IGEM:IMPERIAL/2008/Prototype/Project: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 45: Line 45:


The constructs we have designed are polycistronic, ''B.subtilis'' translation of polycistronic genes  however is not even. The first gene in a polycistron is often more highly expressed than the second even if they have their own individual Ribosome Binding Sites. This may cause issues for our current circuit design but could be easily remedied by using multiple promoters in each construct or by the arrangement of genes along the polycistron.
The constructs we have designed are polycistronic, ''B.subtilis'' translation of polycistronic genes  however is not even. The first gene in a polycistron is often more highly expressed than the second even if they have their own individual Ribosome Binding Sites. This may cause issues for our current circuit design but could be easily remedied by using multiple promoters in each construct or by the arrangement of genes along the polycistron.
Also, the differential codon usage of organisms must be taken into account. Some of our proposed Biomaterials are from other organisms, most notably the elastin exon protein which is from the Human genome. If this protein was expressed in its current form in ''B.subtilis'' the amino acid code would be very different to that observed upon expression in a human. As such, the elastin exon sequence will need to be 'translated' into a sequence that will give the same product in ''B.subtilis'' as the original would in humans.


= Team Divisions =
= Team Divisions =

Revision as of 08:18, 11 August 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><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype><img width=50px src=http://openwetware.org/images/f/f2/Imperial_2008_Logo.png></img</a></html> Home The Project B.subtilis Chassis Wet Lab Dry Lab Notebook

<html> <style type="text/css"> div.Section { font:11pt/16pt Calibri, Verdana, Arial, Geneva, sans-serif; }

/* 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; }

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

/* Subsubheadings */ div.Section h3 { font:16pt Calibri, Verdana, Arial, sans-serif; font-weight:bold; color:#3366FF; margin-left:10px; }

/* Subsubsubheadings */ div.Section h4 { font:12pt Calibri, Verdana, Arial, sans-serif; color:#3366FF; margin-left:15px; }

/* 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; }

  1. sddm {

margin: 0; padding: 0; z-index: 30 }

  1. sddm li {

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

  1. sddm li a {

display: block; margin: 0px 0px 0px 0px; padding: 0 0 12px 0; background: #33bbff; color: #FFFFFF; text-align: center; text-decoration: none; }

  1. sddm li a:hover {

border: 0px }

  1. sddm div {

position: absolute; visibility: hidden; margin: 0; padding: 0; background: #33bbff; border: 1px solid #33bbff } #sddm div a { position: relative; 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: #33bbff; color: #FFFFFF } </style></html>












<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Motility><img width=200px src=http://openwetware.org/images/4/42/Imperial_2008_Motility_Label.png></img></a></html>



<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Biomaterials><img width=200px src=http://openwetware.org/images/0/04/Imperial_2008_Biomaterials_Label.png></img></a></html>

<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Light><img width=200px src=http://openwetware.org/images/3/3d/Imperial_2008_Light_Label.png></img></a></html>
(This is just rough!)


Our project can be broadly divided into three main subsections: The light-sensing pathway, the motility/clutch mechanism and the biomaterial synthesis strategy. To find out more about each part of the project, please click the titles in the image above.
Given below is an overview of B. subtilis; why we chose it as the chassis for our project and what makes it a useful organism for Synthetic Biology (and iGEM in particular!). In addition, there is a regularly-updated "Progress & Problems" section along with information about the distribution of our team.

B. subtilis

B.subtilis as a chassis made its first appearance last year and has many advantages for use at iGEM including but not limited to; a single membrane allowing for easier secretion, its high motility and a large amount of data in the literature. For our project, B.subtilis is vital. E.coli cannot secrete proteins to any high degree and modifications to it to allow this have not always proven particularly succesful. B.subtilis has however been used for this purpose for many decades and though there are some technical issues associated with this, it has performed the job admirably. B.subtilis is also highly motile and posses a unique (at present) system of switching off its motility in the form of a molecular clutch, EpsE (see Motility below).

To make B.subtilis a viable host, it is necessary to produce parts that will work correctly in B.subtilis. When searching the literature, it rapidly became clear that B.subtilis would not be able to use E.coli promoters with any real ability, would happily digest many E.coli produced vectors and also lacked a Ribosome capable of binding Ribosome Binding Sequences from E.coli. As such, we would need new vectors, new promoters and new RBSs in order to get the project off the ground.

To get round modifying a shuttle vector to meet Biobrick standards integration sequences can be used. These sequences are complementary to a gene in B.subtilis and may naturally be incorporated over those genes by the recombination system in B.subtilis. Promoters for B.subtilis were taken from both vectors and the organisms own chromosome via the extensive transcription regulation database for B.subtilis, the DBTBS, to suit the requirements of our system and to give a suitable range of basic promoters for B.subtilis. Although a consensus sequence (5'-AAGGAGGTG-3') for the B.subtilis RBS exists and has been proven to work effectively in the organism, two naturally occuring RBSs from B.subtilis that have been used extensively in the past were chosen as these are the most likey to work.

Progress so Far

Overview of our circuitry for the B. subtilis Bioprinter

The diagram on the right (click for larger image) shows a theoretical overview of the circuitry we'll be adding to B. subtilis in order to control its movement and expression profile.

The top row shows synthetic constructs which we will add that are to be constitutively expressed and the bottom row shows synthetic constructs that we require to be inducible. Genes in green generally upregulate/promote transcription of some target whereas those in red downregulate/repress activity of their targets; lines showing interactions follow the same scheme. The gene for product production (or reporters) is coloured yellow.

The general operation of these constructs should fulfil our specifications, as the simple explanations below demonstrate...

  • No light present: Inducible promoter is not activated, meaning no transcription of product and no production of synthetic EpsE. The cell's own background expression of SinR, should prevent production of native EpsE so the bacteria will remain motile.
  • Light present: Inducible promoter is activated, meaning product is transcribed along with EpsE. Native EpsE is still repressed by the native SinR, but the synthetic EpsE should engage the clutch and halt the bacteria in a short timeframe. The bacteria should, therefore, stop and start producing product.
  • Light removed: Should a bacteria move out of the light, we want it to be able to turn off production and start moving again. When light is absent, the inducible promoter will no longer be active and the bacteria should revert to the "no light" state. The time it takes to do this will be directly linked to the accuracy of deposition, and so we want it to be as fast as possible.

Summary Presentation of work so far - Wet lab

Potential Issues

The constructs we have designed are polycistronic, B.subtilis translation of polycistronic genes however is not even. The first gene in a polycistron is often more highly expressed than the second even if they have their own individual Ribosome Binding Sites. This may cause issues for our current circuit design but could be easily remedied by using multiple promoters in each construct or by the arrangement of genes along the polycistron.

Team Divisions

At the moment we're separated into two teams:

  • The wetlab team (James, Chris, Tom & Krupa) has so far concentrated on the mammoth task of finding sequences and writing protocols, and will move on to setting up and organising experiments.
  • The drylab team (Erika, QQ, Prudence, Clinton & Yanis) is working out a strategy for modelling of behaviour of our bacteria on Matlab, and will be using data from a recorded microscope analysis of subtilis motility to help define parameters. More detail can be found on the Wet Lab and Dry Lab pages.
Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:IMPERIAL/2008/Prototype/Project&oldid=230082"