http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Ttodd&year=&month=2010.igem.org - User contributions [en]2024-03-29T00:26:05ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:BCCS-BristolTeam:BCCS-Bristol2010-10-27T23:26:36Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
__NOTOC__<br />
<center><br />
[[Image:AgrEcoli_bead_logo_new.png|upright= 3.3|frameless|center]]<br />
</center><br />
<br />
<center><br />
==Achievements==<br />
</center><br />
<br />
===Wetlab===<br />
* '''A Well Characterised New BioBrick'''<br />
<br />
:Our new BioBrick ([http://partsregistry.org/Part:BBa_K381001 BBa_K381001]) causes GFP expression in the presence of nitrates.<br />
* '''Elegant Solution to Signal Calibration'''<br />
<br />
:By using constitutive RFP expression as a baseline, we have found a reliable and accurate way of quantifying nitrate levels in soil.<br />
* '''Novel Use of Cell Encapsulation'''<br />
<br />
:By encapsulating our bacteria in gellan beads, we can keep our bacteria contained and concentrated. This improves visibility on soil, and enhances the environmental safety of our device.<br />
* '''Characterising a Pre-Existing BioBrick'''<br />
<br />
:To better inform our own work, and to add knowledge to the BioBrick Registry, we have characterised Edinburgh 2009’s PyeaR BioBrick ([http://partsregistry.org/Part:BBa_K216009 BBa_ K216009]).<br />
===Modelling===<br />
*'''BSim Environmental Interactions'''<br />
<br />
:We have extended BSim, our agent-based modelling framework, to model interactions between bacteria and their environment. We have added 3-Dimensional mesh structures to our simulations, and added an adaptive chemical field routine that can solve partial differential equations in an arbitrary 3-D space without any risk of numerical instability.<br />
*'''BSim Graphical User Interface (GUI)'''<br />
<br />
:We have made BSim more widely accessible by creating a user-friendly and intuitive graphical user interface. This makes BSim accessible to the entire synthetic biology community, rather than just those with JAVA programming knowledge.<br />
*'''Gene Regulatory Network Modelling'''<br />
<br />
:We have investigated the behaviour of our bacteria by creating a mathematical model of their behaviour. This model could then be analysed using numerical and analytic methods.<br />
*'''Collaboration'''<br />
<br />
:Our modelling team have helped to simulate the UCL 2010 team’s new system. Working with another team has also helped to inform the design of the BSim GUI.<br />
*'''agrEcoli Cost estimates'''<br />
<br />
:In support of our human practices work, our modelling team have looked into the cost to farmers of using agrEcoli, and how much money and fertiliser they can expect to save.<br />
===Human Practices===<br />
<br />
*'''Publicising agrEcoli'''<br />
<br />
:Our new approach to human practices, building on previous work by iGEM teams, is a publicity campaign. By presenting our prototype as a functioning and marketable product, we've framed a hypothetical situation in which our project could be released.<br />
<br />
<center><br />
<br />
==Sponsors==<br />
[[Image:BCCS_Sponsors_Logo_Banner.png|upright= 3.3|frameless|center]]<br />
</center></div>Ttoddhttp://2010.igem.org/File:AgrEcoli_bead_logo_new.pngFile:AgrEcoli bead logo new.png2010-10-27T23:24:03Z<p>Ttodd: </p>
<hr />
<div></div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/ModellingTeam:BCCS-Bristol/Modelling2010-10-27T20:47:23Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
=Modelling Strategy=<br />
{{:Team:BCCS-Bristol/newtoc}}<br />
<br />
<br />
Modelling has a key role in synthetic biology. Synthetic biologists use mathematical models to predict how new genetic networks will act, which is crucial when designing a BioBrick construct. It is also important to be able to predict how groups of bacteria will interact with one another and their environment. The primary function of our modelling team is to use computational simulations to assist and inform the work of our biologists.<br />
<br />
<br />
==[[Team:BCCS-Bristol/Modelling/BSIM|BSim Modelling Framework]]==<br />
<br />
<html><center><a href="https://2010.igem.org/Team:BCCS-Bristol/Modelling/BSIM"><br />
<img width="200" src="https://static.igem.org/mediawiki/2010/f/f2/Bsim2010logo.png" border="0"><br />
</a></center></html><br />
<br />
BSim is BCCS Bristol's award winning modelling package. It is an agent-based modelling framework written in Java. BSim's agents operate on the level of individual bacteria, vesicles and particles. The internal state of bacteria can be modelled with ODE's which are solved numerically by BSim. We hope that BSim can be used as a 'virtual microscope', a tool that all biologists can use to investigate almost any microbiological system, from the level of GRN's to multicelluar interaction to environmental interaction. <br />
<br />
<br />
==[[Team:BCCS-Bristol/Modelling/GRN|Modelling the GRN]]==<br />
<br />
<html><center><a href="https://2010.igem.org/Team:BCCS-Bristol/Modelling/GRN"><br />
<img width="200" src="https://static.igem.org/mediawiki/2010/6/6d/Grnicon.png" border="0"><br />
</a></center></html><br />
<br />
Being able to predict how a novel gene regulatory network (GRN) will behave is crucial. Before one commits the time and resources to actually constructing such a network out of BioBricks (or other pieces of genetic material) it is important to know if it is stable, if the time-scales that it operates on are relevant and if it will produce the responses that one is looking for. Many approaches exist to model GRNs, the most popular being coupled ordinary differential equations (ODEs), stochastic networks and graphical models. For the levels of expression considered in this project the coupled ODE approach is the most valid, since it assumes that quantities of repressors, promoters, proteins etc. are high enough to be represented as being well mixed and continuous.<br />
<br />
==[https://2010.igem.org/Team:UCL_London/Genetic_Circuit Collaboration]==<br />
<br />
<html><center><a href="https://2010.igem.org/Team:UCL_London/Genetic_Circuit"><br />
<img width="200" src="https://static.igem.org/mediawiki/2010/f/fa/BCCS_Collaboration_Icon.png"><br />
</a></center></html><br />
<br />
Our team helped to model the UCL team's system this year. Using BSim, we created simulations and visualisations to show that UCL's new 'Feedback Loop' system works faster than the existing IPTG based system. Check out the UCL website for more details.<br />
<br />
During the course of our collaboration, feedback from working on the UCL system helped to inform our Graphical User Interface (GUI) design.</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/SafetyTeam:BCCS-Bristol/Wetlab/Safety2010-10-27T20:01:02Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks/PyeaR|Our BioBrick]] • [[:Team:BCCS-Bristol/Wetlab/Beads|Beads]]• [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] <br />
• [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • </center><br />
<br />
<br />
=Safety=<br />
{{:Team:BCCS-Bristol/newtoc}}<br />
Before even beginning our wetlab work we thought extensively about the safety of our project. We were careful throughout not only to ensure the safety of team members in the lab, but also to highlight and address any safety issues our work itself might raise were it to escape from the lab.<br />
<br />
Finally, as our aim was to produce a working prototype (which we did achieve) we also considered the wider safety implications of using our product in the real world, even basing design decisions on improving safety here.<br />
<br/><br/><br/><br/><br/><br/><br />
<br />
==Lab / Researcher Safety==<br />
<br />
*The agriColi project involved the genetic manipulation and handling of a variety of strains of E.coli and as such falls under specific safety guidelines defined by the University of Bristol (UoB).<br />
<br />
*All students contributing to the laboratory work were registered with the UoB Biological and Genetic Modification Safety Committee, allowing them to work with the variety of biological and chemical agents involved in the agriColi project, under the supervision of Dr. Nigel Savery.<br />
<br />
*All laboratory work performed by registered students fell under the guidelines set out by the UoB Safety Committee and also local rules set out by the laboratories where the work was performed - specifically the DNA-Protein Interactions Unit in the School of Biochemistry (under the supervision of Dr. Nigel Savery) and the Root Development Lab in the School of Biological Sciences (under the supervision of Prof. Claire Grierson).<br />
<br />
*All students involved in the laboratory work were trained to an appropriate level of competence in the application of techniques and use of relevant equipment, and where necessary supervised whilst performing work in the laboratory.<br />
<br />
*All projects were approved and overseen by the iGEM supervisor Dr. Nigel Savery.<br />
<br />
<br />
==Public and Environmental Safety==<br />
<br />
===Distribution===<br />
<br />
Given the nature of the agrEcoli product, certain considerations to the environment had to be factored in to the project. Clearly the aerosol distribution of genetically modified E.coli directly onto soil in the British countryside was not an option available to the team, so an alternative method of delivery had to be devised. See pages on bead design and public engagement for more information regarding the delivery system pioneered by the BCCS-Bristol 2010 iGEM team.<br />
<br />
Experimental observations regarding the Gellan Gel used to create the bead delivery system informed the team's considerations to environmental safety when applying beads to soil. The natural derivation of Gellan Gel, coupled with its degradation over time during various tests means that the gel is biodegradable and harmless to the environment.<br />
<br />
The ''E.coli'' encapsulated within the beads were observed to have died and decayed a significant period of time before the degradation of the gel released them directly into the environment.<br />
<br />
===Toxicity===<br />
<br />
With regards to the toxicity of agrEcoli beads, the Gellan gel used to create the product is a harmless food additive and is used in a wide variety of commercially available foodstuffs. The ''E.coli'' encapsulated within the beads are a lab strain specifically designed to die without the carefully balanced environment created for them in the bead, so and inquisitive children or pets will not by harmed by accidental ingestion of the product.<br />
<br />
As a summary of the above, the observations taken by the team during experiments with the gel encapsulation of genetically modified ''E.coli'' show us that agrEcoli poses little danger to the environment. There is also very little potential danger from the substances or bacteria used to either humans or animals.<br />
<br />
===Does Our New BioBrick Raise Any Safety Issues?===<br />
<br />
We do not believe that our new part [http://partsregistry.org/Part:BBa_K381001 BBa_K381001] raises any safety issues. It's component parts are well characterized and have not raised exceptional safety issues in the past. The PyeaR promoter is naturally occuring in ''E. coli'' and GFP is a well establised biomarker. <br />
<br />
===Local Biosafety===<br />
<br />
All of our work complies with the University of Bristol's local biosafety and bioethics regulations. We have also extensively considered national implications for safety and ethics in our human practices [[Team:BCCS-Bristol/Human_Practices/Marketing_Campaign |public information]] campaign. <br />
<br />
===Safety Issues for Future iGEM Competitions===<br />
<br />
We believe that the cell encapsulation technology that we have applied in our project have significant safety benefits. They provide a route for teams to separate genetically modified bacteria from their environment, which should improve the chances of them becoming accepted for use outside the lab.</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/ImprovementsTeam:BCCS-Bristol/Wetlab/Improvements2010-10-27T20:00:50Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks/PyeaR|Our BioBrick]] • [[:Team:BCCS-Bristol/Wetlab/Beads|Beads]]• [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] <br />
• [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • </center><br />
<br />
<br />
=Improvements=<br />
{{:Team:BCCS-Bristol/newtoc}}<br />
<br />
We’re extremely proud of what we’ve accomplished this year on the wetlab side; first building and well characterising our BioBrick, but then also developing our novel bead approach to improving signal detection. However the project is still far from perfect, below are several things we would like to implement were we to take the project further.<br />
<br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Improving the system==<br />
<br />
===Adding further promoters===<br />
<br />
The most obvious extension to the project is to add the ability to detect multiple nutrients. In terms of improving fertiliser use, it would make most sense to add phosphate and potassium detection, as together with nitrogen these make up the major components of fertiliser.<br />
<br />
There are already promoters available in the parts registry that sense Phosphate (BBa_K116401 submitted by NYMU-Taipei).<br />
<br />
What’s more, the wide variety of fluorescent proteins available in the parts registry makes it easy to add another signal, without requiring different detection technology. These additional signals would also be compatible with our ratio method of calibrating signals. <br />
<br />
===Increasing Signal Strength and Differences===<br />
<br />
As is clear from our results, we were able both to detect our signals, and to distinguish differences between Nitrate levels quite precisely.<br />
<br />
However, our end goal would be a signal system that could be detected by low tech equipment on farmers fields. In this case, having a stronger signal is always better. Increasing the number of bacteria in beads, as listed below, would probably help this, but so too would increasing the amount of GFP produced by our bacteria or even how bright this GFP is. Whilst this was beyond the scope of our project in the time frame, we would hope further work, ideally with collaboration from other teams would make it achievable.<br />
<br />
<br />
==Improving the beads==<br />
<br />
===Adding a Coating===<br />
<br />
One of the major public perception problems highlighted by our human aspects work was fear surrounding spreading 'E. coli' in soil. Whilst our bacteria are already relatively well contained within the beads, this could be improved further by the addition of some kind of coating.<br />
<br />
We think a cellulose coating would be an ideal candidate as it fulfils the main criteria; hopefully allowing Nitrates to diffuse in, but preventing bacteria leaving the bead. It would also be biodegradable along with the rest of the bead, reducing environmental impact.<br />
<br />
===Adding Nutrient Medium===<br />
<br />
Whilst our beads dramatically increase ''E. coli'' survival and thus signal strength, we suspect this could be improved further by the addition of nutrient medium into the beads. This could even be linked to nutrient dependence - for example tryptophan auxotrophy - rendering the 'E. coli' unable to survive at all outside of the beads. This would not only increase the signal strength and thus efficiency of our product, but also make it more palatable to the public. <br />
<br />
Regretfully we didn't have time to experiment in the lab, however, we suspect beads with nutrient media could be made by slurrying cells in Lennox Broth rather than water in the slurry stage of our bead construction procedure.</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/ExperimentsTeam:BCCS-Bristol/Wetlab/Experiments2010-10-27T20:00:34Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks/PyeaR|Our BioBrick]] • [[:Team:BCCS-Bristol/Wetlab/Beads|Beads]]• [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] <br />
• [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • </center><br />
<br />
<br />
==Experiments==<br />
<br />
Here you can find details of all the experiments we conducted, along with the resulting data gathered. In the spirit of scientific co-operation, we've aimed to present our work so that for each experiment, another scientist would be able to replicate it without any prior knowledge or extra information. <br />
<br />
At some points pages may refer to 'standard' procedures. In most cases these are well known techniques, or procedures documented in kits (e.g. Quiagen miniprep kits), however just in case, we've also provided a database of all the standard procedures used.<br />
<br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/standard_protocols| Standard Protocols]] • [[:Team:BCCS-Bristol/Wetlab/K381001_Construction| BBa_K381001 Construction]] • [[:Team:BCCS-Bristol/Wetlab/K381001_Characterisation| BBa_K381001 Characterisation]] • </center> <br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/signal_soil| Signals in Soil]] • [[:Team:BCCS-Bristol/Wetlab/characterising_ratio| Signal Calibration]] • [[:Team:BCCS-Bristol/Wetlab/making_beads| Making Beads]] •</center> <br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/difference_soil| Beads On Soil]] • [[:Team:BCCS-Bristol/Wetlab/difference_solution| Beads In Solution]] • [[:Team:BCCS-Bristol/Wetlab/RFPbead_tests| Final Beads]] • </center><br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/BetaGalactosidaseAssays| Miller Assays ]] • </center> <br />
<br />
<br />
<br />
<!--[[:Team:BCCS-Bristol/Wetlab/K381001_Construction]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/K381001_Characterisation]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/signal_soil]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/making_beads]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/difference_solution]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/difference_soil]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/characterising_ratio]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/RFPbead_tests]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/BetaGalactosidaseAssays]]<br />
<br />
General:<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/standard_protocols]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/signal_bead]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/mesh_data]]<br />
--></div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/AchievementsTeam:BCCS-Bristol/Wetlab/Achievements2010-10-27T20:00:15Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks/PyeaR|Our BioBrick]] • [[:Team:BCCS-Bristol/Wetlab/Beads|Beads]]• [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] <br />
• [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • </center> <br />
<br />
<br />
=Achievements=<br />
{{:Team:BCCS-Bristol/newtoc}}<br />
Despite having an extremely small wet lab team (only two members) we believe we've achieved a lot this year. We've come from our initial ideas stage in July, to possessing a fully functional prototype product, good enough to catch commercial attention by the end of October. Alongside this, we've also helped better characterise a pre-existing BioBrick.<br />
<br />
Below is a brief summary of our biggest achievements<br />
<br />
<br />
==A Well Characterised New BioBrick==<br />
<br />
<br />
[[Image:GFPChar0to10.png|thumbnail|right|400px|Graph 1: BBa_K381001 characterisation]]<br />
<br />
<br />
The focal point of our wetlab work was the construction and characterisation of a new BioBrick [http://partsregistry.org/Part:BBa_K381001 BBa_K381001]. This part is a composite of two pre-existing BioBricks, with details of its design available [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks/PyeaR| here].<br />
<br />
<br />
As shown in graph 1, we mapped its behaviour across the range where it was known to be most sensitive, further details of which can be found [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/K381001_Characterisation| here].<br />
<br />
<br />
We're proud of this part, and hope it will be useful to other teams in the future.<br />
<br/><br/><br/><br/><br/><br/><br />
<br />
==Elegant Solution to Signal Calibration==<br />
<br />
<br />
[[Image:Ratio0to2 quadratic fit.png|thumbnail|left|400px|Graph 2: Characterising our ratio method]]<br />
<br />
<br />
As well as making our BioBrick, we also worked further to develop a product that would actually function. As part of this we altered the traditional signal detection method of measuring output to remove the problem of misleading figures. Our new method of instead detecting the ratio of two outputs removes difficulties in distinguishing between large signals due to high Nitrate levels and those due simply to large colonies.<br />
<br />
<br />
As is evident in graph 2, we well characterised this method and found it to work well, particularly within the range relevant to our project. Our results were even good enough to allow the construction of a formula linking signals detected to levels in the soil.<br />
<br />
<br />
For more information on how the ratio method works and our results from it, click [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/characterising_ratio| here]<br />
<br />
==Novel Use of Cell Encapsulation==<br />
<br />
[[Image:Beads.png|thumbnail|400px|right|Our new beads]]<br />
<br />
<br />
In what we believe is a first in iGEM, we used technology to both allow safe use of our ''E. coli'' in the public and environment dramatically improve signal detection.<br />
<br />
<br />
Our Bead encapsulation method ensures a stronger signal that can actually be detected by our end user (farmers) without high tech equipment. It also reduces the risk of our bacteria escaping unchecked in the environment, and assisted in our human practices side of the project; making our product more appealing to the public.<br />
<br />
<br />
For more information on how the beads work and how they can be made, click [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/Beads| here]. We hope the resources and data we've provided will encourage other iGEM teams to pursue this method in the future.<br />
<br />
<br />
==Characterising A Pre-Existing BioBrick==<br />
<br />
<br />
[[Image:BCCS attempt6betagalgraph.jpg|thumbnail|left|400px|Graph 3: Miller assay results]]<br />
<br />
<br />
As well as developing our own BioBrick, we also worked to better characterise one already in the parts registry [http://partsregistry.org/Part:BBa_K216005| BBa_K216005]. Graph 3 shows just one of many Miller assays we conducted to analyse its performance, more of which can be found [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/BetaGalactosidaseAssays here].</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/AchievementsTeam:BCCS-Bristol/Wetlab/Achievements2010-10-27T19:59:07Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[https://2010.igem.org/Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks/PyeaR|Our BioBrick]] • [[:Team:BCCS-Bristol/Wetlab/Beads|Beads]]• [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] <br />
• [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • </center> <br />
<br />
<br />
=Achievements=<br />
{{:Team:BCCS-Bristol/newtoc}}<br />
Despite having an extremely small wet lab team (only two members) we believe we've achieved a lot this year. We've come from our initial ideas stage in July, to possessing a fully functional prototype product, good enough to catch commercial attention by the end of October. Alongside this, we've also helped better characterise a pre-existing BioBrick.<br />
<br />
Below is a brief summary of our biggest achievements<br />
<br />
<br />
==A Well Characterised New BioBrick==<br />
<br />
<br />
[[Image:GFPChar0to10.png|thumbnail|right|400px|Graph 1: BBa_K381001 characterisation]]<br />
<br />
<br />
The focal point of our wetlab work was the construction and characterisation of a new BioBrick [http://partsregistry.org/Part:BBa_K381001 BBa_K381001]. This part is a composite of two pre-existing BioBricks, with details of its design available [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks/PyeaR| here].<br />
<br />
<br />
As shown in graph 1, we mapped its behaviour across the range where it was known to be most sensitive, further details of which can be found [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/K381001_Characterisation| here].<br />
<br />
<br />
We're proud of this part, and hope it will be useful to other teams in the future.<br />
<br/><br/><br/><br/><br/><br/><br />
<br />
==Elegant Solution to Signal Calibration==<br />
<br />
<br />
[[Image:Ratio0to2 quadratic fit.png|thumbnail|left|400px|Graph 2: Characterising our ratio method]]<br />
<br />
<br />
As well as making our BioBrick, we also worked further to develop a product that would actually function. As part of this we altered the traditional signal detection method of measuring output to remove the problem of misleading figures. Our new method of instead detecting the ratio of two outputs removes difficulties in distinguishing between large signals due to high Nitrate levels and those due simply to large colonies.<br />
<br />
<br />
As is evident in graph 2, we well characterised this method and found it to work well, particularly within the range relevant to our project. Our results were even good enough to allow the construction of a formula linking signals detected to levels in the soil.<br />
<br />
<br />
For more information on how the ratio method works and our results from it, click [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/characterising_ratio| here]<br />
<br />
==Novel Use of Cell Encapsulation==<br />
<br />
[[Image:Beads.png|thumbnail|400px|right|Our new beads]]<br />
<br />
<br />
In what we believe is a first in iGEM, we used technology to both allow safe use of our ''E. coli'' in the public and environment dramatically improve signal detection.<br />
<br />
<br />
Our Bead encapsulation method ensures a stronger signal that can actually be detected by our end user (farmers) without high tech equipment. It also reduces the risk of our bacteria escaping unchecked in the environment, and assisted in our human practices side of the project; making our product more appealing to the public.<br />
<br />
<br />
For more information on how the beads work and how they can be made, click [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/Beads| here]. We hope the resources and data we've provided will encourage other iGEM teams to pursue this method in the future.<br />
<br />
<br />
==Characterising A Pre-Existing BioBrick==<br />
<br />
<br />
[[Image:BCCS attempt6betagalgraph.jpg|thumbnail|left|400px|Graph 3: Miller assay results]]<br />
<br />
<br />
As well as developing our own BioBrick, we also worked to better characterise one already in the parts registry [http://partsregistry.org/Part:BBa_K216005| BBa_K216005]. Graph 3 shows just one of many Miller assays we conducted to analyse its performance, more of which can be found [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/BetaGalactosidaseAssays here].</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/ImprovementsTeam:BCCS-Bristol/Wetlab/Improvements2010-10-27T19:58:20Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[:Team:BCCS-Bristol/Wetlab/Part_Design|Our BioBrick]] • [[:Team:BCCS-Bristol/Wetlab/Beads|Beads]]• [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] <br />
• [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • </center> <br />
<br />
<br />
=Improvements=<br />
{{:Team:BCCS-Bristol/newtoc}}<br />
<br />
We’re extremely proud of what we’ve accomplished this year on the wetlab side; first building and well characterising our BioBrick, but then also developing our novel bead approach to improving signal detection. However the project is still far from perfect, below are several things we would like to implement were we to take the project further.<br />
<br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Improving the system==<br />
<br />
===Adding further promoters===<br />
<br />
The most obvious extension to the project is to add the ability to detect multiple nutrients. In terms of improving fertiliser use, it would make most sense to add phosphate and potassium detection, as together with nitrogen these make up the major components of fertiliser.<br />
<br />
There are already promoters available in the parts registry that sense Phosphate (BBa_K116401 submitted by NYMU-Taipei).<br />
<br />
What’s more, the wide variety of fluorescent proteins available in the parts registry makes it easy to add another signal, without requiring different detection technology. These additional signals would also be compatible with our ratio method of calibrating signals. <br />
<br />
===Increasing Signal Strength and Differences===<br />
<br />
As is clear from our results, we were able both to detect our signals, and to distinguish differences between Nitrate levels quite precisely.<br />
<br />
However, our end goal would be a signal system that could be detected by low tech equipment on farmers fields. In this case, having a stronger signal is always better. Increasing the number of bacteria in beads, as listed below, would probably help this, but so too would increasing the amount of GFP produced by our bacteria or even how bright this GFP is. Whilst this was beyond the scope of our project in the time frame, we would hope further work, ideally with collaboration from other teams would make it achievable.<br />
<br />
<br />
==Improving the beads==<br />
<br />
===Adding a Coating===<br />
<br />
One of the major public perception problems highlighted by our human aspects work was fear surrounding spreading 'E. coli' in soil. Whilst our bacteria are already relatively well contained within the beads, this could be improved further by the addition of some kind of coating.<br />
<br />
We think a cellulose coating would be an ideal candidate as it fulfils the main criteria; hopefully allowing Nitrates to diffuse in, but preventing bacteria leaving the bead. It would also be biodegradable along with the rest of the bead, reducing environmental impact.<br />
<br />
===Adding Nutrient Medium===<br />
<br />
Whilst our beads dramatically increase ''E. coli'' survival and thus signal strength, we suspect this could be improved further by the addition of nutrient medium into the beads. This could even be linked to nutrient dependence - for example tryptophan auxotrophy - rendering the 'E. coli' unable to survive at all outside of the beads. This would not only increase the signal strength and thus efficiency of our product, but also make it more palatable to the public. <br />
<br />
Regretfully we didn't have time to experiment in the lab, however, we suspect beads with nutrient media could be made by slurrying cells in Lennox Broth rather than water in the slurry stage of our bead construction procedure.</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/ExperimentsTeam:BCCS-Bristol/Wetlab/Experiments2010-10-27T19:57:46Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[:Team:BCCS-Bristol/Wetlab/Part_Design|Our BioBrick]] • [[:Team:BCCS-Bristol/Wetlab/Beads|Beads]]• [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] <br />
• [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • </center> <br />
<br />
<br />
==Experiments==<br />
<br />
Here you can find details of all the experiments we conducted, along with the resulting data gathered. In the spirit of scientific co-operation, we've aimed to present our work so that for each experiment, another scientist would be able to replicate it without any prior knowledge or extra information. <br />
<br />
At some points pages may refer to 'standard' procedures. In most cases these are well known techniques, or procedures documented in kits (e.g. Quiagen miniprep kits), however just in case, we've also provided a database of all the standard procedures used.<br />
<br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/standard_protocols| Standard Protocols]] • [[:Team:BCCS-Bristol/Wetlab/K381001_Construction| BBa_K381001 Construction]] • [[:Team:BCCS-Bristol/Wetlab/K381001_Characterisation| BBa_K381001 Characterisation]] • </center> <br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/signal_soil| Signals in Soil]] • [[:Team:BCCS-Bristol/Wetlab/characterising_ratio| Signal Calibration]] • [[:Team:BCCS-Bristol/Wetlab/making_beads| Making Beads]] •</center> <br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/difference_soil| Beads On Soil]] • [[:Team:BCCS-Bristol/Wetlab/difference_solution| Beads In Solution]] • [[:Team:BCCS-Bristol/Wetlab/RFPbead_tests| Final Beads]] • </center><br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/BetaGalactosidaseAssays| Miller Assays ]] • </center> <br />
<br />
<br />
<br />
<!--[[:Team:BCCS-Bristol/Wetlab/K381001_Construction]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/K381001_Characterisation]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/signal_soil]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/making_beads]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/difference_solution]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/difference_soil]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/characterising_ratio]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/RFPbead_tests]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/BetaGalactosidaseAssays]]<br />
<br />
General:<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/standard_protocols]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/signal_bead]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/mesh_data]]<br />
--></div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/ImprovementsTeam:BCCS-Bristol/Wetlab/Improvements2010-10-27T19:57:31Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[:Team:BCCS-Bristol/Wetlab/Part_Design|Our BioBrick]] • [[:Team:BCCS-Bristol/Wetlab/Beads|Beads]]• [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] <br />
• [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • </center> <br />
<br />
<br />
=Improvements=<br />
{{:Team:BCCS-Bristol/newtoc}}<br />
<br />
We’re extremely proud of what we’ve accomplished this year on the wetlab side; first building and well characterising our BioBrick, but then also developing our novel bead approach to improving signal detection. However the project is still far from perfect, below are several things we would like to implement were we to take the project further.<br />
<br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Improving the system==<br />
<br />
===Adding further promoters===<br />
<br />
The most obvious extension to the project is to add the ability to detect multiple nutrients. In terms of improving fertiliser use, it would make most sense to add phosphate and potassium detection, as together with nitrogen these make up the major components of fertiliser.<br />
<br />
There are already promoters available in the parts registry that sense Phosphate (BBa_K116401 submitted by NYMU-Taipei).<br />
<br />
What’s more, the wide variety of fluorescent proteins available in the parts registry makes it easy to add another signal, without requiring different detection technology. These additional signals would also be compatible with our ratio method of calibrating signals. <br />
<br />
===Increasing Signal Strength and Differences===<br />
<br />
As is clear from our results, we were able both to detect our signals, and to distinguish differences between Nitrate levels quite precisely.<br />
<br />
However, our end goal would be a signal system that could be detected by low tech equipment on farmers fields. In this case, having a stronger signal is always better. Increasing the number of bacteria in beads, as listed below, would probably help this, but so too would increasing the amount of GFP produced by our bacteria or even how bright this GFP is. Whilst this was beyond the scope of our project in the time frame, we would hope further work, ideally with collaboration from other teams would make it achievable.<br />
<br />
<br />
==Improving the beads==<br />
<br />
===Adding a Coating===<br />
<br />
One of the major public perception problems highlighted by our human aspects work was fear surrounding spreading 'E. coli' in soil. Whilst our bacteria are already relatively well contained within the beads, this could be improved further by the addition of some kind of coating.<br />
<br />
We think a cellulose coating would be an ideal candidate as it fulfils the main criteria; hopefully allowing Nitrates to diffuse in, but preventing bacteria leaving the bead. It would also be biodegradable along with the rest of the bead, reducing environmental impact.<br />
<br />
===Adding Nutrient Medium===<br />
<br />
Whilst our beads dramatically increase ''E. coli'' survival and thus signal strength, we suspect this could be improved further by the addition of nutrient medium into the beads. This could even be linked to nutrient dependence - for example tryptophan auxotrophy - rendering the 'E. coli' unable to survive at all outside of the beads. This would not only increase the signal strength and thus efficiency of our product, but also make it more palatable to the public. <br />
<br />
Regretfully we didn't have time to experiment in the lab, however, we suspect beads with nutrient media could be made by slurrying cells in Lennox Broth rather than water in the slurry stage of our bead construction procedure.</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/SafetyTeam:BCCS-Bristol/Wetlab/Safety2010-10-27T19:56:32Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[:Team:BCCS-Bristol/Wetlab/Part_Design|Our BioBrick]] • [[:Team:BCCS-Bristol/Wetlab/Beads|Beads]]• [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] <br />
• [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • </center> <br />
<br />
<br />
=Safety=<br />
{{:Team:BCCS-Bristol/newtoc}}<br />
Before even beginning our wetlab work we thought extensively about the safety of our project. We were careful throughout not only to ensure the safety of team members in the lab, but also to highlight and address any safety issues our work itself might raise were it to escape from the lab.<br />
<br />
Finally, as our aim was to produce a working prototype (which we did achieve) we also considered the wider safety implications of using our product in the real world, even basing design decisions on improving safety here.<br />
<br/><br/><br/><br/><br/><br/><br />
<br />
==Lab / Researcher Safety==<br />
<br />
*The agriColi project involved the genetic manipulation and handling of a variety of strains of E.coli and as such falls under specific safety guidelines defined by the University of Bristol (UoB).<br />
<br />
*All students contributing to the laboratory work were registered with the UoB Biological and Genetic Modification Safety Committee, allowing them to work with the variety of biological and chemical agents involved in the agriColi project, under the supervision of Dr. Nigel Savery.<br />
<br />
*All laboratory work performed by registered students fell under the guidelines set out by the UoB Safety Committee and also local rules set out by the laboratories where the work was performed - specifically the DNA-Protein Interactions Unit in the School of Biochemistry (under the supervision of Dr. Nigel Savery) and the Root Development Lab in the School of Biological Sciences (under the supervision of Prof. Claire Grierson).<br />
<br />
*All students involved in the laboratory work were trained to an appropriate level of competence in the application of techniques and use of relevant equipment, and where necessary supervised whilst performing work in the laboratory.<br />
<br />
*All projects were approved and overseen by the iGEM supervisor Dr. Nigel Savery.<br />
<br />
<br />
==Public and Environmental Safety==<br />
<br />
===Distribution===<br />
<br />
Given the nature of the agrEcoli product, certain considerations to the environment had to be factored in to the project. Clearly the aerosol distribution of genetically modified E.coli directly onto soil in the British countryside was not an option available to the team, so an alternative method of delivery had to be devised. See pages on bead design and public engagement for more information regarding the delivery system pioneered by the BCCS-Bristol 2010 iGEM team.<br />
<br />
Experimental observations regarding the Gellan Gel used to create the bead delivery system informed the team's considerations to environmental safety when applying beads to soil. The natural derivation of Gellan Gel, coupled with its degradation over time during various tests means that the gel is biodegradable and harmless to the environment.<br />
<br />
The ''E.coli'' encapsulated within the beads were observed to have died and decayed a significant period of time before the degradation of the gel released them directly into the environment.<br />
<br />
===Toxicity===<br />
<br />
With regards to the toxicity of agrEcoli beads, the Gellan gel used to create the product is a harmless food additive and is used in a wide variety of commercially available foodstuffs. The ''E.coli'' encapsulated within the beads are a lab strain specifically designed to die without the carefully balanced environment created for them in the bead, so and inquisitive children or pets will not by harmed by accidental ingestion of the product.<br />
<br />
As a summary of the above, the observations taken by the team during experiments with the gel encapsulation of genetically modified ''E.coli'' show us that agrEcoli poses little danger to the environment. There is also very little potential danger from the substances or bacteria used to either humans or animals.<br />
<br />
===Does Our New BioBrick Raise Any Safety Issues?===<br />
<br />
We do not believe that our new part [http://partsregistry.org/Part:BBa_K381001 BBa_K381001] raises any safety issues. It's component parts are well characterized and have not raised exceptional safety issues in the past. The PyeaR promoter is naturally occuring in ''E. coli'' and GFP is a well establised biomarker. <br />
<br />
===Local Biosafety===<br />
<br />
All of our work complies with the University of Bristol's local biosafety and bioethics regulations. We have also extensively considered national implications for safety and ethics in our human practices [[Team:BCCS-Bristol/Human_Practices/Marketing_Campaign |public information]] campaign. <br />
<br />
===Safety Issues for Future iGEM Competitions===<br />
<br />
We believe that the cell encapsulation technology that we have applied in our project have significant safety benefits. They provide a route for teams to separate genetically modified bacteria from their environment, which should improve the chances of them becoming accepted for use outside the lab.</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/SafetyTeam:BCCS-Bristol/Wetlab/Safety2010-10-27T19:56:07Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[:Team:BCCS-Bristol/Wetlab/Part_Design|Our BioBrick]] • [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] • <br />
[[:Team:BCCS-Bristol/Wetlab/Beads|Beads]] • [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • </center> <br />
<br />
<br />
=Safety=<br />
{{:Team:BCCS-Bristol/newtoc}}<br />
Before even beginning our wetlab work we thought extensively about the safety of our project. We were careful throughout not only to ensure the safety of team members in the lab, but also to highlight and address any safety issues our work itself might raise were it to escape from the lab.<br />
<br />
Finally, as our aim was to produce a working prototype (which we did achieve) we also considered the wider safety implications of using our product in the real world, even basing design decisions on improving safety here.<br />
<br/><br/><br/><br/><br/><br/><br />
<br />
==Lab / Researcher Safety==<br />
<br />
*The agriColi project involved the genetic manipulation and handling of a variety of strains of E.coli and as such falls under specific safety guidelines defined by the University of Bristol (UoB).<br />
<br />
*All students contributing to the laboratory work were registered with the UoB Biological and Genetic Modification Safety Committee, allowing them to work with the variety of biological and chemical agents involved in the agriColi project, under the supervision of Dr. Nigel Savery.<br />
<br />
*All laboratory work performed by registered students fell under the guidelines set out by the UoB Safety Committee and also local rules set out by the laboratories where the work was performed - specifically the DNA-Protein Interactions Unit in the School of Biochemistry (under the supervision of Dr. Nigel Savery) and the Root Development Lab in the School of Biological Sciences (under the supervision of Prof. Claire Grierson).<br />
<br />
*All students involved in the laboratory work were trained to an appropriate level of competence in the application of techniques and use of relevant equipment, and where necessary supervised whilst performing work in the laboratory.<br />
<br />
*All projects were approved and overseen by the iGEM supervisor Dr. Nigel Savery.<br />
<br />
<br />
==Public and Environmental Safety==<br />
<br />
===Distribution===<br />
<br />
Given the nature of the agrEcoli product, certain considerations to the environment had to be factored in to the project. Clearly the aerosol distribution of genetically modified E.coli directly onto soil in the British countryside was not an option available to the team, so an alternative method of delivery had to be devised. See pages on bead design and public engagement for more information regarding the delivery system pioneered by the BCCS-Bristol 2010 iGEM team.<br />
<br />
Experimental observations regarding the Gellan Gel used to create the bead delivery system informed the team's considerations to environmental safety when applying beads to soil. The natural derivation of Gellan Gel, coupled with its degradation over time during various tests means that the gel is biodegradable and harmless to the environment.<br />
<br />
The ''E.coli'' encapsulated within the beads were observed to have died and decayed a significant period of time before the degradation of the gel released them directly into the environment.<br />
<br />
===Toxicity===<br />
<br />
With regards to the toxicity of agrEcoli beads, the Gellan gel used to create the product is a harmless food additive and is used in a wide variety of commercially available foodstuffs. The ''E.coli'' encapsulated within the beads are a lab strain specifically designed to die without the carefully balanced environment created for them in the bead, so and inquisitive children or pets will not by harmed by accidental ingestion of the product.<br />
<br />
As a summary of the above, the observations taken by the team during experiments with the gel encapsulation of genetically modified ''E.coli'' show us that agrEcoli poses little danger to the environment. There is also very little potential danger from the substances or bacteria used to either humans or animals.<br />
<br />
===Does Our New BioBrick Raise Any Safety Issues?===<br />
<br />
We do not believe that our new part [http://partsregistry.org/Part:BBa_K381001 BBa_K381001] raises any safety issues. It's component parts are well characterized and have not raised exceptional safety issues in the past. The PyeaR promoter is naturally occuring in ''E. coli'' and GFP is a well establised biomarker. <br />
<br />
===Local Biosafety===<br />
<br />
All of our work complies with the University of Bristol's local biosafety and bioethics regulations. We have also extensively considered national implications for safety and ethics in our human practices [[Team:BCCS-Bristol/Human_Practices/Marketing_Campaign |public information]] campaign. <br />
<br />
===Safety Issues for Future iGEM Competitions===<br />
<br />
We believe that the cell encapsulation technology that we have applied in our project have significant safety benefits. They provide a route for teams to separate genetically modified bacteria from their environment, which should improve the chances of them becoming accepted for use outside the lab.</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/SafetyTeam:BCCS-Bristol/Wetlab/Safety2010-10-27T19:55:13Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[:Team:BCCS-Bristol/Wetlab/Part_Design|Part Design]] • [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] • <br />
[[:Team:BCCS-Bristol/Wetlab/Beads|Beads]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • </center> <br />
<br />
<br />
=Safety=<br />
{{:Team:BCCS-Bristol/newtoc}}<br />
Before even beginning our wetlab work we thought extensively about the safety of our project. We were careful throughout not only to ensure the safety of team members in the lab, but also to highlight and address any safety issues our work itself might raise were it to escape from the lab.<br />
<br />
Finally, as our aim was to produce a working prototype (which we did achieve) we also considered the wider safety implications of using our product in the real world, even basing design decisions on improving safety here.<br />
<br/><br/><br/><br/><br/><br/><br />
<br />
==Lab / Researcher Safety==<br />
<br />
*The agriColi project involved the genetic manipulation and handling of a variety of strains of E.coli and as such falls under specific safety guidelines defined by the University of Bristol (UoB).<br />
<br />
*All students contributing to the laboratory work were registered with the UoB Biological and Genetic Modification Safety Committee, allowing them to work with the variety of biological and chemical agents involved in the agriColi project, under the supervision of Dr. Nigel Savery.<br />
<br />
*All laboratory work performed by registered students fell under the guidelines set out by the UoB Safety Committee and also local rules set out by the laboratories where the work was performed - specifically the DNA-Protein Interactions Unit in the School of Biochemistry (under the supervision of Dr. Nigel Savery) and the Root Development Lab in the School of Biological Sciences (under the supervision of Prof. Claire Grierson).<br />
<br />
*All students involved in the laboratory work were trained to an appropriate level of competence in the application of techniques and use of relevant equipment, and where necessary supervised whilst performing work in the laboratory.<br />
<br />
*All projects were approved and overseen by the iGEM supervisor Dr. Nigel Savery.<br />
<br />
<br />
==Public and Environmental Safety==<br />
<br />
===Distribution===<br />
<br />
Given the nature of the agrEcoli product, certain considerations to the environment had to be factored in to the project. Clearly the aerosol distribution of genetically modified E.coli directly onto soil in the British countryside was not an option available to the team, so an alternative method of delivery had to be devised. See pages on bead design and public engagement for more information regarding the delivery system pioneered by the BCCS-Bristol 2010 iGEM team.<br />
<br />
Experimental observations regarding the Gellan Gel used to create the bead delivery system informed the team's considerations to environmental safety when applying beads to soil. The natural derivation of Gellan Gel, coupled with its degradation over time during various tests means that the gel is biodegradable and harmless to the environment.<br />
<br />
The ''E.coli'' encapsulated within the beads were observed to have died and decayed a significant period of time before the degradation of the gel released them directly into the environment.<br />
<br />
===Toxicity===<br />
<br />
With regards to the toxicity of agrEcoli beads, the Gellan gel used to create the product is a harmless food additive and is used in a wide variety of commercially available foodstuffs. The ''E.coli'' encapsulated within the beads are a lab strain specifically designed to die without the carefully balanced environment created for them in the bead, so and inquisitive children or pets will not by harmed by accidental ingestion of the product.<br />
<br />
As a summary of the above, the observations taken by the team during experiments with the gel encapsulation of genetically modified ''E.coli'' show us that agrEcoli poses little danger to the environment. There is also very little potential danger from the substances or bacteria used to either humans or animals.<br />
<br />
===Does Our New BioBrick Raise Any Safety Issues?===<br />
<br />
We do not believe that our new part [http://partsregistry.org/Part:BBa_K381001 BBa_K381001] raises any safety issues. It's component parts are well characterized and have not raised exceptional safety issues in the past. The PyeaR promoter is naturally occuring in ''E. coli'' and GFP is a well establised biomarker. <br />
<br />
===Local Biosafety===<br />
<br />
All of our work complies with the University of Bristol's local biosafety and bioethics regulations. We have also extensively considered national implications for safety and ethics in our human practices [[Team:BCCS-Bristol/Human_Practices/Marketing_Campaign |public information]] campaign. <br />
<br />
===Safety Issues for Future iGEM Competitions===<br />
<br />
We believe that the cell encapsulation technology that we have applied in our project have significant safety benefits. They provide a route for teams to separate genetically modified bacteria from their environment, which should improve the chances of them becoming accepted for use outside the lab.</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/ImprovementsTeam:BCCS-Bristol/Wetlab/Improvements2010-10-27T19:54:53Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[:Team:BCCS-Bristol/Wetlab/Part_Design|Part Design]] • [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] • <br />
[[:Team:BCCS-Bristol/Wetlab/Beads|Beads]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • </center> <br />
<br />
<br />
=Improvements=<br />
{{:Team:BCCS-Bristol/newtoc}}<br />
<br />
We’re extremely proud of what we’ve accomplished this year on the wetlab side; first building and well characterising our BioBrick, but then also developing our novel bead approach to improving signal detection. However the project is still far from perfect, below are several things we would like to implement were we to take the project further.<br />
<br/><br/><br/><br/><br/><br/><br/><br />
<br />
==Improving the system==<br />
<br />
===Adding further promoters===<br />
<br />
The most obvious extension to the project is to add the ability to detect multiple nutrients. In terms of improving fertiliser use, it would make most sense to add phosphate and potassium detection, as together with nitrogen these make up the major components of fertiliser.<br />
<br />
There are already promoters available in the parts registry that sense Phosphate (BBa_K116401 submitted by NYMU-Taipei).<br />
<br />
What’s more, the wide variety of fluorescent proteins available in the parts registry makes it easy to add another signal, without requiring different detection technology. These additional signals would also be compatible with our ratio method of calibrating signals. <br />
<br />
===Increasing Signal Strength and Differences===<br />
<br />
As is clear from our results, we were able both to detect our signals, and to distinguish differences between Nitrate levels quite precisely.<br />
<br />
However, our end goal would be a signal system that could be detected by low tech equipment on farmers fields. In this case, having a stronger signal is always better. Increasing the number of bacteria in beads, as listed below, would probably help this, but so too would increasing the amount of GFP produced by our bacteria or even how bright this GFP is. Whilst this was beyond the scope of our project in the time frame, we would hope further work, ideally with collaboration from other teams would make it achievable.<br />
<br />
<br />
==Improving the beads==<br />
<br />
===Adding a Coating===<br />
<br />
One of the major public perception problems highlighted by our human aspects work was fear surrounding spreading 'E. coli' in soil. Whilst our bacteria are already relatively well contained within the beads, this could be improved further by the addition of some kind of coating.<br />
<br />
We think a cellulose coating would be an ideal candidate as it fulfils the main criteria; hopefully allowing Nitrates to diffuse in, but preventing bacteria leaving the bead. It would also be biodegradable along with the rest of the bead, reducing environmental impact.<br />
<br />
===Adding Nutrient Medium===<br />
<br />
Whilst our beads dramatically increase ''E. coli'' survival and thus signal strength, we suspect this could be improved further by the addition of nutrient medium into the beads. This could even be linked to nutrient dependence - for example tryptophan auxotrophy - rendering the 'E. coli' unable to survive at all outside of the beads. This would not only increase the signal strength and thus efficiency of our product, but also make it more palatable to the public. <br />
<br />
Regretfully we didn't have time to experiment in the lab, however, we suspect beads with nutrient media could be made by slurrying cells in Lennox Broth rather than water in the slurry stage of our bead construction procedure.</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Wetlab/ExperimentsTeam:BCCS-Bristol/Wetlab/Experiments2010-10-27T19:54:36Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[:Team:BCCS-Bristol/Wetlab/Part_Design|Part Design]] • [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] • <br />
[[:Team:BCCS-Bristol/Wetlab/Beads|Beads]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • </center> <br />
<br />
<br />
==Experiments==<br />
<br />
Here you can find details of all the experiments we conducted, along with the resulting data gathered. In the spirit of scientific co-operation, we've aimed to present our work so that for each experiment, another scientist would be able to replicate it without any prior knowledge or extra information. <br />
<br />
At some points pages may refer to 'standard' procedures. In most cases these are well known techniques, or procedures documented in kits (e.g. Quiagen miniprep kits), however just in case, we've also provided a database of all the standard procedures used.<br />
<br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/standard_protocols| Standard Protocols]] • [[:Team:BCCS-Bristol/Wetlab/K381001_Construction| BBa_K381001 Construction]] • [[:Team:BCCS-Bristol/Wetlab/K381001_Characterisation| BBa_K381001 Characterisation]] • </center> <br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/signal_soil| Signals in Soil]] • [[:Team:BCCS-Bristol/Wetlab/characterising_ratio| Signal Calibration]] • [[:Team:BCCS-Bristol/Wetlab/making_beads| Making Beads]] •</center> <br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/difference_soil| Beads On Soil]] • [[:Team:BCCS-Bristol/Wetlab/difference_solution| Beads In Solution]] • [[:Team:BCCS-Bristol/Wetlab/RFPbead_tests| Final Beads]] • </center><br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/BetaGalactosidaseAssays| Miller Assays ]] • </center> <br />
<br />
<br />
<br />
<!--[[:Team:BCCS-Bristol/Wetlab/K381001_Construction]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/K381001_Characterisation]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/signal_soil]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/making_beads]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/difference_solution]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/difference_soil]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/characterising_ratio]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/RFPbead_tests]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/BetaGalactosidaseAssays]]<br />
<br />
General:<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/standard_protocols]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/signal_bead]]<br />
<br />
[[:Team:BCCS-Bristol/Wetlab/mesh_data]]<br />
--></div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/WetlabTeam:BCCS-Bristol/Wetlab2010-10-27T19:54:14Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
=Wet lab=<br />
<br />
<br />
<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[:Team:BCCS-Bristol/Wetlab/Part_Design|Part Design]] • [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] • <br />
[[:Team:BCCS-Bristol/Wetlab/Beads|Beads]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • </center> <br />
<br />
<br />
[[Image:Picture_018.jpg|right|thumbnail|250px|Our Labspace]]<br />
<br />
Our main achievement in the wet lab has been the creation of a new composite BioBrick, engineering ''E. coli'' to produce GFP in response to Nitrates. Not only is this part complete, but it's also well characterised both on its own and working in tandem with a second BioBrick.<br />
<br />
<br />
We also developed a way of encapsulating our bacteria, allowing an easy and safe method of spreading them in the environment, and also dramatically improving signal detection and ''E. coli'' survival in soil.<br />
<br />
<br />
Finally we worked to further characterise the PyeaR promoter submitted by Edinburgh in 2009, improving the quality of information available in the parts registry.<br />
<br />
<br />
In this section you can find out about the design of our parts, including all the decisions we made and why, as well as all the information relating to their construction and characterisation. It also contains material on our new encapsulation methods, and finally lists all our safety considerations both in the lab and on a wider environmental scale.</div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/Human_Practices/Public_Engagement/School_VisitTeam:BCCS-Bristol/Human Practices/Public Engagement/School Visit2010-10-27T19:30:54Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
=School Visit=<br />
<br />
We gave a presentation to each of the classes about iGEM, us and our project, and then did a practical demonstration where the students made their own beads (...without the ''E. coli'').<br />
<br />
==Image Gallery==<br />
<br />
<gallery perrow=5><br />
Image:BCCS-Bristol-ClevedonVisit-03.jpg|Distributing supplies after the presentation<br />
Image:BCCS-Bristol-ClevedonVisit-04.jpg|Students pipetting liquid gellan mix into an ionic solution (salt water)<br />
Image:BCCS-Bristol-ClevedonVisit-05.jpg|We tried to find the best technique to make a perfecty spherical bead<br />
Image:BCCS-Bristol-ClevedonVisit-06.jpg|Testing for consistency <br />
Image:BCCS-Bristol-ClevedonVisit-07.jpg|We asked the students what they would do if they were starting an iGEM project<br />
Image:BCCS-Bristol-ClevedonVisit-08.jpg|Roz discussing studying engineering at university<br />
Image:BCCS-Bristol-ClevedonVisit-09.jpg|Tom explaining what synthetic biology is all about<br />
Image:BCCS-Bristol-ClevedonVisit-10.jpg|The students were interested in hearing where a degree in biology can take you<br />
Image:BCCS-Bristol-ClevedonVisit-11.jpg<br />
Image:BCCS-Bristol-ClevedonVisit-12.jpg|Student experiences problems as stock gellan solution cools and solidifies<br />
Image:BCCS-Bristol-ClevedonVisit-13.jpg|Student showing some of the best beads<br />
Image:BCCS-Bristol-ClevedonVisit-14.jpg<br />
Image:BCCS-Bristol-ClevedonVisit-15.jpg|Kat and Tom explaining that synthetic biology is not all about making jello<br />
Image:BCCS-Bristol-ClevedonVisit-02.jpg|Neeraj reclining after a hard days work<br />
</gallery></div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/TeamTeam:BCCS-Bristol/Team2010-10-27T19:06:27Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
== '''Who we are''' ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
'''Student Team:'''<br />
<br />
*Kira Kowalska, Engineering Mathematics<br />
*Antoni Matyjaszkiewicz, Engineering Mathematics<br />
*Roz Sandwell, Engineering Mathematics<br />
*Katharine Coyte, Biology/ Maths<br />
*Tom Layland, Biochemistry<br />
*Thomas Todd, Complexity Science<br />
*Neeraj Oak, Complexity Science<br />
<br />
'''Staff advisors:'''<br />
<br />
*Nigel Savery, Biochemistry<br />
*Claire Grierson, Biology<br />
*Mario di Bernardo, Engineering Mathematics<br />
*Krasimira Tsaneva-Atanasova, Engineering Mathematics<br />
*Caroline Colijn, Engineering Mathematics<br />
*S J "John" Hogan, Complexity Science<br />
*Paul Verkade, Biochemistry<br />
<br />
<br />
'''Grad student advisors:'''<br />
<br />
*Oli Purcell, Complexity Science<br />
*Thomas Gorochowski, Complexity Science<br />
*Petros Mina, Complexity Science<br />
<br />
<br />
|<br />
[[Image:Bristol_logo.png|right|border|200px]]<br />
[[Image:BCCSLogo.png|right|200px]]<br />
https://static.igem.org/mediawiki/2010/4/4f/OurTeam3.JPG<br />
<br />
|}<br />
<br />
<br />
<br />
==What We've Done==<br />
<br />
All of the work for this iGEM project was carried out by the memebers of the student team. The BSim 2010 software package has built on the 2009 version, which was submitted for the 2009 iGEM competition. All of the new code for environmental interactions and the graphical user interface was written by the 2010 student team members.</div>Ttoddhttp://2010.igem.org/File:BCCSLogo.pngFile:BCCSLogo.png2010-10-27T18:50:15Z<p>Ttodd: </p>
<hr />
<div></div>Ttoddhttp://2010.igem.org/Team:BCCS-Bristol/TeamTeam:BCCS-Bristol/Team2010-10-27T18:48:48Z<p>Ttodd: </p>
<hr />
<div>{{:Team:BCCS-Bristol/Header}}<br />
<br />
== '''Who we are''' ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
'''Student Team:'''<br />
<br />
*Kira Kowalska, Engineering Mathematics<br />
*Antoni Matyjaszkiewicz, Engineering Mathematics<br />
*Roz Sandwell, Engineering Mathematics<br />
*Katharine Coyte, Biology/ Maths<br />
*Tom Layland, Biochemistry<br />
*Thomas Todd, Complexity Science<br />
*Neeraj Oak, Complexity Science<br />
<br />
'''Staff advisors:'''<br />
<br />
*Nigel Savery, Biochemistry<br />
*Claire Grierson, Biology<br />
*Mario di Bernardo, Engineering Mathematics<br />
*Krasimira Tsaneva-Atanasova, Engineering Mathematics<br />
*Caroline Colijn, Engineering Mathematics<br />
*S J "John" Hogan, Complexity Science<br />
*Paul Verkade, Biochemistry<br />
<br />
<br />
'''Grad student advisors:'''<br />
<br />
*Oli Purcell, Complexity Science<br />
*Thomas Gorochowski, Complexity Science<br />
*Petros Mina, Complexity Science<br />
<br />
<br />
|<br />
<br />
https://static.igem.org/mediawiki/2010/4/4f/OurTeam3.JPG<br />
<br />
|}<br />
<br />
[[Image:Bristol_logo.png|right|200px]]<br />
<br />
==What We've Done==<br />
<br />
All of the work for this iGEM project was carried out by the memebers of the student team. The BSim 2010 software package has built on the 2009 version, which was submitted for the 2009 iGEM competition. All of the new code for environmental interactions and the graphical user interface was written by the 2010 student team members.</div>Ttodd