows the critical pathway for our cloning strategy with a huge number of closely-linked steps.
  
  
Line 10: Line 10:
  
  
A summary of the aims of the phases, and constructs that should be produced by the end of each for testing, is given below:
+
A summary of the aims of each phase and constructs to be produced within each phase, is given below:
  
 
====== Phase 1 ======
 
====== Phase 1 ======
Line 18: Line 18:
  
 
====== Phase 2 ======
 
====== Phase 2 ======
Testing and characterisation of inducible promoters; those marked with a 'c' are chemically-inducible and those marked with an 'l' are light-inducible. RFP is used instead of GFP to check output as ytvA responds to blue light - GFP may cause positive feedback. 'Rep' gene signifies a repressor for the chemically-inducible promoter to stop leaky expression.
+
Testing and characterisation of inducible promoters; those marked with a 'c' are chemically-inducible and those marked with an 'l' are light-inducible. RFP is used instead of GFP as a quantifiable output as ytvA responds to blue light - GFP may cause positive feedback. 'Rep' genes encode a repressor for the chemically-inducible promoters to stop leaky expression.
  
 
<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2A.png">
 
<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2A.png">
Line 36: Line 36:
  
 
====== Final Construct ======
 
====== Final Construct ======
Combination of light sensing and light-induced expression of epsE and biomaterial. Each gene has its own promoter because in ''B. subtilis'' it has been shown that levels of expression drop as one moves along an operon.
+
Combination of light sensing and light-induced expression of epsE and biomaterial. Each gene has its own RBS because in ''B. subtilis'', it has been shown that levels of expression decreases as one moves along an operon.
 
<p><html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/S1L.png"></html></p>
 
<p><html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/S1L.png"></html></p>

Revision as of 07:02, 12 September 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>

Cloning Strategy

The Imperial iGEM 2008 team faces the daunting task of working with a chassis that has been rarely used - and never characterised - in the competition to date. While the B. subtilis chassis offers us many advantages, working from the ground up presents many challenges.

Our cloning strategy is highly complex. In order to build increasingly complicated constructs for our final product, we need to build, test and characterise all the parts and devices leading to the final systems. The diagram below shows the critical pathway for our cloning strategy with a huge number of closely-linked steps.


Imperial 2008 Critical Pathway.png


A summary of the aims of each phase and constructs to be produced within each phase, is given below:

Phase 1

Testing and characterisation of constitutive promoters. We will test 4 combinations of 2 promoters and 2 RBSs to characterise them. Antibiotic cassette is placed first on the construct, so that any readthrough from native transcriptase simply boosts production of antibiotic.

<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase1.png"></html>

Phase 2

Testing and characterisation of inducible promoters; those marked with a 'c' are chemically-inducible and those marked with an 'l' are light-inducible. RFP is used instead of GFP as a quantifiable output as ytvA responds to blue light - GFP may cause positive feedback. 'Rep' genes encode a repressor for the chemically-inducible promoters to stop leaky expression.

<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2A.png"> <img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2B.png"></html>

Phase 3

Testing and characterisation of the clutch (epsE) and biomaterial synthesis (SB - signal sequence & biomaterial).

<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase3A.png"> <img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase3B.png"></html>

Phase 4

Combining of light induction and epsE/biomaterial expression, and testing of feasibility.

<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase4A.png"> <img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase4B.png"></html>

Final Construct

Combination of light sensing and light-induced expression of epsE and biomaterial. Each gene has its own RBS because in B. subtilis, it has been shown that levels of expression decreases as one moves along an operon.

<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/S1L.png"></html>