http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=RachelBoyd&year=&month=2010.igem.org - User contributions [en]2024-03-28T17:49:43ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Newcastle/otherTeam:Newcastle/other2010-10-28T02:30:46Z<p>RachelBoyd: /* Random Photos */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
==Photos==<br />
<br />
====Team Photos====<br />
{|<br />
|[[Image:teamnewcastlephoto1.jpg| 300px]]<br />
|[[Image:teamnewcastlephoto2.jpg| 300px]]<br />
|[[Image:Newcastle iGEM Teampic.jpeg| 300px]]<br />
|}<br />
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====Random Photos==== <br />
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[[Image:newcastle_funny1.jpg|200px]] [[Image:newcastle_funny3.jpg|300px]] [[Image:newcastle_funny5.jpg|300px]] [[Image:newcastle_funny4.jpg|300px]] [[Image:newcastle_funny2.jpg|300px]] [[Image:newcastle_funny6.jpg|300px]]<br />
[[Image:newcastle_funny7.jpg|300px]] [[Image:newcastle_funny8.jpg|300px]] [[Image:newcastle_funny9.jpg|300px]]<br />
<br />
====Hoppings Photos====<br />
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{| style="width:100%;" cellpadding="4" cellspacing="4" border="0"<br />
|-<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair.jpeg]]<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair2.jpeg]]<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair3.jpeg]]<br />
|-<br />
|}<br />
<br />
<br />
==Awards==<br />
<br />
[[Team:Newcastle/Medals| Aims and Medals]]<br />
<br />
==Jamboree==<br />
<br />
[[Jamboree| Jamboree]]<br />
<br />
==Poster==<br />
<br />
[[Jamboree#Posters|Poster Requirements]]<br />
<br />
<br />
<br />
<br />
=='''UK iGEM Get Together'''==<br />
[[Image:newcastle_ukget.jpg|800px]]<br />
===Programme===<br />
<br />
[[Media:Programme_for_UK_Gathering_at_Newcastle.pdf|The Programme for UK Gathering at Newcastle]]<br />
<br />
[[UK_Meetup_2010#UK_iGEM_get-together.2C_Newcastle.2C__20th_and_21st_July.2C_2010| UK get together]] <br />
<br />
<br />
<br />
===Presentation===<br />
<br />
[[Media:Final presentation 8.0!.pdf|Our Presentation for the UK Gathering at Newcastle]]<br />
<br />
Questions asked about our presentation: <br />
<br />
#Would this project require us to change the strength of the filamentous cells in relation to different types of concrete? <br />
#What is the strength of filamentous cells? How do filamentous cells actually help to fill up the cracks? <br />
# How can repairing microcracks in concrete help to prevent a building collapsing in an earthquake?<br />
<br />
For answers to these questions see [[Media:Concrete.pdf|Deena's concrete lecture]]<br />
<br />
==Attribution and Contribution==<br />
<br />
[[Team:Newcastle/Attribution and Contribution|Attribution and Contribution]]<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/otherTeam:Newcastle/other2010-10-28T02:29:25Z<p>RachelBoyd: /* Random Photos */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
==Photos==<br />
<br />
====Team Photos====<br />
{|<br />
|[[Image:teamnewcastlephoto1.jpg| 300px]]<br />
|[[Image:teamnewcastlephoto2.jpg| 300px]]<br />
|[[Image:Newcastle iGEM Teampic.jpeg| 300px]]<br />
|}<br />
<br />
====Random Photos==== <br />
<br />
[[Image:newcastle_funny1.jpg|200px]] [[Image:newcastle_funny3.jpg|300px]] [[Image:newcastle_funny5.jpg|200px]] [[Image:newcastle_funny4.jpg|300px]] [[Image:newcastle_funny2.jpg|300px]] [[Image:newcastle_funny6.jpg|300px]]<br />
[[Image:newcastle_funny7.jpg|300px]] [[Image:newcastle_funny8.jpg|300px]] [[Image:newcastle_funny9.jpg|300px]]<br />
<br />
====Hoppings Photos====<br />
<br />
{| style="width:100%;" cellpadding="4" cellspacing="4" border="0"<br />
|-<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair.jpeg]]<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair2.jpeg]]<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair3.jpeg]]<br />
|-<br />
|}<br />
<br />
<br />
==Awards==<br />
<br />
[[Team:Newcastle/Medals| Aims and Medals]]<br />
<br />
==Jamboree==<br />
<br />
[[Jamboree| Jamboree]]<br />
<br />
==Poster==<br />
<br />
[[Jamboree#Posters|Poster Requirements]]<br />
<br />
<br />
<br />
<br />
=='''UK iGEM Get Together'''==<br />
[[Image:newcastle_ukget.jpg|800px]]<br />
===Programme===<br />
<br />
[[Media:Programme_for_UK_Gathering_at_Newcastle.pdf|The Programme for UK Gathering at Newcastle]]<br />
<br />
[[UK_Meetup_2010#UK_iGEM_get-together.2C_Newcastle.2C__20th_and_21st_July.2C_2010| UK get together]] <br />
<br />
<br />
<br />
===Presentation===<br />
<br />
[[Media:Final presentation 8.0!.pdf|Our Presentation for the UK Gathering at Newcastle]]<br />
<br />
Questions asked about our presentation: <br />
<br />
#Would this project require us to change the strength of the filamentous cells in relation to different types of concrete? <br />
#What is the strength of filamentous cells? How do filamentous cells actually help to fill up the cracks? <br />
# How can repairing microcracks in concrete help to prevent a building collapsing in an earthquake?<br />
<br />
For answers to these questions see [[Media:Concrete.pdf|Deena's concrete lecture]]<br />
<br />
==Attribution and Contribution==<br />
<br />
[[Team:Newcastle/Attribution and Contribution|Attribution and Contribution]]<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/otherTeam:Newcastle/other2010-10-28T02:26:17Z<p>RachelBoyd: /* Random Photos */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
==Photos==<br />
<br />
====Team Photos====<br />
{|<br />
|[[Image:teamnewcastlephoto1.jpg| 300px]]<br />
|[[Image:teamnewcastlephoto2.jpg| 300px]]<br />
|[[Image:Newcastle iGEM Teampic.jpeg| 300px]]<br />
|}<br />
<br />
====Random Photos==== <br />
<br />
[[Image:newcastle_funny1.jpg|300px]] [[Image:newcastle_funny3.jpg|300px]] [[Image:newcastle_funny5.jpg|200px]] [[Image:newcastle_funny4.jpg|300px]] [[Image:newcastle_funny2.jpg|300px]] [[Image:newcastle_funny6.jpg|300px]]<br />
[[Image:newcastle_funny7.jpg|300px]] [[Image:newcastle_funny8.jpg|300px]] [[Image:newcastle_funny9.jpg|300px]]<br />
<br />
====Hoppings Photos====<br />
<br />
{| style="width:100%;" cellpadding="4" cellspacing="4" border="0"<br />
|-<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair.jpeg]]<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair2.jpeg]]<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair3.jpeg]]<br />
|-<br />
|}<br />
<br />
<br />
==Awards==<br />
<br />
[[Team:Newcastle/Medals| Aims and Medals]]<br />
<br />
==Jamboree==<br />
<br />
[[Jamboree| Jamboree]]<br />
<br />
==Poster==<br />
<br />
[[Jamboree#Posters|Poster Requirements]]<br />
<br />
<br />
<br />
<br />
=='''UK iGEM Get Together'''==<br />
[[Image:newcastle_ukget.jpg|800px]]<br />
===Programme===<br />
<br />
[[Media:Programme_for_UK_Gathering_at_Newcastle.pdf|The Programme for UK Gathering at Newcastle]]<br />
<br />
[[UK_Meetup_2010#UK_iGEM_get-together.2C_Newcastle.2C__20th_and_21st_July.2C_2010| UK get together]] <br />
<br />
<br />
<br />
===Presentation===<br />
<br />
[[Media:Final presentation 8.0!.pdf|Our Presentation for the UK Gathering at Newcastle]]<br />
<br />
Questions asked about our presentation: <br />
<br />
#Would this project require us to change the strength of the filamentous cells in relation to different types of concrete? <br />
#What is the strength of filamentous cells? How do filamentous cells actually help to fill up the cracks? <br />
# How can repairing microcracks in concrete help to prevent a building collapsing in an earthquake?<br />
<br />
For answers to these questions see [[Media:Concrete.pdf|Deena's concrete lecture]]<br />
<br />
==Attribution and Contribution==<br />
<br />
[[Team:Newcastle/Attribution and Contribution|Attribution and Contribution]]<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/otherTeam:Newcastle/other2010-10-28T02:24:57Z<p>RachelBoyd: /* Random Photos */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
==Photos==<br />
<br />
====Team Photos====<br />
{|<br />
|[[Image:teamnewcastlephoto1.jpg| 300px]]<br />
|[[Image:teamnewcastlephoto2.jpg| 300px]]<br />
|[[Image:Newcastle iGEM Teampic.jpeg| 300px]]<br />
|}<br />
<br />
====Random Photos==== <br />
<br />
[[Image:newcastle_funny1.jpg|300px]] [[Image:newcastle_funny3.jpg|300px]] [[Image:newcastle_funny5.jpg|280px]] [[Image:newcastle_funny4.jpg|300px]] [[Image:newcastle_funny2.jpg|300px]] [[Image:newcastle_funny6.jpg|300px]]<br />
[[Image:newcastle_funny7.jpg|300px]] [[Image:newcastle_funny8.jpg|300px]] [[Image:newcastle_funny9.jpg|300px]]<br />
<br />
====Hoppings Photos====<br />
<br />
{| style="width:100%;" cellpadding="4" cellspacing="4" border="0"<br />
|-<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair.jpeg]]<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair2.jpeg]]<br />
|style="background-repeat: no-repeat; background-color: #ffffff; height:200px; width:300px"|[[Image:Newcastle_iGEM_funfair3.jpeg]]<br />
|-<br />
|}<br />
<br />
<br />
==Awards==<br />
<br />
[[Team:Newcastle/Medals| Aims and Medals]]<br />
<br />
==Jamboree==<br />
<br />
[[Jamboree| Jamboree]]<br />
<br />
==Poster==<br />
<br />
[[Jamboree#Posters|Poster Requirements]]<br />
<br />
<br />
<br />
<br />
=='''UK iGEM Get Together'''==<br />
[[Image:newcastle_ukget.jpg|800px]]<br />
===Programme===<br />
<br />
[[Media:Programme_for_UK_Gathering_at_Newcastle.pdf|The Programme for UK Gathering at Newcastle]]<br />
<br />
[[UK_Meetup_2010#UK_iGEM_get-together.2C_Newcastle.2C__20th_and_21st_July.2C_2010| UK get together]] <br />
<br />
<br />
<br />
===Presentation===<br />
<br />
[[Media:Final presentation 8.0!.pdf|Our Presentation for the UK Gathering at Newcastle]]<br />
<br />
Questions asked about our presentation: <br />
<br />
#Would this project require us to change the strength of the filamentous cells in relation to different types of concrete? <br />
#What is the strength of filamentous cells? How do filamentous cells actually help to fill up the cracks? <br />
# How can repairing microcracks in concrete help to prevent a building collapsing in an earthquake?<br />
<br />
For answers to these questions see [[Media:Concrete.pdf|Deena's concrete lecture]]<br />
<br />
==Attribution and Contribution==<br />
<br />
[[Team:Newcastle/Attribution and Contribution|Attribution and Contribution]]<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/File:Newcastle_funny1.jpgFile:Newcastle funny1.jpg2010-10-28T02:24:27Z<p>RachelBoyd: uploaded a new version of "Image:Newcastle funny1.jpg"</p>
<hr />
<div></div>RachelBoydhttp://2010.igem.org/Template:Team:Newcastle/mainmenuTemplate:Team:Newcastle/mainmenu2010-10-28T02:07:04Z<p>RachelBoyd: </p>
<hr />
<div>{|id="mainmenu" cellspacing=0<br />
|-<br />
|[[Team:Newcastle|Home]]<br />
|[[Team:Newcastle/the_team|Team]]<br />
|[[Team:Newcastle/problem|Problem]]<br />
|[[Team:Newcastle/solution|Solution]]<br />
|[[Team:Newcastle/modelling|Modelling]]<br />
|[[Team:Newcastle/notebook|Lab book]]<br />
|[[Team:Newcastle/meetings|Meetings]]<br />
|[[Team:Newcastle/safety|Ethics]]<br />
|[[Team:Newcastle/Medals|Judging]]<br />
|[[Team:Newcastle/other|Other]]<br />
|[[Team:Newcastle/Sponsors|Sponsors]]<br />
|-<br />
|}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/safetyTeam:Newcastle/safety2010-10-28T02:04:53Z<p>RachelBoyd: </p>
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<div>{{Team:Newcastle/mainbanner}}<br />
==Ethics==<br />
<br />
===Synthetic Biology: Background===<br />
<br />
Synthetic biology is a new research field that has a big potential in coming up with solutions for our everyday problems. However, ethical issues have been raised since the start of development in this field. Here we will discuss the ethical issues that our project will bring.<br />
<br />
====Biohackers====<br />
<br />
In this field, there are always people that view synthetic biology's negative aspects with concern, like worrying about the production of pathogens to be used as weapons. Their fears are that people will hack into systems to obtain data that might be used to do this. Therefore, we have to be very careful with the products of our research so that they remain safe in our hands.<br />
<br />
====Playing God!!!====<br />
<br />
<br />
In synthetic biology, we control the lives of bacteria. We insert proteins into bacteria to make them do what we want them to do. We make them kill themselves at the end of their job because they might be released into the environment and be harmful to other living things. It shows that we do not really treat the single-celled organism as life. However, we are coming up with a novel solution for the environment by healing concrete cracks.<br />
<br />
====Biosecurity====<br />
<br />
Bacteria might be harmful to the environment and living things around. Considering the fact that they are such small organisms and could not be spotted with the naked-eye if they are released in air, we make them kill themselves with the ‘kill switch’ biobrick. This can be very inconsiderate standing in a bacteria’s point of view, but it is the novel thing to do to avoid them hurting other living organisms.<br />
<br />
===Construction: Background===<br />
<br />
Cracks form in concrete structures as soon as they set. These cracks continue to grow in size if they are not repaired due to the weight of the structure, imposed load, freeze-thaw effect and wind loading. The bigger the cracks are, the higher the rate of water seeping into the steel reinforcements, causing them to corrode and thus weakening the structure. Therefore, we would like to use Bacilla Filla to fill up these cracks before things get worse.<br />
<br />
====Environmental Disaster====<br />
<br />
Concrete is a very widely used material in construction. In fact, cement that is used to make concrete is the second most widely used substance in the world after water. Some building structures have to be demolished because the cracks formed appear to be threatening the strength of the structures. These buildings have to be rebuilt in order to reinforce its tensile strength. This is an unsustainable method of recovery because cement is a material that requires a lot of energy to be produced. In the production of cement, the rotating kiln requires the temperature to be between 1350°C-1400°C, which not only uses up a lot of energy resources, but also produces a lot of carbon dioxide. These carbon dioxide contributes to the green house effect which then leads to global warming. Therefore, our project can help to reduce renovation of buildings which in turn lowers the amount of cement that has to be produced each year.<br />
<br />
====Post-Chernobyl Disaster====<br />
<br />
The nuclear power plant combustion that occurred in Chernobyl on 26 April 1986 has allowed radioactive elements to be released into the environment, threatening the health of people living within close proximity of the plant. Therefore, a shelter, the Shelter Implementation Plan (SIP) which will be completed in 2012 is currently being built to cover up the area that where the accident occurred so as to make it environmentally friendly. However, if cracks were to form, something that inevitably occurs for building structures, those radioactive elements will still be dispersed into the environment. With the help of our project, those cracks will be able to be filled-up and the area will be safe again.<br />
<br />
<br />
<br />
==Safety Issues==<br />
<br />
These are the safety questions for the judging form:<br />
<br />
'''1. Would any of your project ideas raise safety issues in terms of:'''<br />
<br />
=====Researcher Safety=====<br />
<br />
We worked in the Centre for Bacterial Cell Biology (CBCB) at Newcastle University for the entire project, where there are clearly defined safety rules and regulations that all laboratory workers must follow. One of our advisors, Dr Wendy Smith, guided us through the safety procedures along with some of the basic techniques during the first [[Team:Newcastle/14_June_2010|introductory]] week, before any laboratory work on the project began. This included carrying out the following risk assessments to determine what control measures would be required.<br />
<br />
(i)'''Chemical Hazards''':<br />
At the beginning of the project written risk assessments were already available in the host laboratories for all procedures that involved potentially hazardous chemicals. These risk assessments were reviewed and the recommended control measures were strictly followed throughout. No additional chemical hazards specific to this project were identified.<br />
<br />
(ii)'''Radioisotopes and carcinogens''':<br />
None of these were used in this project.<br />
<br />
(iii)'''Biological hazards''':<br />
Throughout the project, we used the ''Escherichia coli'' strain DH5α, ''Bacillus subtilis'' strain 3610, ''Bacillus subtilis'' strain 168 and ''Bacillus sphaericus'' strain LMG 22257. Wild-type ''E. coli'' is classified as a hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP). However, ''E. coli'' strain DH5α is derived from a laboratory strain ''E. coli'' K12, which is recognised as disabled and equivalent to an ACDP hazard group 1 organism (i.e. unlikely to cause disease). ''E. coli'' K12 and its derivatives such as strain DH5α are unable to colonise in humans or animals and consequently pose negligible risk to human or animal health. Wild-type ''Bacillus subtilis'' (i.e. strain 3610) is classified as an ACDP hazard group 1 organism and its derivative ''B. subtilis'' strain 168 has disabling auxotrophs mutations (e.g. conferring a requirement for tryptophan, Zeigler ''et al'', 2008) that makes it even less likely to colonise or cause harm to human or animal health. Also ''Bacillus sphaericus'' LMG 22257 is classified as an ADCP hazard group 1 organism making it unlikely to cause any harm to either human or animal health. The potential of any sequences cloned into these bacterial hosts during the project to pose additional hazards was also assessed. None of these sequences were associated with pathogenic traits or traits that might significantly enhance the survival outside the lab. Therefore, no specific safety issues, other than those associated with use of any non-pathogenic microorganism, were identified. It was concluded that containment level 1(CL1) would be sufficient to ensure researcher safety. Nonetheless, all work was carried out in strict compliance with the host laboratory's standard safety procedures, which were more stringent that those required for CL1.<br />
<br />
(iv)'''Other hazards''':<br />
The project also involved conducting some work in the Engineering structures laboratory, where we made and broke concrete 'blocks'. Appropriate safety regulations for the type of work in this laboratory were followed. When we were in the structures lab, we wore safety goggles, steel toe cap boots and gloves.<br />
<br />
=====Public Safety=====<br />
Our project concerns repairing cracks on concrete surfaces by spraying these surfaces with spores of engineered derivatives of ''Bacillus subtilis'' 168. Before releasing engineered bacteria into the environment, considerable further research and testing would be necessary. However, if the project was deemed safe, workers carrying out the spraying should wear an appropriate face mask to minimize inhaling of spores. During the spraying procedure there is a risk that spores could escape into the surrounding environment. As outlined above, ''B. subtilis'' 168 is non-pathogenic and therefore very unlikely to pose a risk to public safety, particularly since any escaping spores would be unable to germinate in the absence of culture media.<br />
<br />
=====Environmental Safety=====<br />
For reasons outlined above, the ''E. coli'' strain DH5α has very limited ability to survive outside the laboratory so taht in the event of escape, it would be unable to survive, disseminate, or displace other organisms. Therefore no specific environmental hazards associated with the ''E. coli'' strain were identified.<br />
<br />
''Bacillus subtilis'' strain 3610 and ''Bacillus sphaericus'' strain LMG22257 are both equivalent to wild type strains that are already widespread in the environment. They have not been modified to enhance their ability to survive, disseminate or displace other organisms. Therefore no specific environmental hazards associated with the ''Bacillus subtilis'' strain 3610 or ''Bacillus sphaericus'' strain LMG22257 were identified.<br />
<br />
GM derivatives of ''Bacillus subtilis'' strain 168 will be released deliberately. This is a strain that has been maintained in the lab for 52 years, during which it has accumulated disabling mutations, such as the tryptophan auxotrophy mentioned above, which greatly diminishes its ability to survive and disseminate outside the laboratory (Zeigler ''et al.'', 2008). It is very unlikely that it could compete effectively and replace wild-type ''B. subtilis''. Nonetheless, the release of any GMO into the environment has been a concern since early days of genetic engineering and one of our instructors (Prof. Anil Wipat) has previously studied such issues (Wipat, 1990). To minimise concerns about environmental safety, in this project we designed the [[Team:Newcastle/Non-target-environment kill switch|Non-target-environment kill switch]] genetic part to prevent dissemination after release into the environment.<br />
<br />
'''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
We do not see any safety issues for the new Biobricks parts that we made this year. <br />
<br />
'''3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
Yes there is a biosafety group at the Centre for Bacterial Cell Biology and Institute of Cell and Molecular Biosciences, which includes the Institute Safety Officer (SSO), Biological Safety Supervisor (BSS), Genetic Modification Chairperson (GMC), Radiation Protection Supervisor (RPS), Laser Protection Officer and Lab Heads. They have reviewed the safety of the lab regularly during the duration of the project.<br />
<br />
* If yes, what does your local biosafety group think about your project?<br />
<br />
They are aware of the project and have reviewed it thoroughly with the team. They discussed about each Biobrick part in detail and found no safety issues with any of them.<br />
<br />
'''4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A full risk assessment should be carried out before the work begins. This should consider: <br />
(i) Consequences of any identifiable hazard and<br />
(ii) The likelihood of the hazard arising.<br />
The risk can then be defined and appropriate control measures can be introduced to minimise the risk. <br />
<br />
The inclusion of a safety kill switch, such as that detailed in our project, could help reduce the risk of accidental release. In addition, strains should be marked with unique barcodes to allow them to be tracked, should they accidently escape.<br />
<br />
<br />
[1] Wipat, A. (1990). "''Release and detection of geneticaly engineered streptomycetes in soil''". PhD thesis, Microbiology Department, John Moores University.<br />
<br />
[2] Zeigler DR, Prágai Z, Rodriguez S, Chevreux B, Muffler A, Albert T et al. (2008). "''The origins of 168, W23, and other Bacillus subtilis legacy strains''". Journal of Bacteriology, 190(21), 6983-95.<br />
<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/Filamentous_CellsTeam:Newcastle/Filamentous Cells2010-10-28T01:53:04Z<p>RachelBoyd: /* Graphs */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
<br />
=Filamentous cell formation by overexpression of ''yneA''=<br />
<br />
''Bacillus subtilis'' cell division is dependent on FtsZ. FtsZ forms a 30 subunit ring at the midpoint of the cell and contracts.<br />
<br />
YneA indirectly stops the formation of the FtsZ ring. In nature, ''yneA'' is expressed during SOS response, allowing the cell to repair DNA damage before continuing with the division cycle.<br />
<br />
It is hypothesized that YneA acts through unknown transmembrane proteins to inhibit FtsZ ring formation; we call these unknown components "Blackbox proteins".<br />
<br />
By expressing YneA and therefore inhibiting FtsZ ring formation, cells will grow filamentous.<br />
<br />
<br />
==Part==<br />
<br />
[[Image:yneA_brick2.png]]<br />
<br />
Our ''IPTG-inducible filamentous cell formation part'' puts ''yneA'' under the control of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302003 strongly LacI-repressible promoter that we designed, hyperspankoid]. In the presence of LacI, induction with IPTG will result in a filamentous cell phenotype. <br />
<br />
The part has no terminator, allowing for transcriptional fusion with ''gfp'' and visualisation under the microscope.<br />
<br />
This is part [http://partsregistry.org/Part:BBa_K302012 BBa_K302012] on the [http://partsregistry.org parts registry]. <br />
<br />
<br />
<br />
[[Image:biochemical_pathway_filamentous.png|700px]]<br />
<br />
==Computational model==<br />
<br />
{|<br />
|<br />
|-<br />
|[[Image:Newcastle_ModelFilamentous.png|600px]]<br />
|We wrote a computational model of our filamentous cell system in SBML and simulated it in COPASI to help us verify our part's behaviour before we built it. The graph on the left shows that FtsZ ring formation is low when ''yneA'' is highly expressed.<br />
|}<br />
<br />
{|<br />
|<br />
|-<br />
|[[Image:Newcastle CellDesigner Filamentous.png|600px]]<br />
|Visualisation of the model's biochemical network in CellDesigner.<br />
|}<br />
<br />
Downloads:<br />
*[[Media:Newcastle_filamentous.mod.txt|SBML-shorthand]]<br />
*[[Media:Newcastle_filamentous.xml.txt|SBML]]<br />
<br />
<br />
==Cloning strategy==<br />
<br />
[[Media:yneA cloning strategy.pdf|yneA cloning strategy]]<br />
<br />
==Characterisation==<br />
<br />
We integrated our part into the ''Bacillus subtilis'' 168 chromosome at ''amyE'' (using the integration vector pGFP-rrnB) and selected for integration by testing for the ability to hydrolyse starch. Homologous recombination at ''amyE'' destroys endogenous expression of amylase. Colonies that are not able to break down starch on agar plate do not have a white halo when exposed to iodine.<br />
<br />
The part was co-transcribed with ''gfp'' fluorescent marker by transcriptional fusion after the ''yneA'' coding sequence.<br />
<br />
We characterised the part first without, and then with, LacI repression (using the integration vector pMutin4 to integrate ''lacI'' into the ''Bacillus subtilis'' 168 chromosome). When testing the part under LacI repression cells were induced with IPTG for two hours.<br />
<br />
<br />
<br />
{|<br />
|[[Image:Newcastle_filamentous_control_pc_expt1.jpg|thumb|Normal ''Bacillus subtilis ''168|280px|centre]]<br />
|[[Image:Newcastle_filamentous_pc_expt1.jpg|thumb|Filamentous cells|280px|centre]]<br />
|[[Image:Newcastle_filamentous_gfp_expt1.jpg|thumb|Filamentous cells showing GFP signal |280px|centre]]<br />
|} <br />
<br />
<center><br />
{|<br />
|[[Image:Newcastle_filamentous_pc_expt2.jpg|thumb|Filamentous cells (integrated at ''amyE'')|300px|centre]]<br />
|<br />
|[[Image:Newcastle_filamentous_gfp_expt2.jpg|thumb|Filamentous cells showing GFP signal(integrated at ''amyE'') | 300px|centre]]<br />
|}<br />
</center><br />
<br />
===Graphs===<br />
<br />
====Table 1:====<br />
{| border="1"<br />
|-<br />
!Stats:<br />
!168<br />
!''yneA''<br />
!pMutin4 0μM IPTG<br />
!pMutin4 1μM IPTG<br />
|-<br />
|Average:<br />
|1.34μm<br />
|3.53μm<br />
|1.74μm<br />
|3.19μm<br />
|-<br />
|Max:<br />
|2.30μm<br />
|6.00μm<br />
|3.62μm<br />
|9.77μm<br />
<br />
|-<br />
|Min:<br />
|0.55μm<br />
|1.31μm<br />
|0.88μm<br />
|1.14μm<br />
|-<br />
|Median:<br />
|1.33μm<br />
|3.27μm<br />
|1.62μm<br />
|2.66μm<br />
|-<br />
|Standard Deviation:<br />
|0.32μm<br />
|1.01μm<br />
|0.80μm<br />
|1.56μm<br />
|}<br />
<br />
<br />
====Figure 1:====<br />
{|<br />
|-<br />
|Distribution of cell lengths is not normal, so the mean is misleading; we are reporting the median instead.<br />
|-<br />
|[[Image:Teamnewcastle_yneA168.png|600px]]<br />
|-<br />
|Figure1: shows statistics for populations of cells<br />
*overexpression of the ''yneA'' construct (Δ''amyE'':pSpac(hy)-oid::''yneA''(cells with YneA construct but no inhibitory regulation) ) leads to a longer cell length compared with our control ''Bacillus subtilis 168''.<br />
*pMT4_0.0: YneA construct in pMutin4 vector with inhibition and no IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4) <br />
*pMT4_1.0: YneA construct in pMutin4 vector with inhibition and 1.0 μM IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4) <br />
|-<br />
|with inhibition cell lengths are comparable to ''Bacillus subtilis 168'' at 0μM IPTG and longer with IPTG induction.<br />
|}<br />
<br />
<br />
====Figure 2:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_yneA1.jpg|300px]][[Image:Teamnewcastle_yneA.jpg|300px]] <br />
|-<br />
|'''Figure2''': ''Bacillus subtilis 168'' cells (left),''Bacillus subtilis'' expressing ''yneA''(centre) and ''Bacillus subtilis'' overexpressing ''yneA''(right)<br />
|-<br />
|The images we have taken this data from had very different numbers of cells, so the cells counts are misleading therefore we are reporting the proportions of cells at a given length. <br />
|}<br />
<br />
<br />
====Figure 3:====<br />
{|<br />
|-<br />
|[[Image:newcastle_no induction.jpg|600px]]<br />
|-<br />
|Figure 3 shows the percentage of cells at different lengths (μm) uninduced<br />
|}<br />
<br />
<br />
====Figure 4:====<br />
{|<br />
|-<br />
|Figure 4:''Bacillus subtilis'' 168 cells (left) and non-induced cells (right)<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_noindBS.jpg|300px]] <br />
|-<br />
|}<br />
<br />
<br />
====Figure 5:====<br />
{|<br />
|-<br />
|[[Image:newcastle_0.2 induction.jpg|600px]]<br />
|-<br />
|Figure 5: shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG <br />
|}<br />
<br />
====Figure 6:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_0.2indBS.jpg|300px]]<br />
|-<br />
|Figure 6: ''Bacillus subtilis 168'' cells (left) and cells induced at 0.2mM IPTG (right)<br />
|}<br />
<br />
<br />
====Figure 7:====<br />
{|<br />
|-<br />
|[[Image:newcastle_1IPTG.jpg|600px]]<br />
|-<br />
|Figure 7: shows the percentage of cells at different lengths (μm) induced at 1mM IPTG <br />
|}<br />
<br />
<br />
====Figure 8:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_1indBS2.jpg|300px]] <br />
|-<br />
|Figure 8: ''Bacillus subtilis'' 168 cells (left) and cells induced at 1mM IPTG(right)<br />
|}<br />
<br />
==Research==<br />
<br />
[[Team:Newcastle/Initial_filamentous|Initial Research]]<br />
<br />
==References==<br />
<br />
Kawai, Y., Moriya, S., & Ogasawara, N. (2003). ''"Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis"''. Molecular microbiology, 47(4), 1113-22.<br />
<br />
Mo, A.H. & Burkholder, W.F., (2010). ''"YneA , an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis , Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity"'' ᰔ †. Society, 192(12), 3159-3173.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/Filamentous_CellsTeam:Newcastle/Filamentous Cells2010-10-28T00:47:02Z<p>RachelBoyd: /* Figure 5: */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
<br />
=Filamentous cell formation by overexpression of ''yneA''=<br />
<br />
''Bacillus subtilis'' cell division is dependent on FtsZ. FtsZ forms a 30 subunit ring at the midpoint of the cell and contracts.<br />
<br />
YneA indirectly stops the formation of the FtsZ ring. In nature, ''yneA'' is expressed during SOS response, allowing the cell to repair DNA damage before continuing with the division cycle.<br />
<br />
It is hypothesized that YneA acts through unknown transmembrane proteins to inhibit FtsZ ring formation; we call these unknown components "Blackbox proteins".<br />
<br />
By expressing YneA and therefore inhibiting FtsZ ring formation, cells will grow filamentous.<br />
<br />
<br />
==Part==<br />
<br />
[[Image:yneA_brick2.png]]<br />
<br />
Our ''IPTG-inducible filamentous cell formation part'' puts ''yneA'' under the control of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302003 strongly LacI-repressible promoter that we designed, hyperspankoid]. In the presence of LacI, induction with IPTG will result in a filamentous cell phenotype. <br />
<br />
The part has no terminator, allowing for transcriptional fusion with ''gfp'' and visualisation under the microscope.<br />
<br />
This is part [http://partsregistry.org/Part:BBa_K302012 BBa_K302012] on the [http://partsregistry.org parts registry]. <br />
<br />
<br />
<br />
[[Image:biochemical_pathway_filamentous.png|700px]]<br />
<br />
==Computational model==<br />
<br />
{|<br />
|<br />
|-<br />
|[[Image:Newcastle_ModelFilamentous.png|600px]]<br />
|We wrote a computational model of our filamentous cell system in SBML and simulated it in COPASI to help us verify our part's behaviour before we built it. The graph on the left shows that FtsZ ring formation is low when ''yneA'' is highly expressed.<br />
|}<br />
<br />
{|<br />
|<br />
|-<br />
|[[Image:Newcastle CellDesigner Filamentous.png|600px]]<br />
|Visualisation of the model's biochemical network in CellDesigner.<br />
|}<br />
<br />
Downloads:<br />
*[[Media:Newcastle_filamentous.mod.txt|SBML-shorthand]]<br />
*[[Media:Newcastle_filamentous.xml.txt|SBML]]<br />
<br />
<br />
==Cloning strategy==<br />
<br />
[[Media:yneA cloning strategy.pdf|yneA cloning strategy]]<br />
<br />
==Characterisation==<br />
<br />
We integrated our part into the ''Bacillus subtilis'' 168 chromosome at ''amyE'' (using the integration vector pGFP-rrnB) and selected for integration by testing for the ability to hydrolyse starch. Homologous recombination at ''amyE'' destroys endogenous expression of amylase. Colonies that are not able to break down starch on agar plate do not have a white halo when exposed to iodine.<br />
<br />
The part was co-transcribed with ''gfp'' fluorescent marker by transcriptional fusion after the ''yneA'' coding sequence.<br />
<br />
We characterised the part first without, and then with, LacI repression (using the integration vector pMutin4 to integrate ''lacI'' into the ''Bacillus subtilis'' 168 chromosome). When testing the part under LacI repression cells were induced with IPTG for two hours.<br />
<br />
<br />
<br />
{|<br />
|[[Image:Newcastle_filamentous_control_pc_expt1.jpg|thumb|Normal ''Bacillus subtilis ''168|280px|centre]]<br />
|[[Image:Newcastle_filamentous_pc_expt1.jpg|thumb|Filamentous cells|280px|centre]]<br />
|[[Image:Newcastle_filamentous_gfp_expt1.jpg|thumb|Filamentous cells showing GFP signal |280px|centre]]<br />
|} <br />
<br />
<center><br />
{|<br />
|[[Image:Newcastle_filamentous_pc_expt2.jpg|thumb|Filamentous cells (integrated at ''amyE'')|300px|centre]]<br />
|<br />
|[[Image:Newcastle_filamentous_gfp_expt2.jpg|thumb|Filamentous cells showing GFP signal(integrated at ''amyE'') | 300px|centre]]<br />
|}<br />
</center><br />
<br />
===Graphs===<br />
<br />
====Table1:====<br />
{| border="1"<br />
|-<br />
!Stats:<br />
!168<br />
!''yneA''<br />
!pMutin4 0μM IPTG<br />
!pMutin4 1μM IPTG<br />
|-<br />
|Average:<br />
|1.34μm<br />
|3.53μm<br />
|1.74μm<br />
|3.19μm<br />
|-<br />
|Max:<br />
|2.30μm<br />
|6.00μm<br />
|3.62μm<br />
|9.77μm<br />
<br />
|-<br />
|Min:<br />
|0.55μm<br />
|1.31μm<br />
|0.88μm<br />
|1.14μm<br />
|-<br />
|Median:<br />
|1.33μm<br />
|3.27μm<br />
|1.62μm<br />
|2.66μm<br />
|-<br />
|Standard Deviation:<br />
|0.32μm<br />
|1.01μm<br />
|0.80μm<br />
|1.56μm<br />
|}<br />
<br />
<br />
====Figure1:====<br />
{|<br />
|-<br />
|Distribution of cell lengths is not normal, so the mean is misleading; we are reporting the median instead.<br />
|-<br />
|[[Image:Teamnewcastle_yneA168.png|600px]]<br />
|-<br />
|Figure1: shows statistics for populations of cells<br />
*overexpression of the ''yneA'' construct (Δ''amyE'':pSpac(hy)-oid::''yneA''(cells with YneA construct but no inhibitory regulation) ) leads to a longer cell length compared with our control ''Bacillus subtilis 168''.<br />
*pMT4_0.0: YneA construct in pMutin4 vector with inhibition and no IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4) <br />
*pMT4_1.0: YneA construct in pMutin4 vector with inhibition and 1.0 μM IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4) <br />
|-<br />
|with inhibition cell lengths are comparable to ''Bacillus subtilis 168'' at 0μM IPTG and longer with IPTG induction.<br />
|}<br />
<br />
<br />
====Figure2:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_yneA1.jpg|300px]][[Image:Teamnewcastle_yneA.jpg|300px]] <br />
|-<br />
|'''Figure2''': ''Bacillus subtilis 168'' cells (left),''Bacillus subtilis'' expressing ''yneA''(centre) and ''Bacillus subtilis'' overexpressing ''yneA''(right)<br />
|-<br />
|The images we have taken this data from had very different numbers of cells, so the cells counts are misleading therefore we are reporting the proportions of cells at a given length. <br />
|}<br />
<br />
<br />
====Figure 3:====<br />
{|<br />
|-<br />
|[[Image:newcastle_no induction.jpg|600px]]<br />
|-<br />
|Figure 3 shows the percentage of cells at different lengths (μm) uninduced<br />
|}<br />
<br />
<br />
====Figure 4:====<br />
{|<br />
|-<br />
|Figure 4:''Bacillus subtilis'' 168 cells (left) and non-induced cells (right)<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_noindBS.jpg|300px]] <br />
|-<br />
|}<br />
<br />
<br />
====Figure 5:====<br />
{|<br />
|-<br />
|[[Image:newcastle_0.2 induction.jpg|600px]]<br />
|-<br />
|Figure 5: shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG <br />
|}<br />
<br />
====Figure 6:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_0.2indBS.jpg|300px]]<br />
|-<br />
|Figure 6: ''Bacillus subtilis 168'' cells (left) and cells induced at 0.2mM IPTG (right)<br />
|}<br />
<br />
<br />
====Figure 7:====<br />
{|<br />
|-<br />
|[[Image:newcastle_1IPTG.jpg|600px]]<br />
|-<br />
|Figure 7: shows the percentage of cells at different lengths (μm) induced at 1mM IPTG <br />
|}<br />
<br />
<br />
====Figure 8:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_1indBS2.jpg|300px]] <br />
|-<br />
|Figure 8: ''Bacillus subtilis'' 168 cells (left) and cells induced at 1mM IPTG(right)<br />
|}<br />
<br />
==Research==<br />
<br />
[[Team:Newcastle/Initial_filamentous|Initial Research]]<br />
<br />
==References==<br />
<br />
Kawai, Y., Moriya, S., & Ogasawara, N. (2003). ''"Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis"''. Molecular microbiology, 47(4), 1113-22.<br />
<br />
Mo, A.H. & Burkholder, W.F., (2010). ''"YneA , an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis , Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity"'' ᰔ †. Society, 192(12), 3159-3173.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/MedalsTeam:Newcastle/Medals2010-10-27T22:08:40Z<p>RachelBoyd: /* iGEM Judging Comments */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
='''iGEM Judging Comments'''=<br />
<br />
This year our team came up with a highly ambitious project and achieved goals in several different areas.<br />
<br />
We successfully modelled, designed, characterised and submitted our IPTG-inducible Filamentous cells BioBrick part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K302012 BBa_K302012]). <br />
<br />
We also developed [https://2010.igem.org/Team:Newcastle/E-Science an e-Science Approach to Synthetic Biology] which focuses on workflows in synthetic biology. This work led to the creation of BBF RFC 66.<br />
<br />
We developed the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302018 Subtilin Immunity] BioBrick, which provides immunity against the lantibiotic subtilin, a quorum sensing molecule for our cell population. Subtilin will help to initiate a population-wide response for concrete repair.<br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302015 Urease] BioBrick increases urea hydrolysis by increasing arginine and arginase production. Arginase breaks down arginine to form urea and ornithine. The overall increase in urea leads to an increase in urease production which hydrolyses urea into carbonate and ammonium ions which are exported out of the cell. The carbonate ions form a bond with calcium ions in the environment, resulting in the production of calcium carbonate.<br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302016 Swarming] BioBrick would help ''Bacillus subtilis'' 168 to swarm on the concrete surface by producing surfactin to reduce surface tension and by initiating flagellum biosynthesis.<br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302030 Levan Glue] BoBrick produces Levan glue in the presence of sucrose. The glue will act as a binding agent for the filamentous cells and the calcium carbonate crystals and will also help in filling up the crack thereby preventing corrosion of the steel reinforcements. <br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302035 ''mazEF'' Kill switch] is built around a stable toxin-antitoxin system for ''Bacillus subtilis''. It would kill bacteria in the absence of sucrose thereby helping to make our project environmentally friendly. <br />
<br />
In total we designed and entered 31 BioBrick parts in the parts registry. <br />
<br />
We also took [https://2010.igem.org/Team:Newcastle/solution#Scanning_Electron_Microscope_Images Scanning Electron Microscope photographs] and found some interesting results: photographs of cells trying to fill up the crack, calcium carbonate crystals and Levan glue covering the cells.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/MedalsTeam:Newcastle/Medals2010-10-27T22:00:33Z<p>RachelBoyd: /* iGEM Judging Comments */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
='''iGEM Judging Comments'''=<br />
<br />
This year our team came up with a highly ambitious project and achieved goals in several different areas.<br />
<br />
We successfully modelled, designed, characterised and submitted our IPTG-inducible Filamentous cells BioBrick part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K302012 BBa_K302012]). <br />
<br />
We also developed [https://2010.igem.org/Team:Newcastle/E-Science An e-Science Approach to Synthetic Biology] which focuses on workflows, to synthetic biology. This led to the creation of BBF RFC 66 based upon our work.<br />
<br />
We developed the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302018 Subtilin Immunity] BioBrick part which provides cells immunity against the lantibiotic subtilin, which is also a quorum sensing molecule for our cell population. It will help in initiating a population wide response for initiating concrete repair.<br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302015 Urease] BioBrick part helps in the increasing urea hydrolysis by increasing arginine and arginase production. Arginase breaks down arginine to form urea and ornithine. The overall increase in urea leads to an increase in urease production which hydrolyses urea into carbonate and ammonium ions which are exported out of the cell. The carbonate ions forms a bond with calcium ions provided in the media and form calcium carbonate.<br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302016 Swarming] BioBrick part would help ''Bacillus subtilis'' 168 to swarm on the concrete surface by producing surfactin to reduce surface tension and by initiating flagellum biosynthesis.<br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302030 Levan Glue] BoBrick part produces Levan glue in the presence of sucrose. The glue will act as a binding agent for the filamentous cells and the calcium carbonate crystals and will also help in filling up the crack thereby preventing corrosion of the steel reinforcements. <br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302035 ''mazEF'' Kill switch] is a stable toxin-antitoxin system for ''Bacillus subtilis''. It would kill bacteria in the absence of sucrose thereby helping to make our project environmentally friendly. <br />
<br />
In total we designed and entered 31 BioBrick parts in the parts registry. <br />
<br />
We also took [https://2010.igem.org/Team:Newcastle/solution#Scanning_Electron_Microscope_Images Scanning Electron Microscope photographs] and found some interesting results: photographs of cells trying to fill up the crack, calcium carbonate crystals and Levan glue covering the cells.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/safetyTeam:Newcastle/safety2010-10-27T21:59:30Z<p>RachelBoyd: /* Safety Issues */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
==Safety Issues==<br />
<br />
These are the safety questions for the judging form:<br />
<br />
'''1. Would any of your project ideas raise safety issues in terms of:'''<br />
<br />
*'''Researcher Safety''':<br />
<br />
We worked in the Centre for Bacterial Cell Biology (CBCB) at Newcastle University for the entire project, where there are clearly defined safety rules and regulations that all laboratory workers must follow. One of our advisors, Dr Wendy Smith, guided us through the safety procedures along with some of the basic techniques during the first [[Team:Newcastle/14_June_2010|introductory]] week, before any laboratory work on the project began. This included carrying out the following risk assessments to determine what control measures would be required.<br />
<br />
(i)'''Chemical Hazards''':<br />
At the beginning of the project written risk assessments were already available in the host laboratories for all procedures that involved potentially hazardous chemicals. These risk assessments were reviewed and the recommended control measures were strictly followed throughout. No additional chemical hazards specific to this project were identified.<br />
<br />
(ii)'''Radioisotopes and carcinogens''':<br />
None of these were used in this project.<br />
<br />
(iii)'''Biological hazards''':<br />
Throughout the project, we used the ''Escherichia coli'' strain DH5α, ''Bacillus subtilis'' strain 3610, ''Bacillus subtilis'' strain 168 and ''Bacillus sphaericus'' strain LMG 22257. Wild-type ''E. coli'' is classified as a hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP). However, ''E. coli'' strain DH5α is derived from a laboratory strain ''E. coli'' K12, which is recognised as disabled and equivalent to an ACDP hazard group 1 organism (i.e. unlikely to cause disease). ''E. coli'' K12 and its derivatives such as strain DH5α are unable to colonise in humans or animals and consequently pose negligible risk to human or animal health. Wild-type ''Bacillus subtilis'' (i.e. strain 3610) is classified as an ACDP hazard group 1 organism and its derivative ''B. subtilis'' strain 168 has disabling auxotrophs mutations (e.g. conferring a requirement for tryptophan, Zeigler ''et al'', 2008) that makes it even less likely to colonise or cause harm to human or animal health. Also ''Bacillus sphaericus'' LMG 22257 is classified as an ADCP hazard group 1 organism making it unlikely to cause any harm to either human or animal health. The potential of any sequences cloned into these bacterial hosts during the project to pose additional hazards was also assessed. None of these sequences were associated with pathogenic traits or traits that might significantly enhance the survival outside the lab. Therefore, no specific safety issues, other than those associated with use of any non-pathogenic microorganism, were identified. It was concluded that containment level 1(CL1) would be sufficient to ensure researcher safety. Nonetheless, all work was carried out in strict compliance with the host laboratory's standard safety procedures, which were more stringent that those required for CL1.<br />
<br />
(iv)'''Other hazards''':<br />
The project also involved conducting some work in the Engineering structures laboratory, where we made and broke concrete 'blocks'. Appropriate safety regulations for the type of work in this laboratory were followed. When we were in the structures lab, we wore safety goggles, steel toe cap boots and gloves.<br />
* '''Public Safety''':<br />
Our project concerns repairing cracks on concrete surfaces by spraying these surfaces with spores of engineered derivatives of ''Bacillus subtilis'' 168. Before releasing engineered bacteria into the environment, considerable further research and testing would be necessary. However, if the project was deemed safe, workers carrying out the spraying should wear an appropriate face mask to minimize inhaling of spores. During the spraying procedure there is a risk that spores could escape into the surrounding environment. As outlined above, ''B. subtilis'' 168 is non-pathogenic and therefore very unlikely to pose a risk to public safety, particularly since any escaping spores would be unable to germinate in the absence of culture media.<br />
<br />
*'''Environmental Safety''':<br />
For reasons outlined above, the ''E. coli'' strain DH5α has very limited ability to survive outside the laboratory so taht in the event of escape, it would be unable to survive, disseminate, or displace other organisms. Therefore no specific environmental hazards associated with the ''E. coli'' strain were identified.<br />
<br />
''Bacillus subtilis'' strain 3610 and ''Bacillus sphaericus'' strain LMG22257 are both equivalent to wild type strains that are already widespread in the environment. They have not been modified to enhance their ability to survive, disseminate or displace other organisms. Therefore no specific environmental hazards associated with the ''Bacillus subtilis'' strain 3610 or ''Bacillus sphaericus'' strain LMG22257 were identified.<br />
<br />
GM derivatives of ''Bacillus subtilis'' strain 168 will be released deliberately. This is a strain that has been maintained in the lab for 52 years, during which it has accumulated disabling mutations, such as the tryptophan auxotrophy mentioned above, which greatly diminishes its ability to survive and disseminate outside the laboratory (Zeigler ''et al.'', 2008). It is very unlikely that it could compete effectively and replace wild-type ''B. subtilis''. Nonetheless, the release of any GMO into the environment has been a concern since early days of genetic engineering and one of our instructors (Prof. Anil Wipat) has previously studied such issues (Wipat, 1990). To minimise concerns about environmental safety, in this project we designed the [[Team:Newcastle/Non-target-environment kill switch|Non-target-environment kill switch]] genetic part to prevent dissemination after release into the environment.<br />
<br />
'''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
We do not see any safety issues for the new Biobricks parts that we made this year. <br />
<br />
'''3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
Yes there is a biosafety group at the Centre for Bacterial Cell Biology and Institute of Cell and Molecular Biosciences, which includes the Institute Safety Officer (SSO), Biological Safety Supervisor (BSS), Genetic Modification Chairperson (GMC), Radiation Protection Supervisor (RPS), Laser Protection Officer and Lab Heads. They have reviewed the safety of the lab regularly during the duration of the project.<br />
<br />
* If yes, what does your local biosafety group think about your project?<br />
<br />
They are aware of the project and have reviewed it thoroughly with the team. They discussed about each Biobrick part in detail and found no safety issues with any of them.<br />
<br />
'''4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A full risk assessment should be carried out before the work begins. This should consider: <br />
(i) Consequences of any identifiable hazard and<br />
(ii) The likelihood of the hazard arising.<br />
The risk can then be defined and appropriate control measures can be introduced to minimise the risk. <br />
<br />
The inclusion of a safety kill switch, such as that detailed in our project, could help reduce the risk of accidental release. In addition, strains should be marked with unique barcodes to allow them to be tracked, should they accidently escape.<br />
<br />
==Ethics==<br />
<br />
===Synthetic Biology: Background===<br />
<br />
Synthetic biology is a new research field that has a big potential in coming up with solutions for our everyday problems. However, ethical issues have been raised since the start of development in this field. Here we will discuss the ethical issues that our project will bring.<br />
<br />
====Biohackers====<br />
<br />
In this field, there are always people that view synthetic biology's negative aspects with concern, like worrying about the production of pathogens to be used as weapons. Their fears are that people will hack into systems to obtain data that might be used to do this. Therefore, we have to be very careful with the products of our research so that they remain safe in our hands.<br />
<br />
====Playing God!!!====<br />
<br />
<br />
In synthetic biology, we control the lives of bacteria. We insert proteins into bacteria to make them do what we want them to do. We make them kill themselves at the end of their job because they might be released into the environment and be harmful to other living things. It shows that we do not really treat the single-celled organism as life. However, we are coming up with a novel solution for the environment by healing concrete cracks.<br />
<br />
====Biosecurity====<br />
<br />
Bacteria might be harmful to the environment and living things around. Considering the fact that they are such small organisms and could not be spotted with the naked-eye if they are released in air, we make them kill themselves with the ‘kill switch’ biobrick. This can be very inconsiderate standing in a bacteria’s point of view, but it is the novel thing to do to avoid them hurting other living organisms.<br />
<br />
===Construction: Background===<br />
<br />
Cracks form in concrete structures as soon as they set. These cracks continue to grow in size if they are not repaired due to the weight of the structure, imposed load, freeze-thaw effect and wind loading. The bigger the cracks are, the higher the rate of water seeping into the steel reinforcements, causing them to corrode and thus weakening the structure. Therefore, we would like to use Bacilla Filla to fill up these cracks before things get worse.<br />
<br />
====Environmental Disaster====<br />
<br />
Concrete is a very widely used material in construction. In fact, cement that is used to make concrete is the second most widely used substance in the world after water. Some building structures have to be demolished because the cracks formed appear to be threatening the strength of the structures. These buildings have to be rebuilt in order to reinforce its tensile strength. This is an unsustainable method of recovery because cement is a material that requires a lot of energy to be produced. In the production of cement, the rotating kiln requires the temperature to be between 1350°C-1400°C, which not only uses up a lot of energy resources, but also produces a lot of carbon dioxide. These carbon dioxide contributes to the green house effect which then leads to global warming. Therefore, our project can help to reduce renovation of buildings which in turn lowers the amount of cement that has to be produced each year.<br />
<br />
====Post-Chernobyl Disaster====<br />
<br />
The nuclear power plant combustion that occurred in Chernobyl on 26 April 1986 has allowed radioactive elements to be released into the environment, threatening the health of people living within close proximity of the plant. Therefore, a shelter, the Shelter Implementation Plan (SIP) which will be completed in 2012 is currently being built to cover up the area that where the accident occurred so as to make it environmentally friendly. However, if cracks were to form, something that inevitably occurs for building structures, those radioactive elements will still be dispersed into the environment. With the help of our project, those cracks will be able to be filled-up and the area will be safe again.<br />
<br />
<br />
<br />
[1] Wipat, A. (1990). "''Release and detection of geneticaly engineered streptomycetes in soil''". PhD thesis, Microbiology Department, John Moores University.<br />
<br />
[2] Zeigler DR, Prágai Z, Rodriguez S, Chevreux B, Muffler A, Albert T et al. (2008). "''The origins of 168, W23, and other Bacillus subtilis legacy strains''". Journal of Bacteriology, 190(21), 6983-95.<br />
<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/safetyTeam:Newcastle/safety2010-10-27T21:49:48Z<p>RachelBoyd: /* Safety Issues */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
==Safety Issues==<br />
<br />
These are the safety questions for the judging form:<br />
<br />
'''1. Would any of your project ideas raise safety issues in terms of:'''<br />
<br />
*'''Researcher Safety''':<br />
<br />
We worked in the Centre for Bacterial Cell Biology (CBCB) at Newcastle University for the entire project, where there are clearly defined safety rules and regulations that all laboratory workers must follow. One of our advisors, Dr Wendy Smith, guided us through the safety procedures along with some of the basic techniques during the first [[Team:Newcastle/14_June_2010|introductory]] week, before any laboratory work on the project began. This included carrying out the following risk assessments to determine what control measures would be required.<br />
<br />
(i)'''Chemical Hazards''':<br />
At the beginning of the project written risk assessments were already available in the host laboratories for all procedures that involved potentially hazardous chemicals. These risk assessments were reviewed and the recommended control measures were strictly followed throughout. No additional chemical hazards specific to this project were identified.<br />
<br />
(ii)'''Radioisotopes and carcinogens''':<br />
None of these were used in this project.<br />
<br />
(iii)'''Biological hazards''':<br />
Throughout the project, we used the ''Escherichia coli'' strain DH5α, ''Bacillus subtilis'' strain 3610, ''Bacillus subtilis'' strain 168 and ''Bacillus sphaericus'' strain LMG 22257. Wild-type ''E. coli'' is classified as a hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP). However, ''E. coli'' strain DH5α is derived from a laboratory strain ''E. coli'' K12, which is recognised as disabled and equivalent to an ACDP hazard group 1 organism (i.e. unlikely to cause disease). ''E. coli'' K12 and its derivatives such as strain DH5α are unable to colonise in humans or animals and consequently pose negligible risk to human or animal health. Wild-type ''Bacillus subtilis'' (i.e. strain 3610) is classified as an ACDP hazard group 1 organism and its derivative ''B. subtilis'' strain 168 has disabling auxotrophs mutations (e.g. conferring a requirement for tryptophan, Zeigler ''et al'', 2008) that makes it even less likely to colonise or cause harm to human or animal health. Also ''Bacillus sphaericus'' LMG 22257 is classified as an ADCP hazard group 1 organism making it unlikely to cause any harm to either human or animal health. The potential of any sequences cloned into these bacterial hosts during the project to pose additional hazards was also assessed. None of these sequences were associated with pathogenic traits or traits that might significantly enhance the survival outside the lab. Therefore, no specific safety issues, other than those associated with use of any non-pathogenic microorganism, were identified. It was concluded that containment level 1(CL1) would be sufficient to ensure researcher safety. Nonetheless, all work was carried out in strict compliance with the host laboratory's standard safety procedures, which were more stringent that those required for CL1.<br />
<br />
(iv)'''Other hazards''':<br />
The project also involved conducting some work in the Engineering structures laboratory, where we made and broke concrete 'blocks'. Appropriate safety regulations for the type of work in this laboratory were followed. When we were in the structures lab, we wore safety goggles, steel toe cap boots and gloves.<br />
* '''Public Safety''':<br />
Our project concerns repairing cracks on concrete surfaces by spraying these surfaces with spores of engineered derivatives of ''Bacillus subtilis'' 168. It is recommended that workers carrying out the spraying should wear an appropriate face mask to minimize inhaling of spores. During the spraying procedure spores could escape into the surrounding environment, but their concentration will diluted very rapidly with distance, greatly reducing potential hazards away from the immediate area of spraying. As outlined above, ''B. subtilis'' 168 is non-pathogenic and therefore very unlikely to pose a risk to public safety, particularly since any escaping spores will be unable to germinate in the absence of culture media.<br />
<br />
*'''Environmental Safety''':<br />
For reasons outlined above, the ''E. coli'' strain DH5α has very limited ability to survive outside the laboratory so taht in the very unlikely event of escape, it will be unable to survive, disseminate with and/or displace other organisms. Therefore no specific environmental hazards associated with the ''E. coli'' strain were identified.<br />
<br />
''Bacillus subtilis'' strain 3610 and ''Bacillus sphaericus'' strain LMG22257 are both equivalent to wild type strain that are already prevalent in the environment. They have not been modified to enhance their ability to survive, disseminate or displace with other organisms. Therefore no specific environmental hazards associated with the ''Bacillus subtilis'' strain 3610 and ''Bacillus sphaericus'' strain LMG22257 were identified.<br />
<br />
GM derivatives of ''Bacillus subtilis'' strain 168 will be released deliberately. This is a strain that has been maintained in the lab for 52 years, during which it has accumulated disabling mutations, such as tryptophan auxotrophy mentioned above, which greatly diminishes its ability to survive and disseminate outside the laboratory (Zeigler ''et al.'', 2008). It seems very unlikely that it could compete effectively and replace wild-type ''B. subtilis''. Nonetheless, the release of any GMO into the environment has been a concern since early days of genetic engineering and one of our instructors (Prof. Anil Wipat) has previously studied such issues (Wipat, 1990). To minimise concerns about environmental safety, in this project we designed the [[Team:Newcastle/Non-target-environment kill switch|Non-target-environment kill switch]] genetic part to prevent dissemination after release into the environment.<br />
<br />
'''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
We do not see any safety issues for the new Biobricks parts that we made this year. <br />
<br />
'''3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
Yes there is a biosafety group at the Centre for Bacterial Cell Biology and Institute of Cell and Molecular Biosciences, which includes the Institute Safety Officer (SSO), Biological Safety Supervisor (BSS), Genetic Modification Chairperson (GMC), Radiation Protection Supervisor (RPS), Laser Protection Officer and Lab Heads. They have reviewed the safety of the lab from time to time during the duration of the project.<br />
<br />
* If yes, what does your local biosafety group think about your project?<br />
<br />
They are aware about the whole project and they reviewed it thoroughly with the whole team. They discussed about each and every Biobrick part in detail and found no safety issues with it.<br />
<br />
'''4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A full risk assessment should be carried out before the work begins. This should consider: <br />
(i) Consequences of any identifiable hazard and<br />
(ii) The likelihood of the hazard arising.<br />
The risk can then be defined and appropriate control measures can be introduced to minimise the risk. <br />
<br />
The inclusion of a safety kill switch, such as that detailed in our project, could help reduce the risk of accidental release. In addition, strains should be marked with unique barcodes to allow them to be tracked, should they accidently escape.<br />
<br />
==Ethics==<br />
<br />
===Synthetic Biology: Background===<br />
<br />
Synthetic biology is a new research field that has a big potential in coming up with solutions for our everyday problems. However, ethical issues have been raised since the start of development in this field. Here we will discuss the ethical issues that our project will bring.<br />
<br />
====Biohackers====<br />
<br />
In this field, there are always people that view synthetic biology's negative aspects with concern, like worrying about the production of pathogens to be used as weapons. Their fears are that people will hack into systems to obtain data that might be used to do this. Therefore, we have to be very careful with the products of our research so that they remain safe in our hands.<br />
<br />
====Playing God!!!====<br />
<br />
<br />
In synthetic biology, we control the lives of bacteria. We insert proteins into bacteria to make them do what we want them to do. We make them kill themselves at the end of their job because they might be released into the environment and be harmful to other living things. It shows that we do not really treat the single-celled organism as life. However, we are coming up with a novel solution for the environment by healing concrete cracks.<br />
<br />
====Biosecurity====<br />
<br />
Bacteria might be harmful to the environment and living things around. Considering the fact that they are such small organisms and could not be spotted with the naked-eye if they are released in air, we make them kill themselves with the ‘kill switch’ biobrick. This can be very inconsiderate standing in a bacteria’s point of view, but it is the novel thing to do to avoid them hurting other living organisms.<br />
<br />
===Construction: Background===<br />
<br />
Cracks form in concrete structures as soon as they set. These cracks continue to grow in size if they are not repaired due to the weight of the structure, imposed load, freeze-thaw effect and wind loading. The bigger the cracks are, the higher the rate of water seeping into the steel reinforcements, causing them to corrode and thus weakening the structure. Therefore, we would like to use Bacilla Filla to fill up these cracks before things get worse.<br />
<br />
====Environmental Disaster====<br />
<br />
Concrete is a very widely used material in construction. In fact, cement that is used to make concrete is the second most widely used substance in the world after water. Some building structures have to be demolished because the cracks formed appear to be threatening the strength of the structures. These buildings have to be rebuilt in order to reinforce its tensile strength. This is an unsustainable method of recovery because cement is a material that requires a lot of energy to be produced. In the production of cement, the rotating kiln requires the temperature to be between 1350°C-1400°C, which not only uses up a lot of energy resources, but also produces a lot of carbon dioxide. These carbon dioxide contributes to the green house effect which then leads to global warming. Therefore, our project can help to reduce renovation of buildings which in turn lowers the amount of cement that has to be produced each year.<br />
<br />
====Post-Chernobyl Disaster====<br />
<br />
The nuclear power plant combustion that occurred in Chernobyl on 26 April 1986 has allowed radioactive elements to be released into the environment, threatening the health of people living within close proximity of the plant. Therefore, a shelter, the Shelter Implementation Plan (SIP) which will be completed in 2012 is currently being built to cover up the area that where the accident occurred so as to make it environmentally friendly. However, if cracks were to form, something that inevitably occurs for building structures, those radioactive elements will still be dispersed into the environment. With the help of our project, those cracks will be able to be filled-up and the area will be safe again.<br />
<br />
<br />
<br />
[1] Wipat, A. (1990). "''Release and detection of geneticaly engineered streptomycetes in soil''". PhD thesis, Microbiology Department, John Moores University.<br />
<br />
[2] Zeigler DR, Prágai Z, Rodriguez S, Chevreux B, Muffler A, Albert T et al. (2008). "''The origins of 168, W23, and other Bacillus subtilis legacy strains''". Journal of Bacteriology, 190(21), 6983-95.<br />
<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/safetyTeam:Newcastle/safety2010-10-27T21:48:56Z<p>RachelBoyd: /* Safety Issues */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
==Safety Issues==<br />
<br />
These are the safety questions for the judging form:<br />
<br />
'''1. Would any of your project ideas raise safety issues in terms of:'''<br />
<br />
*'''Researcher Safety''':<br />
<br />
We worked in the Centre for Bacterial Cell Biology (CBCB) at Newcastle University for the entire project, where there are clearly defined safety rules and regulations that all laboratory workers must follow. One of our advisors, Dr Wendy Smith, guided us through the safety procedures along with some of the basic techniques during the first [[Team:Newcastle/14_June_2010|introductory]] week, before any laboratory work on the project began. This included carrying out the following risk assessments to determine what control measures would be required.<br />
<br />
(i)'''Chemical Hazards''':<br />
At the beginning of the project written risk assessments were already available in the host laboratories for all procedures that involved potentially hazardous chemicals. These risk assessments were reviewed and the recommended control measures were strictly followed throughout. No additional chemical hazards specific to this project were identified.<br />
<br />
(ii)'''Radioisotopes and carcinogens''':<br />
None of these were used in this project.<br />
<br />
(iii)'''Biological hazards''':<br />
Throughout the project, we used the ''Escherichia coli'' strain DH5α, ''Bacillus subtilis'' strain 3610, ''Bacillus subtilis'' strain 168 and ''Bacillus sphaericus'' strain LMG 22257. Wild-type ''E. coli'' is classified as a hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP). However, ''E. coli'' strain DH5α is derived from a laboratory strain ''E. coli'' K12, which is recognised as disabled and equivalent to an ACDP hazard group 1 organism (i.e. unlikely to cause disease). ''E. coli'' K12 and its derivatives such as strain DH5α are unable to colonise in humans or animals and consequently pose negligible risk to human or animal health. Wild-type ''Bacillus subtilis'' (i.e. strain 3610) is classified as an ACDP hazard group 1 organism and its derivative ''B. subtilis'' strain 168 has disabling auxotrophs mutations (e.g. conferring a requirement for tryptophan, Zeigler ''et al'', 2008) that makes it even less likely to colonise or cause harm to human or animal health. Also ''Bacillus sphaericus'' LMG 22257 is classified as an ADCP hazard group 1 organism making it unlikely to cause any harm to either human or animal health. The potential of any sequences cloned into these bacterial hosts during the project to pose additional hazards was also assessed. None of these sequences were associated with pathogenic traits or traits that might significantly enhance the survival outside the lab. Therefore, no specific safety issues, other than those associated with use of any non-pathogenic microorganism, were identified. It was concluded that containment level 1(CL1) would be sufficient to ensure researcher safety. Nonetheless, all work was carried out in strict compliance with the host laboratory's standard safety procedures, which were more stringent that those required for CL1.<br />
<br />
(iv)'''Other hazards''':<br />
The project also involved conducting some work in the Engineering structures laboratory, where we made and broke concrete 'blocks'. Appropriate safety regulations for the type of work in this laboratory were followed. When we were in the structures lab, we wore safety goggles, steel toe cap boots and gloves, which will be able to protect us from lab based accidents.<br />
<br />
* '''Public Safety''':<br />
Our project concerns repairing cracks on concrete surfaces by spraying these surfaces with spores of engineered derivatives of ''Bacillus subtilis'' 168. It is recommended that workers carrying out the spraying should wear an appropriate face mask to minimize inhaling of spores. During the spraying procedure spores could escape into the surrounding environment, but their concentration will diluted very rapidly with distance, greatly reducing potential hazards away from the immediate area of spraying. As outlined above, ''B. subtilis'' 168 is non-pathogenic and therefore very unlikely to pose a risk to public safety, particularly since any escaping spores will be unable to germinate in the absence of culture media.<br />
<br />
*'''Environmental Safety''':<br />
For reasons outlined above, the ''E. coli'' strain DH5α has very limited ability to survive outside the laboratory so taht in the very unlikely event of escape, it will be unable to survive, disseminate with and/or displace other organisms. Therefore no specific environmental hazards associated with the ''E. coli'' strain were identified.<br />
<br />
''Bacillus subtilis'' strain 3610 and ''Bacillus sphaericus'' strain LMG22257 are both equivalent to wild type strain that are already prevalent in the environment. They have not been modified to enhance their ability to survive, disseminate or displace with other organisms. Therefore no specific environmental hazards associated with the ''Bacillus subtilis'' strain 3610 and ''Bacillus sphaericus'' strain LMG22257 were identified.<br />
<br />
GM derivatives of ''Bacillus subtilis'' strain 168 will be released deliberately. This is a strain that has been maintained in the lab for 52 years, during which it has accumulated disabling mutations, such as tryptophan auxotrophy mentioned above, which greatly diminishes its ability to survive and disseminate outside the laboratory (Zeigler ''et al.'', 2008). It seems very unlikely that it could compete effectively and replace wild-type ''B. subtilis''. Nonetheless, the release of any GMO into the environment has been a concern since early days of genetic engineering and one of our instructors (Prof. Anil Wipat) has previously studied such issues (Wipat, 1990). To minimise concerns about environmental safety, in this project we designed the [[Team:Newcastle/Non-target-environment kill switch|Non-target-environment kill switch]] genetic part to prevent dissemination after release into the environment.<br />
<br />
'''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
We do not see any safety issues for the new Biobricks parts that we made this year. <br />
<br />
'''3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
Yes there is a biosafety group at the Centre for Bacterial Cell Biology and Institute of Cell and Molecular Biosciences, which includes the Institute Safety Officer (SSO), Biological Safety Supervisor (BSS), Genetic Modification Chairperson (GMC), Radiation Protection Supervisor (RPS), Laser Protection Officer and Lab Heads. They have reviewed the safety of the lab from time to time during the duration of the project.<br />
<br />
* If yes, what does your local biosafety group think about your project?<br />
<br />
They are aware about the whole project and they reviewed it thoroughly with the whole team. They discussed about each and every Biobrick part in detail and found no safety issues with it.<br />
<br />
'''4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A full risk assessment should be carried out before the work begins. This should consider: <br />
(i) Consequences of any identifiable hazard and<br />
(ii) The likelihood of the hazard arising.<br />
The risk can then be defined and appropriate control measures can be introduced to minimise the risk. <br />
<br />
The inclusion of a safety kill switch, such as that detailed in our project, could help reduce the risk of accidental release. In addition, strains should be marked with unique barcodes to allow them to be tracked, should they accidently escape.<br />
<br />
==Ethics==<br />
<br />
===Synthetic Biology: Background===<br />
<br />
Synthetic biology is a new research field that has a big potential in coming up with solutions for our everyday problems. However, ethical issues have been raised since the start of development in this field. Here we will discuss the ethical issues that our project will bring.<br />
<br />
====Biohackers====<br />
<br />
In this field, there are always people that view synthetic biology's negative aspects with concern, like worrying about the production of pathogens to be used as weapons. Their fears are that people will hack into systems to obtain data that might be used to do this. Therefore, we have to be very careful with the products of our research so that they remain safe in our hands.<br />
<br />
====Playing God!!!====<br />
<br />
<br />
In synthetic biology, we control the lives of bacteria. We insert proteins into bacteria to make them do what we want them to do. We make them kill themselves at the end of their job because they might be released into the environment and be harmful to other living things. It shows that we do not really treat the single-celled organism as life. However, we are coming up with a novel solution for the environment by healing concrete cracks.<br />
<br />
====Biosecurity====<br />
<br />
Bacteria might be harmful to the environment and living things around. Considering the fact that they are such small organisms and could not be spotted with the naked-eye if they are released in air, we make them kill themselves with the ‘kill switch’ biobrick. This can be very inconsiderate standing in a bacteria’s point of view, but it is the novel thing to do to avoid them hurting other living organisms.<br />
<br />
===Construction: Background===<br />
<br />
Cracks form in concrete structures as soon as they set. These cracks continue to grow in size if they are not repaired due to the weight of the structure, imposed load, freeze-thaw effect and wind loading. The bigger the cracks are, the higher the rate of water seeping into the steel reinforcements, causing them to corrode and thus weakening the structure. Therefore, we would like to use Bacilla Filla to fill up these cracks before things get worse.<br />
<br />
====Environmental Disaster====<br />
<br />
Concrete is a very widely used material in construction. In fact, cement that is used to make concrete is the second most widely used substance in the world after water. Some building structures have to be demolished because the cracks formed appear to be threatening the strength of the structures. These buildings have to be rebuilt in order to reinforce its tensile strength. This is an unsustainable method of recovery because cement is a material that requires a lot of energy to be produced. In the production of cement, the rotating kiln requires the temperature to be between 1350°C-1400°C, which not only uses up a lot of energy resources, but also produces a lot of carbon dioxide. These carbon dioxide contributes to the green house effect which then leads to global warming. Therefore, our project can help to reduce renovation of buildings which in turn lowers the amount of cement that has to be produced each year.<br />
<br />
====Post-Chernobyl Disaster====<br />
<br />
The nuclear power plant combustion that occurred in Chernobyl on 26 April 1986 has allowed radioactive elements to be released into the environment, threatening the health of people living within close proximity of the plant. Therefore, a shelter, the Shelter Implementation Plan (SIP) which will be completed in 2012 is currently being built to cover up the area that where the accident occurred so as to make it environmentally friendly. However, if cracks were to form, something that inevitably occurs for building structures, those radioactive elements will still be dispersed into the environment. With the help of our project, those cracks will be able to be filled-up and the area will be safe again.<br />
<br />
<br />
<br />
[1] Wipat, A. (1990). "''Release and detection of geneticaly engineered streptomycetes in soil''". PhD thesis, Microbiology Department, John Moores University.<br />
<br />
[2] Zeigler DR, Prágai Z, Rodriguez S, Chevreux B, Muffler A, Albert T et al. (2008). "''The origins of 168, W23, and other Bacillus subtilis legacy strains''". Journal of Bacteriology, 190(21), 6983-95.<br />
<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/28_July_2010Team:Newcastle/28 July 20102010-10-27T21:48:43Z<p>RachelBoyd: /* Discussion */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
[[Image:Newcastle_Lab_4.jpeg|260px|right]]<br />
<br />
=PCR Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to prove that ''Bacillus subtilis'' 168 and 3610 chromosomal DNA extraction worked by amplifying P''araE'' using PCR.<br />
<br />
[[Image:Newcastle_Thermo.JPG|200px|thumb|right|Thermocycler]]<br />
[[Image:Newcastle Loading Gel.jpg|200px|thumb|right|Loading the gel]]<br />
[[Image:Newcastle Prep Chr Gel.jpg|200px|thumb|right|Gel]]<br />
<br />
==Materials and Protocol==<br />
<br />
Please refer to: [[Team:Newcastle/PCR#GoTaq_PCR|GoTaq PCR protocol]].<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle 280710 PCR.png|150px]]<br />
<br />
'''Figure 1''': Gel electrophoresis of the PCR products<br />
<br />
* '''Lane 1''': 1 Kb DNA ladder<br />
* '''Lane 2''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 3''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 4''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE'' <br />
* '''Lane 5''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE''<br />
<br />
==Discussion==<br />
We found bands in lanes 2, 3, 4 and 5 of around 200 bp in size which is an approximate size of the P''araE'' which is found on the chromosome of both ''B. subtilis'' 168 and 3610.<br />
<br />
==Conclusion==<br />
This experiment proves that the DNA extraction from both ''B. subtilis'' 168 and 3610 done on 27th July, 2010 was successful.<br />
<br />
=Plasmid Miniprep Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing ''lacI'' Biobrick from ''E. coli'' DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of nanodrop experiment.<br />
<br />
==Materials and Protocol==<br />
[[Image:Newcastle_overnight_culture.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_plasmids.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_pSB1C3.jpg|200px|thumb|right]]<br />
<br />
Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol and [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol.<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle_280710_miniprep.png|250px]]<br />
<br />
'''Figure 2''': Gel electrophoresis of the plasmid after restriction digestion with EcoR1.<br />
<br />
* '''Lane 1''': 1 kb DNA ladder<br />
* '''Lane 2''': Extraction of pSB1C3 plasmid<br />
* '''Lane 3''': Extraction of pSB1C3 plasmid<br />
* '''Lane 4''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 5''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 6''': Extraction of pSB1AK3 plasmid containing double terminator<br />
* '''Lane 7''': Extraction of pSB1AK3 plasmid containing double terminator<br />
<br />
<br />
<br />
{|border=1<br />
|-<br />
!'''Lane 1'''<br />
!'''Lane 2'''<br />
!'''Lane 3'''<br />
!'''Lane 4'''<br />
!'''Lane 5'''<br />
!'''Lane 6'''<br />
!'''Lane 7'''<br />
|-<br />
|N/A<br />
|29.9 µl/ml<br />
|28.9 µl/ml<br />
|34.0 µl/ml<br />
|29.8 µl/ml<br />
|6.1 µl/ml<br />
|6.7 µl/ml<br />
|}<br />
'''Table 1''': Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.<br />
<br />
==Discussion==<br />
We found bands in the lane 2, 3, 4, 5, and 6 showing the presence of plasmid in ''E. coli'' DH5α cells. <br />
However, the concentration was very low, therefore this will have to be repeated.<br />
<br />
==Conclusion==<br />
This experiment shows that there is plasmid present in the ''E. coli'' DH5α cells but they are present in a very low amount possibly due to the ommission of antibiotic in the media. <br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/28_July_2010Team:Newcastle/28 July 20102010-10-27T21:39:33Z<p>RachelBoyd: /* Plasmid Miniprep Experiment */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
[[Image:Newcastle_Lab_4.jpeg|260px|right]]<br />
<br />
=PCR Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to prove that ''Bacillus subtilis'' 168 and 3610 chromosomal DNA extraction worked by amplifying P''araE'' using PCR.<br />
<br />
[[Image:Newcastle_Thermo.JPG|200px|thumb|right|Thermocycler]]<br />
[[Image:Newcastle Loading Gel.jpg|200px|thumb|right|Loading the gel]]<br />
[[Image:Newcastle Prep Chr Gel.jpg|200px|thumb|right|Gel]]<br />
<br />
==Materials and Protocol==<br />
<br />
Please refer to: [[Team:Newcastle/PCR#GoTaq_PCR|GoTaq PCR protocol]].<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle 280710 PCR.png|150px]]<br />
<br />
'''Figure 1''': Gel electrophoresis of the PCR products<br />
<br />
* '''Lane 1''': 1 Kb DNA ladder<br />
* '''Lane 2''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 3''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 4''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE'' <br />
* '''Lane 5''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE''<br />
<br />
==Discussion==<br />
We found bands in lanes 2, 3, 4 and 5 of around 200 bp in size which is an approximate size of the P''araE'' which is found on the chromosome of both ''B. subtilis'' 168 and 3610.<br />
<br />
==Conclusion==<br />
This experiment proves that the DNA extraction from both ''B. subtilis'' 168 and 3610 done on 27th July, 2010 was successful.<br />
<br />
=Plasmid Miniprep Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing ''lacI'' Biobrick from ''E. coli'' DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of nanodrop experiment.<br />
<br />
==Materials and Protocol==<br />
[[Image:Newcastle_overnight_culture.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_plasmids.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_pSB1C3.jpg|200px|thumb|right]]<br />
<br />
Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol and [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol.<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle_280710_miniprep.png|250px]]<br />
<br />
'''Figure 2''': Gel electrophoresis of the plasmid after restriction digestion with EcoR1.<br />
<br />
* '''Lane 1''': 1 kb DNA ladder<br />
* '''Lane 2''': Extraction of pSB1C3 plasmid<br />
* '''Lane 3''': Extraction of pSB1C3 plasmid<br />
* '''Lane 4''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 5''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 6''': Extraction of pSB1AK3 plasmid containing double terminator<br />
* '''Lane 7''': Extraction of pSB1AK3 plasmid containing double terminator<br />
<br />
<br />
<br />
{|border=1<br />
|-<br />
!'''Lane 1'''<br />
!'''Lane 2'''<br />
!'''Lane 3'''<br />
!'''Lane 4'''<br />
!'''Lane 5'''<br />
!'''Lane 6'''<br />
!'''Lane 7'''<br />
|-<br />
|N/A<br />
|29.9 µl/ml<br />
|28.9 µl/ml<br />
|34.0 µl/ml<br />
|29.8 µl/ml<br />
|6.1 µl/ml<br />
|6.7 µl/ml<br />
|}<br />
'''Table 1''': Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.<br />
<br />
==Discussion==<br />
We found bands in the lane 2, 3, 4, 5, and 6 showing the presence of plasmid in ''E. coli'' DH5α cells. <br />
However, the concentration was very low. Therefore this will have to be repeated. <br />
<br />
==Conclusion==<br />
This experiment shows that there is plasmid present in the ''E. coli'' DH5α cells but they are present in a very low amount possibly due to the ommission of antibiotic in the media. <br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/28_July_2010Team:Newcastle/28 July 20102010-10-27T21:34:40Z<p>RachelBoyd: /* Result */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
[[Image:Newcastle_Lab_4.jpeg|260px|right]]<br />
<br />
=PCR Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to prove that ''Bacillus subtilis'' 168 and 3610 chromosomal DNA extraction worked by amplifying P''araE'' using PCR.<br />
<br />
[[Image:Newcastle_Thermo.JPG|200px|thumb|right|Thermocycler]]<br />
[[Image:Newcastle Loading Gel.jpg|200px|thumb|right|Loading the gel]]<br />
[[Image:Newcastle Prep Chr Gel.jpg|200px|thumb|right|Gel]]<br />
<br />
==Materials and Protocol==<br />
<br />
Please refer to: [[Team:Newcastle/PCR#GoTaq_PCR|GoTaq PCR protocol]].<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle 280710 PCR.png|150px]]<br />
<br />
'''Figure 1''': Gel electrophoresis of the PCR products<br />
<br />
* '''Lane 1''': 1 Kb DNA ladder<br />
* '''Lane 2''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 3''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 4''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE'' <br />
* '''Lane 5''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE''<br />
<br />
==Discussion==<br />
We found bands in lanes 2, 3, 4 and 5 of around 200 bp in size which is an approximate size of the P''araE'' which is found on the chromosome of both ''B. subtilis'' 168 and 3610.<br />
<br />
==Conclusion==<br />
This experiment proves that the DNA extraction from both ''B. subtilis'' 168 and 3610 done on 27th July, 2010 was successful.<br />
<br />
=Plasmid Miniprep Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing ''lacI'' Biobrick from ''E. coli'' DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of nanodrop experiment.<br />
<br />
==Materials and Protocol==<br />
[[Image:Newcastle_overnight_culture.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_plasmids.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_pSB1C3.jpg|200px|thumb|right]]<br />
<br />
Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol and [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol.<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle_280710_miniprep.png|250px]]<br />
<br />
Figure 2:Gel electrophoresis of the plasmid after restriction digestion with EcoR1.<br />
<br />
* '''Lane 1''': 1 kb DNA ladder<br />
* '''Lane 2''': Extraction of pSB1C3 plasmid<br />
* '''Lane 3''': Extraction of pSB1C3 plasmid<br />
* '''Lane 4''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 5''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 6''': Extraction of pSB1AK3 plasmid containing double terminator<br />
* '''Lane 7''': Extraction of pSB1AK3 plasmid containing double terminator<br />
<br />
<br />
<br />
{|border=1<br />
|-<br />
!'''Lane 1'''<br />
!'''Lane 2'''<br />
!'''Lane 3'''<br />
!'''Lane 4'''<br />
!'''Lane 5'''<br />
!'''Lane 6'''<br />
!'''Lane 7'''<br />
|-<br />
|N/A<br />
|29.9 µl/ml<br />
|28.9 µl/ml<br />
|34.0 µl/ml<br />
|29.8 µl/ml<br />
|6.1 µl/ml<br />
|6.7 µl/ml<br />
|}<br />
'''Table 1''': Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.<br />
<br />
==Discussion==<br />
We found bands in the lane 2, 3, 4, 5, and 6 showing the presence of plasmid in ''E. coli'' DH5α cells. The ideal concentration of DNA calculated using nanodrop experiment is 150 µg/ml but in the table 1, where all the values have been less than 150 µg/ml which shows that even though there is plasmid present in the cells but it is present in very low amount. One possible explanation for this to happen could be that when the transformed ''E. coli'' DH5α cells were grown overnight for the plasmid extraction protocol, the medium in which they were grown did not contain any antibiotics and because of this the cells did not require plasmid which conferred bacteria with antibiotic resistance and this process is called as plasmid shuffle.<br />
<br />
==Conclusion==<br />
This experiment shows that there is plasmid present in the ''E. coli'' DH5α cells but they are present in a very low amount possibly due to plasmid shuffle which could have occurred during overnight growth in the cultures which did not contain antibiotics against which plasmid provides resistance to the cell.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/28_July_2010Team:Newcastle/28 July 20102010-10-27T21:34:15Z<p>RachelBoyd: /* PCR Experiment */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
[[Image:Newcastle_Lab_4.jpeg|260px|right]]<br />
<br />
=PCR Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to prove that ''Bacillus subtilis'' 168 and 3610 chromosomal DNA extraction worked by amplifying P''araE'' using PCR.<br />
<br />
[[Image:Newcastle_Thermo.JPG|200px|thumb|right|Thermocycler]]<br />
[[Image:Newcastle Loading Gel.jpg|200px|thumb|right|Loading the gel]]<br />
[[Image:Newcastle Prep Chr Gel.jpg|200px|thumb|right|Gel]]<br />
<br />
==Materials and Protocol==<br />
<br />
Please refer to: [[Team:Newcastle/PCR#GoTaq_PCR|GoTaq PCR protocol]].<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle 280710 PCR.png|150px]]<br />
<br />
Figure 1:Gel electrophoresis of the PCR products<br />
<br />
* '''Lane 1''': 1 Kb DNA ladder<br />
* '''Lane 2''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 3''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 4''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE'' <br />
* '''Lane 5''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE''<br />
<br />
==Discussion==<br />
We found bands in lanes 2, 3, 4 and 5 of around 200 bp in size which is an approximate size of the P''araE'' which is found on the chromosome of both ''B. subtilis'' 168 and 3610.<br />
<br />
==Conclusion==<br />
This experiment proves that the DNA extraction from both ''B. subtilis'' 168 and 3610 done on 27th July, 2010 was successful.<br />
<br />
=Plasmid Miniprep Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing ''lacI'' Biobrick from ''E. coli'' DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of nanodrop experiment.<br />
<br />
==Materials and Protocol==<br />
[[Image:Newcastle_overnight_culture.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_plasmids.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_pSB1C3.jpg|200px|thumb|right]]<br />
<br />
Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol and [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol.<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle_280710_miniprep.png|250px]]<br />
<br />
Figure 2:Gel electrophoresis of the plasmid after restriction digestion with EcoR1.<br />
<br />
* '''Lane 1''': 1 kb DNA ladder<br />
* '''Lane 2''': Extraction of pSB1C3 plasmid<br />
* '''Lane 3''': Extraction of pSB1C3 plasmid<br />
* '''Lane 4''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 5''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 6''': Extraction of pSB1AK3 plasmid containing double terminator<br />
* '''Lane 7''': Extraction of pSB1AK3 plasmid containing double terminator<br />
<br />
<br />
<br />
{|border=1<br />
|-<br />
!'''Lane 1'''<br />
!'''Lane 2'''<br />
!'''Lane 3'''<br />
!'''Lane 4'''<br />
!'''Lane 5'''<br />
!'''Lane 6'''<br />
!'''Lane 7'''<br />
|-<br />
|N/A<br />
|29.9 µl/ml<br />
|28.9 µl/ml<br />
|34.0 µl/ml<br />
|29.8 µl/ml<br />
|6.1 µl/ml<br />
|6.7 µl/ml<br />
|}<br />
'''Table 1''': Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.<br />
<br />
==Discussion==<br />
We found bands in the lane 2, 3, 4, 5, and 6 showing the presence of plasmid in ''E. coli'' DH5α cells. The ideal concentration of DNA calculated using nanodrop experiment is 150 µg/ml but in the table 1, where all the values have been less than 150 µg/ml which shows that even though there is plasmid present in the cells but it is present in very low amount. One possible explanation for this to happen could be that when the transformed ''E. coli'' DH5α cells were grown overnight for the plasmid extraction protocol, the medium in which they were grown did not contain any antibiotics and because of this the cells did not require plasmid which conferred bacteria with antibiotic resistance and this process is called as plasmid shuffle.<br />
<br />
==Conclusion==<br />
This experiment shows that there is plasmid present in the ''E. coli'' DH5α cells but they are present in a very low amount possibly due to plasmid shuffle which could have occurred during overnight growth in the cultures which did not contain antibiotics against which plasmid provides resistance to the cell.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/MedalsTeam:Newcastle/Medals2010-10-27T21:34:08Z<p>RachelBoyd: /* iGEM Judging Comments */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
='''iGEM Judging Comments'''=<br />
<br />
This year our team came up with a highly ambitious project and achieved goals in several different areas.<br />
<br />
We successfully modelled, designed, characterised and entered our IPTG-inducible Filamentous cells BioBrick part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K302012 BBa_K302012]). <br />
<br />
We also developed [https://2010.igem.org/Team:Newcastle/E-Science An e-Science Approach to Synthetic Biology] which focuses on workflows, to synthetic biology. This led to the creation of BBF RFC 66 based upon our work.<br />
<br />
We developed the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302018 Subtilin Immunity] BioBrick part which provides cells immunity against the lantibiotic subtilin, which is also a quorum sensing molecule for our cell population. It will help in initiating a population wide response for initiating concrete repair.<br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302015 Urease] BioBrick part helps in the increasing urea hydrolysis by increasing arginine and arginase production. Arginase breaks down arginine to form urea and ornithine. The overall increase in urea leads to an increase in urease production which hydrolyses urea into carbonate and ammonium ions which are exported out of the cell. The carbonate ions forms a bond with calcium ions provided in the media and form calcium carbonate.<br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302016 Swarming] BioBrick part would help ''Bacillus subtilis'' 168 to swarm on the concrete surface by producing surfactin to reduce surface tension and by initiating flagellum biosynthesis.<br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302030 Levan Glue] BoBrick part produces Levan glue in the presence of sucrose. The glue will act as a binding agent for the filamentous cells and the calcium carbonate crystals and will also help in filling up the crack thereby preventing corrosion of the steel reinforcements. <br />
<br />
Our [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302035 ''mazEF'' Kill switch] is a stable toxin-antitoxin system for ''Bacillus subtilis''. It would kill bacteria in the absence of sucrose thereby helping to make our project environmentally friendly. <br />
<br />
In total we designed and entered 34 BioBrick parts in the parts registry. <br />
<br />
We also took [https://2010.igem.org/Team:Newcastle/solution#Scanning_Electron_Microscope_Images Scanning Electron Microscope photographs] and found some interesting results: photographs of cells trying to fill up the crack, calcium carbonate crystals and Levan glue covering the cells.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:NewcastleTeam:Newcastle2010-10-27T21:32:46Z<p>RachelBoyd: /* Project Description */</p>
<hr />
<div>__NOTOC__{{Team:Newcastle/mainbanner}}<br />
=BacillaFilla: Fixing Cracks in Concrete= <br />
<br />
<br />
<br />
[[Image:Newcastle_iGEM_Teampic.jpeg|centre|468px]]<br />
<br />
<br />
<br />
<div style="text-align:justify;padding:5px"><br />
===Project Description===<br />
<br />
BacillaFilla, an engineered ''Bacillus subtilis'', aims to repair [[Team:Newcastle/problem|cracks in concrete]] which can cause catastrophic structural failure. BacillaFilla would be applied to structures by spraying onto their surfaces.<br />
<br />
BacillaFilla would swim deep into the cracks. Repair would be effected by production of [[Team:Newcastle/Urease|CaCO<sub>3</sub>]], [[Team:Newcastle/Filamentous_Cells|filamentous ''Bacillus subtilis'' cells]] and [[Team:Newcastle/glue|levansucrose glue]]. CaCO<sub>3</sub> expands at the same rate as concrete, making it an ideal filler. Filamentous ''Bacillus subtilis'' cells have similar tensile strength to the synthetic fibres used in fibre-reinforced concrete, and provide reinforcement. Levansucrose glues CaCO<sub>3</sub> and filamentous cells in place.<br />
<br />
''B. subtilis'' 168 sporulates, making it ideal for storage and transportation. The cells can be [[Team:Newcastle/solution#Alkalinity_resistance|made to be tolerant to concrete's high pH]].<br />
<br />
We designed a [[Team:Newcastle/Swarming|swarming]] BioBrick part for repairing ''B. subtilis'' 168's defective ''swrA'' and ''sfp'' genes, regaining motility. At the end of the crack the quorum sensing peptide [[Team:Newcastle/End_of_crack_%26_signalling_system|subtilin]] triggers a co-ordinated population response from a [[Team:Newcastle/End_of_crack_%26_signalling_system#2008Brick|subtilin-inducible promoter]]. Upregulating ''SR1'' and ''rocF'' promotes arginine and urea production, increasing exogenous CaCO<sub>3</sub> deposition. Over-producing YneA induces the filamentous cell phenotype, while SacB converts extracellular sucrose to levan glue.<br />
<br />
To protect the environment our project also includes a design for a [[Team:Newcastle/Non-target-environment_kill_switch| kill switch]].<br />
<br />
<br />
[[Image:newcastle_summary.png|850px]]<br />
<br />
<br />
{|style cellpadding="20" cellspacing="0"<br />
! colspan="2" |<font size=4> <center>'''Summary of achievements:'''</center></font><br />
|-<br />
|<span style="color:Sienna">'''BRONZE:'''</span><br />
|From the beginning of the project, we have successfully registered the team of two instructors, six advisors and eight members. We have completed and submitted a Project Summary form, developed ideas and shared them on our iGEM wikipedia page. We also entered 31 new BioBrick parts into the Registry of Parts. One part, [http://partsregistry.org/Part:BBa_K302012 IPTG-induced filamentous cell formation], the BioBrick for the IPTG-induced filamentous cell formation, was demonstrated to work as expected.<br />
|-<br />
|<span style="color:Silver">'''SILVER:'''</span><br />
|Our new BioBrick, the IPTG-inducible filamentous cell formation part works, so we [[Team:Newcastle/Filamentous_Cells#Characterisation|characterised]] it and included the information, [http://partsregistry.org/Part:BBa_K302012:Experience BBa_K302012], on the Parts Registry. <br />
|-<br />
|<span style="color:Goldenrod">'''GOLD:'''</span><br />
|In order to obtain a gold, we investigated the benefits of an e-Science approach, focusing on workflows, to synthetic biology. Details can be found at [[Team:Newcastle/E-Science|here]]. This part of the project resulted in us proposing a new standard for a RESTful API which facilitates the discovery and publication of models of functional biological units. The standard has been submitted to the BioBricks Foundation as [[BBFRFC66|BBF RFC 66]]. We have also improved on existing BioBrick parts to produce hyperspankoid: [http://partsregistry.org/Part:BBa_K302003 BBa_K302003].<br />
|-<br />
|}<br />
<br />
<br />
<br />
<!---[http://twitter.com/newcastle_igem Follow us on Twitter] or join our [http://www.facebook.com/pages/Newcastle-iGEM-2010/140948965930577| Facebook Fan page!] ---><br />
<!-- <br />
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!align="center"|[[Team:Newcastle|Home]]<br />
!align="center"|[[Team:Newcastle/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Newcastle Official Team Profile]<br />
!align="center"|[[Team:Newcastle/Project|Project]]<br />
!align="center"|[[Team:Newcastle/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Newcastle/Modelling|Modelling]]<br />
!align="center"|[[Team:Newcastle/Notebook|Notebook]]<br />
!align="center"|[[Team:Newcastle/Safety|Safety]]<br />
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<br />
|}-->{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:NewcastleTeam:Newcastle2010-10-27T21:32:18Z<p>RachelBoyd: /* BacillaFilla: Fixing cracks in Concrete */</p>
<hr />
<div>__NOTOC__{{Team:Newcastle/mainbanner}}<br />
=BacillaFilla: Fixing Cracks in Concrete= <br />
<br />
<br />
<br />
[[Image:Newcastle_iGEM_Teampic.jpeg|centre|468px]]<br />
<br />
<br />
<br />
<div style="text-align:justify;padding:5px"><br />
===Project Description===<br />
<br />
BacillaFilla, an engineered ''Bacillus subtilis'', aims to repair [[Team:Newcastle/problem|cracks in concrete]] which can cause catastrophic structural failure. BacillaFilla would be applied to structures by spraying onto their surfaces.<br />
<br />
BacillaFilla would swim deep into the cracks. Repair would be effected by production of [[Team:Newcastle/Urease|CaCO<sub>3</sub>]], [[Team:Newcastle/Filamentous_Cells|filamentous ''Bacillus subtilis'' cells]] and [[Team:Newcastle/glue|levansucrose glue]]. CaCO<sub>3</sub> expands at the same rate as concrete, making it an ideal filler. Filamentous ''Bacillus subtilis'' cells have similar tensile strength to the synthetic fibres used in fibre-reinforced concrete, and provide reinforcement. Levansucrose glues CaCO<sub>3</sub> and filamentous cells in place.<br />
<br />
''B. subtilis'' 168 sporulates, making it ideal for storage and transportation. The cells can be [[Team:Newcastle/solution#Alkalinity_resistance|made to be tolerant to concrete's high pH]].<br />
<br />
We designed a [[Team:Newcastle/Swarming|swarming]] BioBrick part for repairing ''B. subtilis'' 168's defective ''swrA'' and ''sfp'' genes, regaining motility. At the end of the crack the quorum sensing peptide [[Team:Newcastle/End_of_crack_%26_signalling_system|subtilin]] triggers a co-ordinated population response from a [[Team:Newcastle/End_of_crack_%26_signalling_system#2008Brick|subtilin-inducible promoter]]. Upregulating ''SR1'' and ''rocF'' promotes arginine and urea production, increasing exogenous CaCO<sub>3</sub> deposition. Over-producing YneA induces the filamentous cell phenotype, while SacB converts extracellular sucrose to levan glue.<br />
<br />
To protect the environment our project also includes a design for a [[Team:Newcastle/Non-target-environment_kill_switch| kill switch]].<br />
<br />
<br />
[[Image:newcastle_summary.png|850px]]<br />
<br />
<br />
{|style cellpadding="20" cellspacing="0"<br />
! colspan="2" |<font size=4> <center>'''Summary of achievements:'''</center></font><br />
|-<br />
|<span style="color:Sienna">'''BRONZE:'''</span><br />
|From the beginning of the project, we have successfully registered the team of two instructors, six advisors and eight members. We have completed and submitted a Project Summary form, developed ideas and shared them on our iGEM wikipedia page. We also entered 23 new BioBrick parts into the Registry of Parts. One part, [http://partsregistry.org/Part:BBa_K302012 IPTG-induced filamentous cell formation], the BioBrick for the IPTG-induced filamentous cell formation, was demonstrated to work as expected.<br />
|-<br />
|<span style="color:Silver">'''SILVER:'''</span><br />
|Our new BioBrick, the IPTG-inducible filamentous cell formation part works, so we [[Team:Newcastle/Filamentous_Cells#Characterisation|characterised]] it and included the information, [http://partsregistry.org/Part:BBa_K302012:Experience BBa_K302012], on the Parts Registry. <br />
|-<br />
|<span style="color:Goldenrod">'''GOLD:'''</span><br />
|In order to obtain a gold, we investigated the benefits of an e-Science approach, focusing on workflows, to synthetic biology. Details can be found at [[Team:Newcastle/E-Science|here]]. This part of the project resulted in us proposing a new standard for a RESTful API which facilitates the discovery and publication of models of functional biological units. The standard has been submitted to the BioBricks Foundation as [[BBFRFC66|BBF RFC 66]]. We have also improved on existing BioBrick parts to produce hyperspankoid: [http://partsregistry.org/Part:BBa_K302003 BBa_K302003].<br />
|-<br />
|}<br />
<br />
<br />
<br />
<!---[http://twitter.com/newcastle_igem Follow us on Twitter] or join our [http://www.facebook.com/pages/Newcastle-iGEM-2010/140948965930577| Facebook Fan page!] ---><br />
<!-- <br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="2" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Newcastle|Home]]<br />
!align="center"|[[Team:Newcastle/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Newcastle Official Team Profile]<br />
!align="center"|[[Team:Newcastle/Project|Project]]<br />
!align="center"|[[Team:Newcastle/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Newcastle/Modelling|Modelling]]<br />
!align="center"|[[Team:Newcastle/Notebook|Notebook]]<br />
!align="center"|[[Team:Newcastle/Safety|Safety]]<br />
<br />
<br />
|}-->{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:NewcastleTeam:Newcastle2010-10-27T21:31:11Z<p>RachelBoyd: /* Project Description */</p>
<hr />
<div>__NOTOC__{{Team:Newcastle/mainbanner}}<br />
=BacillaFilla: Fixing cracks in Concrete= <br />
<br />
<br />
<br />
[[Image:Newcastle_iGEM_Teampic.jpeg|centre|468px]]<br />
<br />
<br />
<br />
<div style="text-align:justify;padding:5px"><br />
===Project Description===<br />
<br />
BacillaFilla, an engineered ''Bacillus subtilis'', aims to repair [[Team:Newcastle/problem|cracks in concrete]] which can cause catastrophic structural failure. BacillaFilla would be applied to structures by spraying onto their surfaces.<br />
<br />
BacillaFilla would swim deep into the cracks. Repair would be effected by production of [[Team:Newcastle/Urease|CaCO<sub>3</sub>]], [[Team:Newcastle/Filamentous_Cells|filamentous ''Bacillus subtilis'' cells]] and [[Team:Newcastle/glue|levansucrose glue]]. CaCO<sub>3</sub> expands at the same rate as concrete, making it an ideal filler. Filamentous ''Bacillus subtilis'' cells have similar tensile strength to the synthetic fibres used in fibre-reinforced concrete, and provide reinforcement. Levansucrose glues CaCO<sub>3</sub> and filamentous cells in place.<br />
<br />
''B. subtilis'' 168 sporulates, making it ideal for storage and transportation. The cells can be [[Team:Newcastle/solution#Alkalinity_resistance|made to be tolerant to concrete's high pH]].<br />
<br />
We designed a [[Team:Newcastle/Swarming|swarming]] BioBrick part for repairing ''B. subtilis'' 168's defective ''swrA'' and ''sfp'' genes, regaining motility. At the end of the crack the quorum sensing peptide [[Team:Newcastle/End_of_crack_%26_signalling_system|subtilin]] triggers a co-ordinated population response from a [[Team:Newcastle/End_of_crack_%26_signalling_system#2008Brick|subtilin-inducible promoter]]. Upregulating ''SR1'' and ''rocF'' promotes arginine and urea production, increasing exogenous CaCO<sub>3</sub> deposition. Over-producing YneA induces the filamentous cell phenotype, while SacB converts extracellular sucrose to levan glue.<br />
<br />
To protect the environment our project also includes a design for a [[Team:Newcastle/Non-target-environment_kill_switch| kill switch]].<br />
<br />
<br />
[[Image:newcastle_summary.png|850px]]<br />
<br />
<br />
{|style cellpadding="20" cellspacing="0"<br />
! colspan="2" |<font size=4> <center>'''Summary of achievements:'''</center></font><br />
|-<br />
|<span style="color:Sienna">'''BRONZE:'''</span><br />
|From the beginning of the project, we have successfully registered the team of two instructors, six advisors and eight members. We have completed and submitted a Project Summary form, developed ideas and shared them on our iGEM wikipedia page. We also entered 23 new BioBrick parts into the Registry of Parts. One part, [http://partsregistry.org/Part:BBa_K302012 IPTG-induced filamentous cell formation], the BioBrick for the IPTG-induced filamentous cell formation, was demonstrated to work as expected.<br />
|-<br />
|<span style="color:Silver">'''SILVER:'''</span><br />
|Our new BioBrick, the IPTG-inducible filamentous cell formation part works, so we [[Team:Newcastle/Filamentous_Cells#Characterisation|characterised]] it and included the information, [http://partsregistry.org/Part:BBa_K302012:Experience BBa_K302012], on the Parts Registry. <br />
|-<br />
|<span style="color:Goldenrod">'''GOLD:'''</span><br />
|In order to obtain a gold, we investigated the benefits of an e-Science approach, focusing on workflows, to synthetic biology. Details can be found at [[Team:Newcastle/E-Science|here]]. This part of the project resulted in us proposing a new standard for a RESTful API which facilitates the discovery and publication of models of functional biological units. The standard has been submitted to the BioBricks Foundation as [[BBFRFC66|BBF RFC 66]]. We have also improved on existing BioBrick parts to produce hyperspankoid: [http://partsregistry.org/Part:BBa_K302003 BBa_K302003].<br />
|-<br />
|}<br />
<br />
<br />
<br />
<!---[http://twitter.com/newcastle_igem Follow us on Twitter] or join our [http://www.facebook.com/pages/Newcastle-iGEM-2010/140948965930577| Facebook Fan page!] ---><br />
<!-- <br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="2" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Newcastle|Home]]<br />
!align="center"|[[Team:Newcastle/Team|Team]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Newcastle Official Team Profile]<br />
!align="center"|[[Team:Newcastle/Project|Project]]<br />
!align="center"|[[Team:Newcastle/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Newcastle/Modelling|Modelling]]<br />
!align="center"|[[Team:Newcastle/Notebook|Notebook]]<br />
!align="center"|[[Team:Newcastle/Safety|Safety]]<br />
<br />
<br />
|}-->{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/28_July_2010Team:Newcastle/28 July 20102010-10-27T21:26:21Z<p>RachelBoyd: /* Result */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
'''28 July 2010'''<br />
<br />
[[Image:Newcastle_Lab_4.jpeg|260px|right]]<br />
<br />
=PCR Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to prove that ''Bacillus subtilis'' 168 and 3610 chromosomal DNA extraction worked by amplifying P''araE'' using PCR.<br />
<br />
[[Image:Newcastle_Thermo.JPG|200px|thumb|right|Thermocycler]]<br />
[[Image:Newcastle Loading Gel.jpg|200px|thumb|right|Loading the gel]]<br />
[[Image:Newcastle Prep Chr Gel.jpg|200px|thumb|right|Gel]]<br />
<br />
==Materials and Protocol==<br />
<br />
Please refer to: [[Team:Newcastle/PCR#GoTaq_PCR|GoTaq PCR protocol]].<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle 280710 PCR.png|150px]]<br />
<br />
Figure 1:Gel electrophoresis of the PCR products<br />
<br />
* '''Lane 1''': 1 Kb DNA ladder<br />
* '''Lane 2''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 3''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 4''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE'' <br />
* '''Lane 5''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE''<br />
<br />
==Discussion==<br />
We found bands in the lane 2, 3, 4 and 5 of around 200 bp size which is an approximate size of the P''araE'' which is found on the chromosome of both ''B. subtilis'' 168 and 3610.<br />
<br />
==Conclusion==<br />
This experiment proves that the DNA extraction from both ''B. subtilis'' 168 and 3610 done on 27th July, 2010 was successful.<br />
<br />
=Plasmid Miniprep Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing ''lacI'' Biobrick from ''E. coli'' DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of nanodrop experiment.<br />
<br />
==Materials and Protocol==<br />
[[Image:Newcastle_overnight_culture.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_plasmids.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_pSB1C3.jpg|200px|thumb|right]]<br />
<br />
Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol and [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol.<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle_280710_miniprep.png|250px]]<br />
<br />
Figure 2:Gel electrophoresis of the plasmid after restriction digestion with EcoR1.<br />
<br />
* '''Lane 1''': 1 kb DNA ladder<br />
* '''Lane 2''': Extraction of pSB1C3 plasmid<br />
* '''Lane 3''': Extraction of pSB1C3 plasmid<br />
* '''Lane 4''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 5''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 6''': Extraction of pSB1AK3 plasmid containing double terminator<br />
* '''Lane 7''': Extraction of pSB1AK3 plasmid containing double terminator<br />
<br />
<br />
<br />
{|border=1<br />
|-<br />
!'''Lane 1'''<br />
!'''Lane 2'''<br />
!'''Lane 3'''<br />
!'''Lane 4'''<br />
!'''Lane 5'''<br />
!'''Lane 6'''<br />
!'''Lane 7'''<br />
|-<br />
|N/A<br />
|29.9 µl/ml<br />
|28.9 µl/ml<br />
|34.0 µl/ml<br />
|29.8 µl/ml<br />
|6.1 µl/ml<br />
|6.7 µl/ml<br />
|}<br />
'''Table 1''': Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.<br />
<br />
==Discussion==<br />
We found bands in the lane 2, 3, 4, 5, and 6 showing the presence of plasmid in ''E. coli'' DH5α cells. The ideal concentration of DNA calculated using nanodrop experiment is 150 µg/ml but in the table 1, where all the values have been less than 150 µg/ml which shows that even though there is plasmid present in the cells but it is present in very low amount. One possible explanation for this to happen could be that when the transformed ''E. coli'' DH5α cells were grown overnight for the plasmid extraction protocol, the medium in which they were grown did not contain any antibiotics and because of this the cells did not require plasmid which conferred bacteria with antibiotic resistance and this process is called as plasmid shuffle.<br />
<br />
==Conclusion==<br />
This experiment shows that there is plasmid present in the ''E. coli'' DH5α cells but they are present in a very low amount possibly due to plasmid shuffle which could have occurred during overnight growth in the cultures which did not contain antibiotics against which plasmid provides resistance to the cell.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/28_July_2010Team:Newcastle/28 July 20102010-10-27T21:26:02Z<p>RachelBoyd: /* PCR Experiment */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
'''28 July 2010'''<br />
<br />
[[Image:Newcastle_Lab_4.jpeg|260px|right]]<br />
<br />
=PCR Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to prove that ''Bacillus subtilis'' 168 and 3610 chromosomal DNA extraction worked by amplifying P''araE'' using PCR.<br />
<br />
[[Image:Newcastle_Thermo.JPG|200px|thumb|right|Thermocycler]]<br />
[[Image:Newcastle Loading Gel.jpg|200px|thumb|right|Loading the gel]]<br />
[[Image:Newcastle Prep Chr Gel.jpg|200px|thumb|right|Gel]]<br />
<br />
==Materials and Protocol==<br />
<br />
Please refer to: [[Team:Newcastle/PCR#GoTaq_PCR|GoTaq PCR protocol]].<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle 280710 PCR.png|150px]]<br />
<br />
Figure 1:Gel electrophoresis of the PCR products<br />
<br />
* '''Lane 1''': 1 kb DNA ladder<br />
* '''Lane 2''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 3''': ''B. subtilis'' 168 chromosomal DNA containing P''araE'' <br />
* '''Lane 4''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE'' <br />
* '''Lane 5''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE'' <br />
<br />
==Discussion==<br />
We found bands in the lane 2, 3, 4 and 5 of around 200 bp size which is an approximate size of the P''araE'' which is found on the chromosome of both ''B. subtilis'' 168 and 3610.<br />
<br />
==Conclusion==<br />
This experiment proves that the DNA extraction from both ''B. subtilis'' 168 and 3610 done on 27th July, 2010 was successful.<br />
<br />
=Plasmid Miniprep Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing ''lacI'' Biobrick from ''E. coli'' DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of nanodrop experiment.<br />
<br />
==Materials and Protocol==<br />
[[Image:Newcastle_overnight_culture.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_plasmids.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_pSB1C3.jpg|200px|thumb|right]]<br />
<br />
Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol and [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol.<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle_280710_miniprep.png|250px]]<br />
<br />
Figure 2:Gel electrophoresis of the plasmid after restriction digestion with EcoR1.<br />
<br />
* '''Lane 1''': 1 kb DNA ladder<br />
* '''Lane 2''': Extraction of pSB1C3 plasmid<br />
* '''Lane 3''': Extraction of pSB1C3 plasmid<br />
* '''Lane 4''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 5''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 6''': Extraction of pSB1AK3 plasmid containing double terminator<br />
* '''Lane 7''': Extraction of pSB1AK3 plasmid containing double terminator<br />
<br />
<br />
<br />
{|border=1<br />
|-<br />
!'''Lane 1'''<br />
!'''Lane 2'''<br />
!'''Lane 3'''<br />
!'''Lane 4'''<br />
!'''Lane 5'''<br />
!'''Lane 6'''<br />
!'''Lane 7'''<br />
|-<br />
|N/A<br />
|29.9 µl/ml<br />
|28.9 µl/ml<br />
|34.0 µl/ml<br />
|29.8 µl/ml<br />
|6.1 µl/ml<br />
|6.7 µl/ml<br />
|}<br />
'''Table 1''': Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.<br />
<br />
==Discussion==<br />
We found bands in the lane 2, 3, 4, 5, and 6 showing the presence of plasmid in ''E. coli'' DH5α cells. The ideal concentration of DNA calculated using nanodrop experiment is 150 µg/ml but in the table 1, where all the values have been less than 150 µg/ml which shows that even though there is plasmid present in the cells but it is present in very low amount. One possible explanation for this to happen could be that when the transformed ''E. coli'' DH5α cells were grown overnight for the plasmid extraction protocol, the medium in which they were grown did not contain any antibiotics and because of this the cells did not require plasmid which conferred bacteria with antibiotic resistance and this process is called as plasmid shuffle.<br />
<br />
==Conclusion==<br />
This experiment shows that there is plasmid present in the ''E. coli'' DH5α cells but they are present in a very low amount possibly due to plasmid shuffle which could have occurred during overnight growth in the cultures which did not contain antibiotics against which plasmid provides resistance to the cell.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/Attribution_and_ContributionTeam:Newcastle/Attribution and Contribution2010-10-27T21:18:35Z<p>RachelBoyd: /* Filamentous cells */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
The work on this project (including part design, computational modelling, lab work and graphic design) was completed entirely by a [[Team:Newcastle/the_team|multidisciplinary team of students]]. <br />
<br />
===Sub Projects===<br />
<br />
====Filamentous cells====<br />
1. '''Research and Design'''<br />
<br />
Rachel, Phil, Deena and Jannetta<br />
<br />
2. '''Lab work'''<br />
<br />
Steven, Alan, Rachel and Deena<br />
<br />
3. '''Characterisation and Testing'''<br />
<br />
Harsh and Rachel<br />
<br />
4. '''Modelling'''<br />
<br />
Steven and Jannetta<br />
<br />
====Urease====<br />
1. '''Research and Design'''<br />
<br />
Alan and Steven<br />
<br />
2. '''Lab and Testing'''<br />
<br />
Alan, Harsh and Steven<br />
<br />
3. '''Modelling'''<br />
<br />
Steven<br />
<br />
====Glue====<br />
<br />
1. '''Research and Design'''<br />
<br />
Phil<br />
<br />
2. '''Lab and Testing'''<br />
<br />
Every student<br />
<br />
====Subtilin Production and Immunity====<br />
1. '''Research and Design'''<br />
<br />
Alan, Steven and Rachel<br />
<br />
2. '''Lab'''<br />
<br />
Phil and Younus<br />
<br />
===Chassis Testing===<br />
<br />
====Extreme Base Resistance====<br />
1. '''Research'''<br />
<br />
Harsh and Steven<br />
<br />
2. '''Lab'''<br />
<br />
Steven and Harsh<br />
<br />
====Swarming====<br />
1. '''Research'''<br />
<br />
Harsh<br />
<br />
2. '''Lab'''<br />
<br />
Phil<br />
<br />
===Workflows===<br />
Jannetta developed an [https://2010.igem.org/Team:Newcastle/E-Science e-Science Approach to Synthetic Biology] with a focus on workflows. She proposed a new standard for a RESTful API which facilitates the discovery and publication of models of functional biological units. The RFC has been submitted to the BioBrick Foundation as BBF RFC 66.<br />
<br />
===T-shirt Design===<br />
The design was formed by Younus, Jannetta and Harsh and the whole team approved the design for the current t-shirt.<br />
<br />
===Poster Design===<br />
The design was put forward by Younus and Rachel and the whole team approved the design.<br />
<br />
===Presentation Design===<br />
The design was put forward by Younus and Jannetta and the whole team approved the design.<br />
<br />
===Instructors and Advisors===<br />
All of our Instructors and Advisors have provided us with their invaluable help and guidance throughout the span of the project.<br />
<br />
#Dr Wendy Smith gave us introductory lab session and taught us about the lab safety. She also supervised us throughout our stay in the lab (often into the the early hours :-)<br />
#Prof. Colin Harwood and Dr Jem Stach suggested the antisense RNA approach for the ''SR1'' BioBrick Part. They also helped us in writing the cloning strategies.<br />
#Dr Colin Davie provided advice and access to the Civil Engineering labs to prepare and crack concrete.<br />
#Mr Goksel Misirili provided help and expertise with modelling.<br />
#Dr Matthew Pocock helped us with the presentation design and talk. <br />
<br />
<br />
Details of this work can be found on the [[Team:Newcastle/solution|Solution]], [[Team:Newcastle/modelling|Modelling]] and [[Team:Newcastle/notebook|Lab book]] pages. This work is unrelated to the work done by the labs of our instructors and advisors.<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/28_July_2010Team:Newcastle/28 July 20102010-10-27T21:16:53Z<p>RachelBoyd: /* Result */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
'''28 July 2010'''<br />
<br />
[[Image:Newcastle_Lab_4.jpeg|260px|right]]<br />
<br />
=PCR Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to prove that ''Bacillus subtilis'' 168 and 3610 chromosomal DNA extraction worked by amplifying ''anaR'' gene using PCR.<br />
<br />
[[Image:Newcastle_Thermo.JPG|200px|thumb|right|Thermocycler]]<br />
[[Image:Newcastle Loading Gel.jpg|200px|thumb|right|Loading the gel]]<br />
[[Image:Newcastle Prep Chr Gel.jpg|200px|thumb|right|Gel]]<br />
<br />
==Materials and Protocol==<br />
<br />
Please refer to: [[Team:Newcastle/PCR#GoTaq_PCR|GoTaq PCR protocol]].<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle 280710 PCR.png|150px]]<br />
<br />
Figure 1:Gel electrophoresis of the PCR products<br />
<br />
* '''Lane 1''': 1 kb DNA ladder<br />
* '''Lane 2''': ''B. subtilis'' 168 chromosomal DNA containing ''anaR'' gene<br />
* '''Lane 3''': ''B. subtilis'' 168 chromosomal DNA containing ''anaR'' gene<br />
* '''Lane 4''': ''B. subtilis'' 3610 chromosomal DNA containing ''anaR'' gene<br />
* '''Lane 5''': ''B. subtilis'' 3610 chromosomal DNA containing ''anaR'' gene<br />
<br />
==Discussion==<br />
We found bands in the lane 2, 3, 4 and 5 of around 200 bp size which is an approximate size of the ''anaR'' gene which is found on the chromosome of both ''B. subtilis'' 168 and 3610.<br />
<br />
==Conclusion==<br />
This experiment proves that the DNA extraction from both ''B. subtilis'' 168 and 3610 done on 27th July, 2010 was successful.<br />
<br />
=Plasmid Miniprep Experiment=<br />
<br />
==Aims==<br />
The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing ''lacI'' Biobrick from ''E. coli'' DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of nanodrop experiment.<br />
<br />
==Materials and Protocol==<br />
[[Image:Newcastle_overnight_culture.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_plasmids.jpg|200px|thumb|right]]<br />
[[Image:Newcastle_pSB1C3.jpg|200px|thumb|right]]<br />
<br />
Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol and [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol.<br />
<br />
==Result==<br />
<br />
[[Image:Newcastle_280710_miniprep.png|250px]]<br />
<br />
Figure 2:Gel electrophoresis of the plasmid after restriction digestion with EcoR1.<br />
<br />
* '''Lane 1''': 1kb DNA ladder<br />
* '''Lane 2''': Extraction of pSB1C3 plasmid<br />
* '''Lane 3''': Extraction of pSB1C3 plasmid<br />
* '''Lane 4''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 5''': Extraction of plasmid containing ''lacI''<br />
* '''Lane 6''': Extraction of pSB1AK3 plasmid containing double terminator<br />
* '''Lane 7''': Extraction of pSB1AK3 plasmid containing double terminator<br />
<br />
<br />
<br />
{|border=1<br />
|-<br />
!'''Lane 1'''<br />
!'''Lane 2'''<br />
!'''Lane 3'''<br />
!'''Lane 4'''<br />
!'''Lane 5'''<br />
!'''Lane 6'''<br />
!'''Lane 7'''<br />
|-<br />
|N/A<br />
|29.9 µl/ml<br />
|28.9 µl/ml<br />
|34.0 µl/ml<br />
|29.8 µl/ml<br />
|6.1 µl/ml<br />
|6.7 µl/ml<br />
|}<br />
'''Table 1''': Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.<br />
<br />
==Discussion==<br />
We found bands in the lane 2, 3, 4, 5, and 6 showing the presence of plasmid in ''E. coli'' DH5α cells. The ideal concentration of DNA calculated using nanodrop experiment is 150 µg/ml but in the table 1, where all the values have been less than 150 µg/ml which shows that even though there is plasmid present in the cells but it is present in very low amount. One possible explanation for this to happen could be that when the transformed ''E. coli'' DH5α cells were grown overnight for the plasmid extraction protocol, the medium in which they were grown did not contain any antibiotics and because of this the cells did not require plasmid which conferred bacteria with antibiotic resistance and this process is called as plasmid shuffle.<br />
<br />
==Conclusion==<br />
This experiment shows that there is plasmid present in the ''E. coli'' DH5α cells but they are present in a very low amount possibly due to plasmid shuffle which could have occurred during overnight growth in the cultures which did not contain antibiotics against which plasmid provides resistance to the cell.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/Attribution_and_ContributionTeam:Newcastle/Attribution and Contribution2010-10-27T21:14:44Z<p>RachelBoyd: /* Instructors and Advisors */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
The work on this project (including part design, computational modelling, lab work and graphic design) was completed entirely by a [[Team:Newcastle/the_team|multidisciplinary team of students]]. <br />
<br />
===Sub Projects===<br />
<br />
====Filamentous cells====<br />
1. '''Research and Design'''<br />
<br />
Rachel, Phil, Deena and Jannetta<br />
<br />
2. '''Lab work'''<br />
<br />
Alan, Rachel and Deena<br />
<br />
3. '''Characterisation and Testing'''<br />
<br />
Harsh and Rachel<br />
<br />
4. '''Modelling'''<br />
<br />
Steven and Jannetta<br />
<br />
====Urease====<br />
1. '''Research and Design'''<br />
<br />
Alan and Steven<br />
<br />
2. '''Lab and Testing'''<br />
<br />
Alan, Harsh and Steven<br />
<br />
3. '''Modelling'''<br />
<br />
Steven<br />
<br />
====Glue====<br />
<br />
1. '''Research and Design'''<br />
<br />
Phil<br />
<br />
2. '''Lab and Testing'''<br />
<br />
Every student<br />
<br />
====Subtilin Production and Immunity====<br />
1. '''Research and Design'''<br />
<br />
Alan, Steven and Rachel<br />
<br />
2. '''Lab'''<br />
<br />
Phil and Younus<br />
<br />
===Chassis Testing===<br />
<br />
====Extreme Base Resistance====<br />
1. '''Research'''<br />
<br />
Harsh and Steven<br />
<br />
2. '''Lab'''<br />
<br />
Steven and Harsh<br />
<br />
====Swarming====<br />
1. '''Research'''<br />
<br />
Harsh<br />
<br />
2. '''Lab'''<br />
<br />
Phil<br />
<br />
===Workflows===<br />
Jannetta developed an [https://2010.igem.org/Team:Newcastle/E-Science e-Science Approach to Synthetic Biology] with a focus on workflows. She proposed a new standard for a RESTful API which facilitates the discovery and publication of models of functional biological units. The RFC has been submitted to the BioBrick Foundation as BBF RFC 66.<br />
<br />
===T-shirt Design===<br />
The design was formed by Younus, Jannetta and Harsh and the whole team approved the design for the current t-shirt.<br />
<br />
===Poster Design===<br />
The design was put forward by Younus and Rachel and the whole team approved the design.<br />
<br />
===Presentation Design===<br />
The design was put forward by Younus and Jannetta and the whole team approved the design.<br />
<br />
===Instructors and Advisors===<br />
All of our Instructors and Advisors have provided us with their invaluable help and guidance throughout the span of the project.<br />
<br />
#Dr Wendy Smith gave us introductory lab session and taught us about the lab safety. She also supervised us throughout our stay in the lab (often into the the early hours :-)<br />
#Prof. Colin Harwood and Dr Jem Stach suggested the antisense RNA approach for the ''SR1'' BioBrick Part. They also helped us in writing the cloning strategies.<br />
#Dr Colin Davie provided advice and access to the Civil Engineering labs to prepare and crack concrete.<br />
#Mr Goksel Misirili provided help and expertise with modelling.<br />
#Dr Matthew Pocock helped us with the presentation design and talk. <br />
<br />
<br />
Details of this work can be found on the [[Team:Newcastle/solution|Solution]], [[Team:Newcastle/modelling|Modelling]] and [[Team:Newcastle/notebook|Lab book]] pages. This work is unrelated to the work done by the labs of our instructors and advisors.<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/27_July_2010Team:Newcastle/27 July 20102010-10-27T21:14:05Z<p>RachelBoyd: /* Conclusion */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
=Genomic DNA extraction experiment=<br />
<br />
[[Image:Newcastle alan chromosome.jpg|thumb|200px|right]]<br />
[[Image:Newcastle ice chromosome.jpg|thumb|200px|right]]<br />
==Aims==<br />
The aim of today's experiment is to extract genomic DNA from both ''B. subtilis'' strains 168 and 3610. The genes necessary for the [[Team:Newcastle/Swarming|swarming BioBrick]] and [[Team:Newcastle/Urease|''rocF'' BioBrick]] will then hopefully be obtained from the genomic DNA using PCR.<br />
<br />
==Protocol==<br />
* Please refer to: [[Team:Newcastle/DNA extraction| DNA extraction of ''B. subtilis'']] for materials required and protocol.<br />
<br />
==Discussion==<br />
At the end of the DNA precipitation step, we observed a small white pellet in all the eppendorf tubes.<br />
<br />
==Conclusion==<br />
The experiment was a success! The quality of the extracted DNA will be checked by using PCR on 28th July, 2010.<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/26_July_2010Team:Newcastle/26 July 20102010-10-27T21:12:29Z<p>RachelBoyd: /* Results: */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
='''Preparation for cloning of the ''rocF'' BioBrick'''=<br />
<br />
==Aim==<br />
In preparation for Gibson cloning of the ''rocF'' BioBrick we started work on mini preps of plasmid DNA, and on ''B. subtilis'' 168 chromosomal DNA extraction.<br />
<br />
==Re-hydration of registry parts==<br />
[[Image:Newcastlehydration.jpg|200px|thumb|right|Re-hydration of dried parts registry DNA]]<br />
We re-hydrated using sterile distill water: <br />
#[http://partsregistry.org/Part:pSB1C3 pSB1C3] (the plasmid we will be submitting our BioBricks to the registry in) and<br />
#[http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] (the double terminator we will be using for the ''rocF'' BioBrick) from the parts distribution.<br />
<br />
==Transformation of ''E. coli''==<br />
We transformed and plated separate tubes of ''E. coli'' DH5α with:<br />
<br />
# The above two re-hydrated plasmids<br />
# [http://partsregistry.org/Part:BBa_K143062 BBa_K143062], a LacI BioBrick sent to us by Imperial College, London, UK which we will use to help characterise many of our BioBricks, including ''rocF''.<br />
# A positive control which we had already prepared during our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with ''rfp'' insert.<br />
# A negative control (no vector), to verify the antibiotic plates are working (no growth should be observed on this plate).<br />
<br />
<br />
Please refer to the transformation protocol for ''E. coli'' DH5α here: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']].<br />
<br />
==Overnight cultures of ''B. subtilis'' 168 for chromosomal DNA extraction==<br />
The ''rocF'' coding sequence is to be amplified from the ''B. subtilis'' 168 genome by PCR. Before we can do this we need to extract 168 chromosomal DNA.<br />
<br />
Today we plated up overnight cultures of ''B. subtilis'' 168 so that we can do chromosome extraction tomorrow.<br />
<br />
='''PCR of Genomic DNA'''=<br />
<br />
==Aim:==<br />
<br />
To determine whether the genomic DNA has been extracted from ''B. subtilis'' strains 168 and 3610.<br />
<br />
==Materials:==<br />
<br />
* Pipette<br />
* Microfuge<br />
* Microtubes<br />
* Distilled H<sub>2</sub>O<br />
* Nucleotide DNTP<br />
* 5x GoTaq buffer<br />
* Template DNA<br />
* Forward and reverse primers<br />
<br />
==Protocol:==<br />
<br />
* For the full protocol, please refer to [[Team:Newcastle/PCR|PCR]].<br />
<br />
===Conditions in ThermoCycler:===<br />
* Melting temperature, Tm used for anneal step is 59°C.<br />
<br />
==Results:==<br />
Gel electrophoresis will be undertaken tomorrow to determine the results.<br />
<br />
==Conclusion:==<br />
<br />
Please refer to Lab book dated [[Team:Newcastle/27_July_2010|27th July 2010]].<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/26_July_2010Team:Newcastle/26 July 20102010-10-27T21:11:20Z<p>RachelBoyd: /* PCR of Genomic DNA */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
='''Preparation for cloning of the ''rocF'' BioBrick'''=<br />
<br />
==Aim==<br />
In preparation for Gibson cloning of the ''rocF'' BioBrick we started work on mini preps of plasmid DNA, and on ''B. subtilis'' 168 chromosomal DNA extraction.<br />
<br />
==Re-hydration of registry parts==<br />
[[Image:Newcastlehydration.jpg|200px|thumb|right|Re-hydration of dried parts registry DNA]]<br />
We re-hydrated using sterile distill water: <br />
#[http://partsregistry.org/Part:pSB1C3 pSB1C3] (the plasmid we will be submitting our BioBricks to the registry in) and<br />
#[http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] (the double terminator we will be using for the ''rocF'' BioBrick) from the parts distribution.<br />
<br />
==Transformation of ''E. coli''==<br />
We transformed and plated separate tubes of ''E. coli'' DH5α with:<br />
<br />
# The above two re-hydrated plasmids<br />
# [http://partsregistry.org/Part:BBa_K143062 BBa_K143062], a LacI BioBrick sent to us by Imperial College, London, UK which we will use to help characterise many of our BioBricks, including ''rocF''.<br />
# A positive control which we had already prepared during our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with ''rfp'' insert.<br />
# A negative control (no vector), to verify the antibiotic plates are working (no growth should be observed on this plate).<br />
<br />
<br />
Please refer to the transformation protocol for ''E. coli'' DH5α here: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']].<br />
<br />
==Overnight cultures of ''B. subtilis'' 168 for chromosomal DNA extraction==<br />
The ''rocF'' coding sequence is to be amplified from the ''B. subtilis'' 168 genome by PCR. Before we can do this we need to extract 168 chromosomal DNA.<br />
<br />
Today we plated up overnight cultures of ''B. subtilis'' 168 so that we can do chromosome extraction tomorrow.<br />
<br />
='''PCR of Genomic DNA'''=<br />
<br />
==Aim:==<br />
<br />
To determine whether the genomic DNA has been extracted from ''B. subtilis'' strains 168 and 3610.<br />
<br />
==Materials:==<br />
<br />
* Pipette<br />
* Microfuge<br />
* Microtubes<br />
* Distilled H<sub>2</sub>O<br />
* Nucleotide DNTP<br />
* 5x GoTaq buffer<br />
* Template DNA<br />
* Forward and reverse primers<br />
<br />
==Protocol:==<br />
<br />
* For the full protocol, please refer to [[Team:Newcastle/PCR|PCR]].<br />
<br />
===Conditions in ThermoCycler:===<br />
* Melting temperature, Tm used for anneal step is 59°C.<br />
<br />
==Results:==<br />
Gel electrophoresis will be run tomorrow to determine the results.<br />
<br />
==Conclusion:==<br />
<br />
Please refer to Lab book dated [[Team:Newcastle/27_July_2010|27th July 2010]].<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/E-ScienceTeam:Newcastle/E-Science2010-10-27T21:10:55Z<p>RachelBoyd: /* Motivation: */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
=An e-Science Approach to Synthetic Biology=<br />
<br />
=Motivation:=<br />
<br />
{|style="text-align:justify"<br />
|Synthetic Biology is the engineering of biological entities to perform novel and desirable functions. Synthetic biologists already make use of computational resources to a large extent which can be seen by the software tools such as CLONEQC, Biskit and Internet based repositories such as the MIT Biobrick library at http://partsregistry.org/Main_Page. In both Systems and Synthetic Biology analysis consists of researchers taking the outputs from some software and feeding it to the inputs of other software. This pipeline or workflow can be computerised by using software such as [http://www.taverna.org.uk/ Taverna]. Research into the application of computerised workflows to Synthetic Biology has revealed the paucity of published material in this area. Therefore, this has been identified as an opportunity for research and possible development of tools, which could greatly enhance methodologies and workflows in Synthetic Biology. We show how an e-Science approach, i.e. the utilisation of advanced computing resources and technologies to support scientists, benefits the Synthetic Biology engineering process. We further propose a development life-cycle and show how this approach can improve the design of synthetic biological entities.<br />
|[[Image:Newcastel_workflows_Synbio001.png|thumb|Motivation ]]<br />
|}<br />
<br />
=Introduction:=<br />
<br />
{|style="text-align:justify" <br />
|-<br />
|style="width:150px" rowspan=2|[[Image:Newcastle workflows Aim.jpg|thumb|left]]<br />
|''Aim:'' <br />
The aim of this project was to investigate the application of e-Science approaches in Synthetic Biology, particularly workflows.<br />
|-<br />
|''Objectives:''<br />
# To produce a proof-of-concept web service that can be used by a workflow to automate the process of designing a BioBrick.<br />
# To produce one or more Taverna workflows, that use this web service, to show how this e-Science approach will benefit the design process (Oinn et al., 2006; Hull et al., 2006) and reduce development time.<br />
# To produce simulatable model parts in CellML, that could eventually be compiled into a complex model.<br />
# To identify design patterns or motifs which are recognisable patterns in models that can be re-used and simulated independently of the rest of the model. Identifying these motifs should aid the design process and produce re-usable parts to simplify future designs. In principle, the motifs are similar to and serves the same purpose as design patterns in computer software.<br />
|-<br />
|}<br />
<br />
==Synthetic Biology:==<br />
<br />
{|style="text-align:justify"<br />
|-<br />
|style="width:150px"|[[Image:Newcastle workflows Synthetic.jpg|thumb|left]]<br />
|Chopra defines Syntehtic Biology as a field that involves the synthesis of novel biological systems which are not generally found in nature (Chopra and Kamma, 2006).<br />
|-<br />
|}<br />
<br />
==Engineering Principles:==<br />
<br />
{|style="text-align:justify"<br />
|-<br />
|style="width:150px"|[[Image:Newcastle workflows Engineering.jpg|thumb|left]]<br />
|Two design approaches to Synthetic biology are emerging, namely top-down and bottom up.<br />
<br />
The top-down approach starts with an overview of a system. The system is studied and iteratively broken into modules until the complete system can be described in terms of minimal elements. In the bottom-up approach design starts with minimal elements that, according to certain rules, can fit together like Lego c bricks (http://lego.com) until a complete system is built (Heinemann and Panke, 2006).<br />
<br />
Synthetic biologists are adopting principles from established fields of engineering to increase tractability and speed of design (Andrianantoandro et al., 2006).<br />
<br />
We define Synthetic Biology as: Synthetic Biology is the engineering of biological systems to perform desirable and predictable functions.<br />
|-<br />
|}<br />
<br />
==Modelling languages:==<br />
<br />
{|style="text-align:justify"<br />
|-<br />
|style="width:150px"|[[Image:Newcastle_workflows_Modelling.jpg|thumb|left]]<br />
|Systems Biology Markup Language (SBML) was developed to represent biochemical reaction networks in a way that can be used by different software systems to exchange models ().<br />
<br />
CellML is a mark-up language used to store and exchange computer based mathematical models. Originally it was intended for the description of biological models, but it has been adopted by several other fields of study.<br />
<br />
Both CellML and SBML are based on the XML (Hucka, 2003; Cuellar et al., 2003). The main difference between SMBL and CellML is that in CellML the underlying mathematics of cellular models are described in a very general way.<br />
|-<br />
|}<br />
<br />
==Virtual Parts:==<br />
<br />
{|style="text-align:justify"<br />
|-<br />
|style="width:150px"|[[Image:Newcastel_workflows_Virtualparts.jpg|thumb|left]]<br />
|Cooling et al. (2010) introduce the concept of Standard Virtual Biological Parts (further on referred to as virtual parts). Virtual parts are mathematical models used to represent biological parts that can be combined to inform system design.<br />
<br />
The use of virtual parts, however, goes beyond just the representation of biological parts. The virtual parts can also be used to represent bioenvironmental elements which are intracellular events occurring in cells or chassis. These bioenvironmental elements become interfacing parts that act as "glue" for the aggregation of the standard biological parts.<br />
<br />
We refer to the standard biological parts as physical parts and the interfacing parts as non-physical parts.<br />
|-<br />
|}<br />
<br />
==Development Life Cycle:==<br />
<br />
Another concept, that can be borrowed from software engineering, is the software development life cycle (SDLC). We used, as a basis, what is known as the classic life-cycle paradigm or waterfall model, see Figure 2.<br />
<br />
[[Image:Newcastle workflows waterfall.png|Figure 2: Waterfall Model]]<br />
<br />
==e-Science:==<br />
<br />
{|style="text-align:justify"<br />
|-<br />
|style="width:150px"|[[Image:Newcastle workflows Synthetic.jpg|thumb|left]]<br />
|"The term 'e-Science' denotes the systematic development of research methods that exploit advanced computational thinking"<br />
Professor Malcolm Atkinson, eScience Envoy<br />
<br />
e-Science is an enabling concept. It aims to allow the sharing of resources required in science across administrative borders.<br />
<br />
Sharing is accomplished by tapping into techniques such as cloud and grid computing.<br />
&nbsp;<br/><br />
&nbsp;<br/><br />
|-<br />
|}<br />
<br />
==Web Services:==<br />
<br />
{|style="text-align:justify"<br />
|-<br />
|style="width:150px"|[[Image:Newcastle workflows Webservice.jpg|thumb|left]]<br />
|Web services are set apart from other types of services by the communications protocol they use. To be part of what is known as the Web, using HyperText Transfer Protocol (HTTP) is a prerequisite.<br />
<br />
Web services, regardless of the architecture chosen, enable programmatic access to on-line resources and therefore more and more services are developed and become available on-line.<br />
<br />
SOAP web services: The Simple Object Access Protocol, or SOAP as it is popularly known was originally designed to be a RPC (Remote Procedure Call) protocol using HTTP as the transport protocol. It was later extended to allow for other transport protocols such as the Simple Mail Transport Protocol (SMTP) (Kennard and Stiver, 2000). SOAP is a packaging protocol for sharing messages between applications (Snell, 2001).<br />
<br />
RESTful web services: Roy Fielding described REST, Representational State Transfer, a software architectural style (Fielding and Fielding, 2000). REST uses the WWW as a starting point. Fielding refers to the WWW as the Null Style. As such it is an ideal architecture for web services.<br />
|-<br />
|}<br />
<br />
==Workflows:==<br />
<br />
{|style="text-align:justify"<br />
|-<br />
|style="width:150px"|[[Image:Newcastle_workflows_Workflow.jpg|thumb|left]]<br />
|A workflow can be described as a sequence of connected steps or work activities that are followed to produce a required outcome. The sequence and the ways the steps impact on each other are regulated by a set of rules (DiCaterino et al., 1997 ).<br />
<br />
The processing of the information drawn from all the distributed sources can be accomplished using workflows.<br />
To manage the complex and heterogenous nature of such a distributed environment, scientist have turned to computerised workflows. <br />
|-<br />
|}<br />
<br />
=Methods:=<br />
==The Synthetic Biology Development Lifecycle==<br />
<br />
Unlike the waterfall model, there is currently no need for the maintenance phase in synthetic biology.<br />
<br />
We propose the model in Figure 1 to fit Synthetic Biology. It is important to note the iterative nature of the software development life cycle which was also inherited by our proposed Synthetic Biology life cycle.<br />
<br />
[[Image:Newcastle_workflows_Sbdlc.png]]<br />
<br />
Figure 1: A proposed development life cycle for Synthetic Biology. <br />
<br />
==Using CellML==<br />
<br />
The modelling language of choice for virtual parts is CellML. One of the main reasons for choosing CellML over SBML (or any of the other modelling language available) is its modular nature and its proven track record in representing intracellular processes in systems biology (Cooling et al., 2010).<br />
Identifying motifs<br />
<br />
To identify motifs and the parts they were composed of, the CellML model of the subtilin receiver was converted to a graphical representation (Figure 2).<br />
<br />
[[Image:Newcastle_workflows_SubtilinReceiver.png]]<br />
<br />
Figure 2: A graphic respresentation of the Subtilin Receiver model. <br />
<br />
The virtual parts for these parts were extracted from the CellML Subtilin receiver model and assembled again in a new file.<br />
<br />
The model in the file was checked using [http://cor.physiol.ox.ac.uk/ COR] because it was quick to reload changed files and to return informative errors<br />
Database<br />
<br />
A MySQL relational database was designed and created to hold both physical and non-physical virtual parts, required to build a simulatable model of a BioBrick. Both types of parts are saved in the same table but are distinguished by a boolean flag.<br />
<br />
The database was populated to hold at least one generic part for each type of part that we used. A naming convention was adopted allows for the retrieval of parts based on the part name.<br />
<br />
The structure of the database is shown in the E-R diagram in Figure 3.<br />
<br />
[[Image:Newcastle_workflows_Erdiagram.png|800px]]<br />
<br />
Figure 3: An E-R diagram of the database created for the BioBrickIt service, to hold virtual parts and motifs. (Click on image for larger format)<br />
<br />
==Web Service==<br />
<br />
A web service, which we called BioBrickIt, was developed in Java and served with the Apache Tomcat web server. The web service provides the functionality to populate the database with virtual parts and other information required to create simulatable CellML models. It further provides the functionality to simulate models retrieved.<br />
<br />
A choice had to be made between offering a SOAP or a RESTful web service. Time constraints and simplicity of implementation favoured a RESTful service. Resources are exposed as URLs and results are returned as plain text rather than in XML, as would be the case if it was a SOAP service (Pautasso and Leymann, 2008).<br />
<br />
The web service accesses the relational database discussed in section 2.4 using Java DataBase Connectivity (JDBC).<br />
<br />
The web service can retrieve a simulatable model of a motif by firstly retrieving its rules.<br />
<br />
==Simulation==<br />
<br />
Simulation of models were provided by connecting the web service to an instance of JSim. JSim (http://www.physiome.org/jsim/) is a software application, written in Java, for simulating quantitative numeric models. It is possible to run JSim as a service, a stand alone application or an applet.<br />
<br />
The JSim server was installed on the same server as the Apache Tomcat web server.<br />
<br />
Simulation is requested from the BioBrickIt service by passing the parameter simulation=true with the URL when a model of a motif is requested.<br />
<br />
==Taverna workflows==<br />
<br />
To create the workflows that utilised the web service, the decision was made to use [http://www.taverna.org.uk/ Taverna Workbench]. Taverna is an open source application for managing workflows. It can be used to integrate molecular biology tools and databases available on the Web. It is especially useful for web services (Hull et al., 2006). Taverna was chosen because of familiarity with the product, but also because time restrictions did not allow for the evaluation and comparison of other tools.<br />
<br />
=Results:=<br />
<br />
==Motifs==<br />
<br />
We identified two motifs, a constitutive promoter motif and a coding sequence motif (see Figures 1 and 2)<br />
<br />
[[Image:Newcastle_workflows_Motif1.png|800px]]<br />
<br />
Figure 1: The constitutive promoter motif. <br />
<br />
[[Image:Newcastle_workflows_Motif2.png|800px]]<br />
<br />
Figure 2: The coding sequence motif.<br />
<br />
The coding sequence motif illustrates how a modular approach can be beneficial to the design process. The constitutive promoter motif that is embedded in the coding sequence motif is shown with the red dotted line.<br />
<br />
The motifs were identified using the Subtilin receiver model created by the Newcastle University iGEM team of 2008.<br />
<br />
==The BioBrickIt Web Service==<br />
<br />
We developed a RESTful web service which we called BioBrickIt. It is available online at http://msc.jannetta.com:8080/BioBrickIt. BioBrickIt was developed using J2EE web technologies, JAXP for XML handling, the Apache Tomcat web server and a mySQL database. The service and its source code can also be downloaded as a .war file from http://msc.jannetta.com:8080/BioBrickIt/BioBrickIt.war.<br />
<br />
The web service exposes the following resources:<br />
Adding a component (or virtual part) to the database from a file containing the part in cellML format<br />
<br />
* Listing all the components in the database<br />
* Listing all the component types in the database<br />
* Listing all the variables for a specified component<br />
* Retrieving a specified component<br />
* Listing the sequence for a physical component<br />
* Listing all the motifs in the database<br />
* Listing the rules for a specified motif<br />
* Retrieving the model for a specified motif<br />
* Simulating the retrieved model<br />
<br />
==Database==<br />
<br />
A MySQL relational database was designed and implemented to hold both physical and non-physical parts. The database was populated manually and using the file upload feature of the BioBrickIt web service. A set of generic parts was all that was required for this research, but the database contains several other parts which are available from the 2008 iGEM Subtilin receiver. The database is online and can be accessed via the BioBrickIt Service at http://msc.jannetta.com:8080/BioBrickIt/.<br />
<br />
==JSim server==<br />
<br />
When accessing the BioBrickIt service on http://msc.jannetta.com:8080/BioBrickIt/, a simulation of the generated motif can be requested. This is done by adding simulate=yes as a parameter to the URL. For example requesting a simulation of the generic constitutive promotor can be obtained with: http://msc.jannetta.com:8080/BioBrickIt/GetMotifModel?motif_name=motif_constitutive_promoter&simulate=yes.<br />
<br />
==Taverna workflows==<br />
<br />
We used Taverna Workbench to create 3 workflows. The first workflow (Figure 3) makes use of the BioBrickIt Webservice to determine the presence of restriction sites in a provided sequence.<br />
<br />
The second workflow (Figure 4) retrieves the sequences of provided physical parts using the BioBrickIt service. It then concatenates the sequences. The intension is that this workflow be extended or embedded in a larger workflow that could conver a CellML model to a sequence.<br />
<br />
[[Image:Newcastle_workflows_Workflow1.png|800px]]<br />
<br />
Figure 3: Workflow 1, Restriction site finder. <br />
<br />
[[Image:Newcastle_workflows_Workflow2.png]]<br />
<br />
Figure 4: Workflow 2, Retrieve sequences of virtual parts. <br />
<br />
[[Image:Newcastle_workflows_Workflow3.png]]<br />
<br />
Figure 5: Workflow 3, Retrieve simulatable CellML model of a motif. <br />
<br />
=Discussion:=<br />
<br />
Synthetic Biology is a very young field of study and still lacks the methodologies already established in other fields of engineering. To speed up the process of putting these methodologies in place, it makes sense to borrow from the other engineering disciplines. Systems biology already makes use of an e-Science approach using computerised workflows with great success. The approach serves to avoid tedious cutting and pasting of retrieved result from one software package to the next and thus saves significant amounts of time and effort to improve efficiency and accuracy.<br />
<br />
Using the bottom-up approach to design, means that design starts with the most simple of identifiable parts, working its way up to a complex system composed of these parts (Cooling et al., 2010). This approach relates very closely to the development of software and fits in very well with a design and development approach similar to the software development life cycle. Thus, we proposed a Synthetic Biology life cycle which, apart from one phase, is almost identical to the SDLC.<br />
<br />
The subtilin receiver model created by Cooling et al. (2010) provided all the virtual parts necessary for this research project. Creating a graphical representation of the model allowed us to identify the motifs which provided the first level of abstraction and encapsulation. The principles of abstraction and encapsulation were borrowed from software and serve the purpose of hiding the complexity of a design (Henderson-Sellers, 1997). Models consist not only of physical parts, but also need non-physical parts that glue the physical parts together for simulation purposes. These physical and non-physical parts provide the minimal parts required in the bottom-up approach. The rules for putting these parts together were captured in a database which allowed us to query the database for a series of parts in the correct order almost ready for simulation. Once the parts are extracted from the database according to the rules, programmatically extract the units that need to be specified in the model and the connections that need to be made between parts. A naming convention which allowed us to determine which variables from which parts need connecting. An alternative approach would be to extend the database to hold the information required for mapping parts and variables. This approach would simplify the creation of the CellML parts, but would complicate the design of the software and database.<br />
<br />
With the database in place and populated we developed a RESTful web service that extracts the parts required for a requested motif, assemble the motif and returns it in CellML as a simulatable model. The service can return the CellML to the client, but it can also simulate the model using JSim, a Java based software application. Three services, a database service, a web service and a simulating service were established. Finally a Taverna workflow was created that use these services in order to create a generic simulatable model. The parts of the generic model can, for the moment, be replaced manually with specific parts. Ideally the service should be extended to replace parts automatically so that minimal input is required from the user.<br />
<br />
Although all of the above mentioned services were installed on one server it is possible to develop these services such that they can be run on different servers and off course in different locations on the Internet.<br />
<br />
A variation on the waterfall software development life cycle was suggested and it was illustrated how this method can successfully be used to guide the design process. It is, however, a very old method and software engineering has produced many alternatives since. It would be worth while investigating some of the newer methods that might prove to be even more successful if applied to the Synthetic Biology development life cycle.<br />
<br />
==Putting it all together==<br />
<br />
Figure 1 shows, from start to finish, the internal flow of information in an ideal workflow that would produce a complete and simulated model of the requested part or motif. The workflow includes the workflows already created as well as the services provided by BioBrickIt. To actually be able to implement the complete workflow both BioBrickIt and the Taverna workflows created will need extending.<br />
<br />
The process is started with input by the user (Figure 1(a)), specifying which motif and which specific parts are required.<br />
<br />
The next step. (Figure 1(b)), is to retrieve the motif with generic parts from the database. The motif is composed of virtual part and assembled according to the rules which are also capture in the database. This motif is simulatable as is, but contains no specific parts as yet.<br />
<br />
Step 3, (Figure 1(c)), uses a service, such as BioBrickIt, that can use the generated motif and replace generic virtual parts with specified parts of the same type. For instance, a generic constitutive promoter can be replaced by pspaRK.<br />
<br />
Step 4, (Figure 1(d)), uses a service such as JSim to simulate the model. To make sense of the simulation results it is usually required to visualise it in some form. Visualisation is done in step 5.<br />
<br />
Step 5, (Figure 1(e)), produces human readable formats of the simulation results. Visualisation is usually in the format of graphs.<br />
<br />
Step 6, (Figure 1(f)), is the production of a BioBrick sequence from the produced model produced in step 3. Newcastle University is currently researching such a model-to-sequence conversion algorithm. This step would make use of the GetVPSequence and GetSites workflows, to retrieve physical part sequences and check them for specified restriction sites.<br />
<br />
Step 7, (Figure 1(g)), show the possible results that can be produced by the workflow.<br />
<br />
[[Image:Newcastle_workflows_Workflow.gif]]<br />
<br />
Figure 1 (This image is 1.1 MB and might take a while to download.)<br />
<br />
[[E-Science_References]]<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/26_July_2010Team:Newcastle/26 July 20102010-10-27T21:08:03Z<p>RachelBoyd: /* PCR of Genomic DNA */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
='''Preparation for cloning of the ''rocF'' BioBrick'''=<br />
<br />
==Aim==<br />
In preparation for Gibson cloning of the ''rocF'' BioBrick we started work on mini preps of plasmid DNA, and on ''B. subtilis'' 168 chromosomal DNA extraction.<br />
<br />
==Re-hydration of registry parts==<br />
[[Image:Newcastlehydration.jpg|200px|thumb|right|Re-hydration of dried parts registry DNA]]<br />
We re-hydrated using sterile distill water: <br />
#[http://partsregistry.org/Part:pSB1C3 pSB1C3] (the plasmid we will be submitting our BioBricks to the registry in) and<br />
#[http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] (the double terminator we will be using for the ''rocF'' BioBrick) from the parts distribution.<br />
<br />
==Transformation of ''E. coli''==<br />
We transformed and plated separate tubes of ''E. coli'' DH5α with:<br />
<br />
# The above two re-hydrated plasmids<br />
# [http://partsregistry.org/Part:BBa_K143062 BBa_K143062], a LacI BioBrick sent to us by Imperial College, London, UK which we will use to help characterise many of our BioBricks, including ''rocF''.<br />
# A positive control which we had already prepared during our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with ''rfp'' insert.<br />
# A negative control (no vector), to verify the antibiotic plates are working (no growth should be observed on this plate).<br />
<br />
<br />
Please refer to the transformation protocol for ''E. coli'' DH5α here: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']].<br />
<br />
==Overnight cultures of ''B. subtilis'' 168 for chromosomal DNA extraction==<br />
The ''rocF'' coding sequence is to be amplified from the ''B. subtilis'' 168 genome by PCR. Before we can do this we need to extract 168 chromosomal DNA.<br />
<br />
Today we plated up overnight cultures of ''B. subtilis'' 168 so that we can do chromosome extraction tomorrow.<br />
<br />
='''PCR of Genomic DNA'''=<br />
<br />
==Aim:==<br />
<br />
To determine whether the genomic DNA has been extracted from ''B. subtilis'' strain 3610.<br />
<br />
==Materials:==<br />
<br />
* Pipette<br />
* Microfuge<br />
* Microtubes<br />
* Distilled H<sub>2</sub>O<br />
* Nucleotide DNTP<br />
* 5x GoTaq buffer<br />
* Template DNA (''B. subtilis'' ATCC 6633, 1:1 and 1:2)<br />
* Forward and reverse primers<br />
<br />
==Protocol:==<br />
<br />
* For the full protocol, please refer to [[Team:Newcastle/PCR|PCR]].<br />
<br />
===Conditions in ThermoCycler:===<br />
* Melting temperature, Tm used for anneal step is 59°C.<br />
<br />
==Results:==<br />
Gel electrophoresis will be run tomorrow to determine the results.<br />
<br />
==Conclusion:==<br />
<br />
Please refer to Lab book dated [[Team:Newcastle/27_July_2010|27th July 2010]].<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:NewcastleTeam:Newcastle2010-10-27T21:08:02Z<p>RachelBoyd: /* Project Description */</p>
<hr />
<div>__NOTOC__{{Team:Newcastle/mainbanner}}<br />
=BacillaFilla: Fixing cracks in Concrete= <br />
<br />
<br />
<br />
[[Image:Newcastle_iGEM_Teampic.jpeg|centre|468px]]<br />
<br />
<br />
<br />
<div style="text-align:justify;padding:5px"><br />
===Project Description===<br />
<br />
BacillaFilla, an engineered ''Bacillus subtilis'', aims to repair [[Team:Newcastle/problem|cracks in concrete]] which can cause catastrophic structural failure. BacillaFilla would be applied to structures by spraying onto their surface.<br />
<br />
BacillaFilla swims deep into the cracks. Repair is effected by production of [[Team:Newcastle/Urease|CaCO<sub>3</sub>]], [[Team:Newcastle/Filamentous_Cells|filamentous ''Bacillus subtilis'' cells]] and [[Team:Newcastle/glue|levan]]. CaCO<sub>3</sub> expands at the same rate as concrete, making it the ideal filler. Filamentous ''Bacillus subtilis'' cells have similar tensile strength to the synthetic fibres used in fibre-reinforced concrete, and provide reinforcement. Levan glues CaCO<sub>3</sub> and filamentous cells in place.<br />
<br />
''B. subtilis'' 168 sporulates, making it ideal for storage and transportation. The cells can be [[Team:Newcastle/solution#Alkalinity_resistance|made to be tolerant to concrete's high pH]].<br />
<br />
We designed a [[Team:Newcastle/Swarming|swarming]] BioBrick part for repairing ''B. subtilis'' 168's defective ''swrA'' and ''sfp'' genes and regaining motility. At the end of the crack the quorum sensing peptide [[Team:Newcastle/End_of_crack_%26_signalling_system|subtilin]] triggers a co-ordinated population response from a [[Team:Newcastle/End_of_crack_%26_signalling_system#2008Brick|subtilin-inducible promoter]]. Upregulating ''SR1'' and ''rocF'' promotes arginine and urea production, increasing exogenous CaCO<sub>3</sub> deposition. Over-producing YneA induces the filamentous cell phenotype, while SacB converts extracellular sucrose to levan glue.<br />
<br />
To protect the environment our project also includes a design for a [[Team:Newcastle/Non-target-environment_kill_switch| kill switch]].<br />
<br />
<br />
[[Image:newcastle_summary.png|850px]]<br />
<br />
<br />
{|style cellpadding="20" cellspacing="0"<br />
! colspan="2" |<font size=4> <center>'''Summary of achievements:'''</center></font><br />
|-<br />
|<span style="color:Sienna">'''BRONZE:'''</span><br />
|From the beginning of the project, we have successfully registered the team of two instructors, six advisors and eight members. We have completed and submitted a Project Summary form, developed ideas and shared them on our iGEM wikipedia page. We also entered 23 new BioBrick parts into the Registry of Parts. One part, [http://partsregistry.org/Part:BBa_K302012 IPTG-induced filamentous cell formation], the BioBrick for the IPTG-induced filamentous cell formation, was demonstrated to work as expected.<br />
|-<br />
|<span style="color:Silver">'''SILVER:'''</span><br />
|Our new BioBrick, the IPTG-inducible filamentous cell formation part works, so we [[Team:Newcastle/Filamentous_Cells#Characterisation|characterised]] it and included the information, [http://partsregistry.org/Part:BBa_K302012:Experience BBa_K302012], on the Parts Registry. <br />
|-<br />
|<span style="color:Goldenrod">'''GOLD:'''</span><br />
|In order to obtain a gold, we investigated the benefits of an e-Science approach, focusing on workflows, to synthetic biology. Details can be found at [[Team:Newcastle/E-Science|here]]. This part of the project resulted in us proposing a new standard for a RESTful API which facilitates the discovery and publication of models of functional biological units. The standard has been submitted to the BioBricks Foundation as [[BBFRFC66|BBF RFC 66]]. We have also improved on existing BioBrick parts to produce hyperspankoid: [http://partsregistry.org/Part:BBa_K302003 BBa_K302003].<br />
|-<br />
|}<br />
<br />
<br />
<br />
<!---[http://twitter.com/newcastle_igem Follow us on Twitter] or join our [http://www.facebook.com/pages/Newcastle-iGEM-2010/140948965930577| Facebook Fan page!] ---><br />
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!align="center"|[[Team:Newcastle/Team|Team]]<br />
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|}-->{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/26_July_2010Team:Newcastle/26 July 20102010-10-27T21:05:47Z<p>RachelBoyd: /* Materials: */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
='''Preparation for cloning of the ''rocF'' BioBrick'''=<br />
<br />
==Aim==<br />
In preparation for Gibson cloning of the ''rocF'' BioBrick we started work on mini preps of plasmid DNA, and on ''B. subtilis'' 168 chromosomal DNA extraction.<br />
<br />
==Re-hydration of registry parts==<br />
[[Image:Newcastlehydration.jpg|200px|thumb|right|Re-hydration of dried parts registry DNA]]<br />
We re-hydrated using sterile distill water: <br />
#[http://partsregistry.org/Part:pSB1C3 pSB1C3] (the plasmid we will be submitting our BioBricks to the registry in) and<br />
#[http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] (the double terminator we will be using for the ''rocF'' BioBrick) from the parts distribution.<br />
<br />
==Transformation of ''E. coli''==<br />
We transformed and plated separate tubes of ''E. coli'' DH5α with:<br />
<br />
# The above two re-hydrated plasmids<br />
# [http://partsregistry.org/Part:BBa_K143062 BBa_K143062], a LacI BioBrick sent to us by Imperial College, London, UK which we will use to help characterise many of our BioBricks, including ''rocF''.<br />
# A positive control which we had already prepared during our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with ''rfp'' insert.<br />
# A negative control (no vector), to verify the antibiotic plates are working (no growth should be observed on this plate).<br />
<br />
<br />
Please refer to the transformation protocol for ''E. coli'' DH5α here: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']].<br />
<br />
==Overnight cultures of ''B. subtilis'' 168 for chromosomal DNA extraction==<br />
The ''rocF'' coding sequence is to be amplified from the ''B. subtilis'' 168 genome by PCR. Before we can do this we need to extract 168 chromosomal DNA.<br />
<br />
Today we plated up overnight cultures of ''B. subtilis'' 168 so that we can do chromosome extraction tomorrow.<br />
<br />
='''PCR of Genomic DNA'''=<br />
<br />
==Aim:==<br />
<br />
To determine whether the genomic DNA has been extracted from ''B. subtilis'' strain 3610.<br />
<br />
==Materials:==<br />
<br />
* Pipette<br />
* Microfuge<br />
* Microtubes<br />
* Distilled H<sub>2</sub>O<br />
* Nucleotide DNTP<br />
* 5x GoTaq buffer<br />
* Template DNA (B. subtilis ATCC 6633, 1:1 and 1:2)<br />
* Forward and reverse primers<br />
<br />
==Protocol:==<br />
<br />
* For the full protocol, please refer to [[Team:Newcastle/PCR|PCR]].<br />
<br />
===Conditions in ThermoCycler:===<br />
* Melting temperature, Tm used for Anneal step is 59°C.<br />
<br />
==Results:==<br />
Gel electrophoresis will be run tomorrow to determine the results.<br />
<br />
==Conclusion:==<br />
<br />
Please refer to Lab book dated [[Team:Newcastle/27_July_2010|27th July 2010]].<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/safetyTeam:Newcastle/safety2010-10-27T21:05:22Z<p>RachelBoyd: /* Post-Chernobyl Disaster */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
==Safety Issues==<br />
<br />
These are the safety questions for the judging form:<br />
<br />
'''1. Would any of your project ideas raise safety issues in terms of:'''<br />
<br />
*'''Researcher Safety''':<br />
<br />
We worked in the Centre for Bacterial Cell Biology (CBCB) at Newcastle University for the entire project, where there are clearly defined safety rules and regulations that all laboratory workers must follow. One of our advisors, Dr Wendy Smith, guided us through the first [[Team:Newcastle/14_June_2010|introductory]] week, before any laboratory work began. This included carrying out the following risk assessments to determine what control measures would be required.<br />
<br />
(i)'''Chemical Hazards''':<br />
At the beginning of the project written risk assessments were already available in the host laboratories for all procedures that involved potentially hazardous chemicals. These risk assessments were reviewed and the recommended control measures were strictly followed throughout. No additional chemical hazards specific to this project were identified.<br />
<br />
(ii)'''Radioisotopes and carcinogens''':<br />
None of them were used in this project.<br />
<br />
(iii)'''Biological hazards''':<br />
Throughout the project, we used the ''Escherichia coli'' strain DH5α, ''Bacillus subtilis'' strain 3610, ''Bacillus subtilis'' strain 168 and ''Bacillus sphaericus'' strain LMG 22257. Wild-type ''E. coli'' is classified as a hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP). However, ''E. coli'' strain DH5α is derived from a laboratory strain ''E. coli'' K12 strain, which is recognised as disabled and equivalent to an ACDP hazard group 1 organism (i.e. unlikely to cause disease). ''E. coli'' K12 and its derivatives such as strain DH5α are unable to colonise in humans or animals and consequently pose negligible risk to human or animal health. Wild-type ''Bacillus subtilis'' (i.e. strain 3610) is classified as an ACDP hazard group 1 organism and its derivative ''B. subtilis'' strain 168 has disabling auxotrophs mutations (e.g. conferring a requirement for tryptophan, Zeigler ''et al'', 2008) that makes it even less likely to colonise or cause harm to human or animal health. Also ''Bacillus sphaericus'' LMG 22257 is classified as an ADCP hazard group 1 organism making it unlikely to cause any harm to either human or animal health. The potential of any sequences cloned into these bacterial hosts during the project to pose additional hazards was also assessed. None of these sequences were associated with pathogenic traits or traits that might significantly enhance the survival outside the lab. Therefore, no specific safety issues, other than those associated with use of any non-pathogenic microorganism, were identified. It was concluded that containment level 1(CL1) would be sufficient to ensure researcher safety. Nonetheless, all work was carried out in strict compliance with the host laboratory's standard safety procedures, which were more stringent that those required for CL1.<br />
<br />
(iv)'''Other hazards''':<br />
The project also involved conducting some work in the Engineering structures laboratory, where we made and broke concrete 'blocls'. Appropriate safety regulations for the type of work in this laboratory were followed. When we were in the structures lab, we wore safety goggles, steel toe cap boots and gloves, which will be able to protect us from lab based accidents.<br />
<br />
* '''Public Safety''':<br />
Our project concerns repairing cracks on concrete surfaces by spraying these surfaces with spores of engineered derivatives of ''Bacillus subtilis'' 168. It is recommended that workers carrying out the spraying should wear an appropriate face mask to minimize inhaling of spores. During the spraying procedure spores could escape into the surrounding environment, but their concentration will diluted very rapidly with distance, greatly reducing potential hazards away from the immediate area of spraying. As outlined above, ''B. subtilis'' 168 is non-pathogenic and therefore very unlikely to pose a risk to public safety, particularly since any escaping spores will be unable to germinate in the absence of culture media.<br />
<br />
*'''Environmental Safety''':<br />
For reasons outlined above, the ''E. coli'' strain DH5α has very limited ability to survive outside the laboratory so taht in the very unlikely event of escape, it will be unable to survive, disseminate with and/or displace other organisms. Therefore no specific environmental hazards associated with the ''E. coli'' strain were identified.<br />
<br />
GM derivatives of ''Bacillus subtilis'' strain 168 will be released deliberately. This is a strain that has been maintained in the lab for 52 years, during which it has accumulated disabling mutations, such as tryptophan auxotrophy mentioned above, which greatly diminishes its ability to survive and disseminate outside the laboratory (Zeigler ''et al.'', 2008). It seems very unlikely that it could compete effectively and replace wild-type ''B. subtilis''. Nonetheless, the release of any GMO into the environment has been a concern since early days of genetic engineering and one of our instructors (Prof. Anil Wipat) has previously studied such issues (Wipat, 1990). To minimise concerns about environmental safety, in this project we designed the [[Team:Newcastle/Non-target-environment kill switch|Non-target-environment kill switch]] genetic part to prevent dissemination after release into the environment.<br />
<br />
'''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
We do not see any safety issues for the new Biobricks parts that we made this year. <br />
<br />
'''3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
Yes there is a biosafety group at the Centre for Bacterial Cell Biology and Institute of Cell and Molecular Biosciences, which includes the Institute Safety Officer (SSO), Biological Safety Supervisor (BSS), Genetic Modification Chairperson (GMC), Radiation Protection Supervisor (RPS), Laser Protection Officer and Lab Heads. They have reviewed the safety of the lab from time to time during the duration of the project.<br />
<br />
* If yes, what does your local biosafety group think about your project?<br />
<br />
They are aware about the whole project and they reviewed it thoroughly with the whole team. They discussed about each and every Biobrick part in detail and found no safety issues with it.<br />
<br />
'''4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A full risk assessment should be carried out before the work begins. This should consider: <br />
(i) Consequences of any identifiable hazard and<br />
(ii) The likelihood of the hazard arising.<br />
The risk can then be defined and appropriate control measures can be introduced to minimise the risk. <br />
<br />
The inclusion of a safety kill switch, such as that detailed in our project, could help reduce the risk of accidental release. In addition, strains should be marked with unique barcodes to allow them to be tracked, should they accidently escape.<br />
<br />
==Ethics==<br />
<br />
===Synthetic Biology: Background===<br />
<br />
Synthetic biology is a new research field that has a big potential in coming up with solutions for our everyday problems. However, ethical issues have been raised since the start of development in this field. Here we will discuss the ethical issues that our project will bring.<br />
<br />
====Biohackers====<br />
<br />
In this field, there are always people that view synthetic biology's negative aspects with concern, like worrying about the production of pathogens to be used as weapons. Their fears are that people will hack into systems to obtain data that might be used to do this. Therefore, we have to be very careful with the products of our research so that they remain safe in our hands.<br />
<br />
====Playing God!!!====<br />
<br />
<br />
In synthetic biology, we control the lives of bacteria. We insert proteins into bacteria to make them do what we want them to do. We make them kill themselves at the end of their job because they might be released into the environment and be harmful to other living things. It shows that we do not really treat the single-celled organism as life. However, we are coming up with a novel solution for the environment by healing concrete cracks.<br />
<br />
====Biosecurity====<br />
<br />
Bacteria might be harmful to the environment and living things around. Considering the fact that they are such small organisms and could not be spotted with the naked-eye if they are released in air, we make them kill themselves with the ‘kill switch’ biobrick. This can be very inconsiderate standing in a bacteria’s point of view, but it is the novel thing to do to avoid them hurting other living organisms.<br />
<br />
===Construction: Background===<br />
<br />
Cracks form in concrete structures as soon as they set. These cracks continue to grow in size if they are not repaired due to the weight of the structure, imposed load, freeze-thaw effect and wind loading. The bigger the cracks are, the higher the rate of water seeping into the steel reinforcements, causing them to corrode and thus weakening the structure. Therefore, we would like to use Bacilla Filla to fill up these cracks before things get worse.<br />
<br />
====Environmental Disaster====<br />
<br />
Concrete is a very widely used material in construction. In fact, cement that is used to make concrete is the second most widely used substance in the world after water. Some building structures have to be demolished because the cracks formed appear to be threatening the strength of the structures. These buildings have to be rebuilt in order to reinforce its tensile strength. This is an unsustainable method of recovery because cement is a material that requires a lot of energy to be produced. In the production of cement, the rotating kiln requires the temperature to be between 1350°C-1400°C, which not only uses up a lot of energy resources, but also produces a lot of carbon dioxide. These carbon dioxide contributes to the green house effect which then leads to global warming. Therefore, our project can help to reduce renovation of buildings which in turn lowers the amount of cement that has to be produced each year.<br />
<br />
====Post-Chernobyl Disaster====<br />
<br />
The nuclear power plant combustion that occurred in Chernobyl on 26 April 1986 has allowed radioactive elements to be released into the environment, threatening the health of people living within close proximity of the plant. Therefore, a shelter, the Shelter Implementation Plan (SIP) which will be completed in 2012 is currently being built to cover up the area that where the accident occurred so as to make it environmentally friendly. However, if cracks were to form, something that inevitably occurs for building structures, those radioactive elements will still be dispersed into the environment. With the help of our project, those cracks will be able to be filled-up and the area will be safe again.<br />
<br />
<br />
<br />
[1] Wipat, A. (1990). "''Release and detection of geneticaly engineered streptomycetes in soil''". PhD thesis, Microbiology Department, John Moores University.<br />
<br />
[2] Zeigler DR, Prágai Z, Rodriguez S, Chevreux B, Muffler A, Albert T et al. (2008). "''The origins of 168, W23, and other Bacillus subtilis legacy strains''". Journal of Bacteriology, 190(21), 6983-95.<br />
<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/26_July_2010Team:Newcastle/26 July 20102010-10-27T21:05:16Z<p>RachelBoyd: /* Materials: */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
='''Preparation for cloning of the ''rocF'' BioBrick'''=<br />
<br />
==Aim==<br />
In preparation for Gibson cloning of the ''rocF'' BioBrick we started work on mini preps of plasmid DNA, and on ''B. subtilis'' 168 chromosomal DNA extraction.<br />
<br />
==Re-hydration of registry parts==<br />
[[Image:Newcastlehydration.jpg|200px|thumb|right|Re-hydration of dried parts registry DNA]]<br />
We re-hydrated using sterile distill water: <br />
#[http://partsregistry.org/Part:pSB1C3 pSB1C3] (the plasmid we will be submitting our BioBricks to the registry in) and<br />
#[http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] (the double terminator we will be using for the ''rocF'' BioBrick) from the parts distribution.<br />
<br />
==Transformation of ''E. coli''==<br />
We transformed and plated separate tubes of ''E. coli'' DH5α with:<br />
<br />
# The above two re-hydrated plasmids<br />
# [http://partsregistry.org/Part:BBa_K143062 BBa_K143062], a LacI BioBrick sent to us by Imperial College, London, UK which we will use to help characterise many of our BioBricks, including ''rocF''.<br />
# A positive control which we had already prepared during our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with ''rfp'' insert.<br />
# A negative control (no vector), to verify the antibiotic plates are working (no growth should be observed on this plate).<br />
<br />
<br />
Please refer to the transformation protocol for ''E. coli'' DH5α here: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']].<br />
<br />
==Overnight cultures of ''B. subtilis'' 168 for chromosomal DNA extraction==<br />
The ''rocF'' coding sequence is to be amplified from the ''B. subtilis'' 168 genome by PCR. Before we can do this we need to extract 168 chromosomal DNA.<br />
<br />
Today we plated up overnight cultures of ''B. subtilis'' 168 so that we can do chromosome extraction tomorrow.<br />
<br />
='''PCR of Genomic DNA'''=<br />
<br />
==Aim:==<br />
<br />
To determine whether the genomic DNA has been extracted from ''B. subtilis'' strain 3610.<br />
<br />
==Materials:==<br />
<br />
* Pipette<br />
* Microfuge<br />
* Microtubes<br />
* Distilled H2O<br />
* Nucleotide DNTP<br />
* 5x GoTaq buffer<br />
* Template DNA (B. subtilis ATCC 6633, 1:1 and 1:2)<br />
* Forward and reverse primers<br />
<br />
==Protocol:==<br />
<br />
* For the full protocol, please refer to [[Team:Newcastle/PCR|PCR]].<br />
<br />
===Conditions in ThermoCycler:===<br />
* Melting temperature, Tm used for Anneal step is 59°C.<br />
<br />
==Results:==<br />
Gel electrophoresis will be run tomorrow to determine the results.<br />
<br />
==Conclusion:==<br />
<br />
Please refer to Lab book dated [[Team:Newcastle/27_July_2010|27th July 2010]].<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/safetyTeam:Newcastle/safety2010-10-27T21:04:48Z<p>RachelBoyd: /* Post-Chernobyl Disaster */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
==Safety Issues==<br />
<br />
These are the safety questions for the judging form:<br />
<br />
'''1. Would any of your project ideas raise safety issues in terms of:'''<br />
<br />
*'''Researcher Safety''':<br />
<br />
We worked in the Centre for Bacterial Cell Biology (CBCB) at Newcastle University for the entire project, where there are clearly defined safety rules and regulations that all laboratory workers must follow. One of our advisors, Dr Wendy Smith, guided us through the first [[Team:Newcastle/14_June_2010|introductory]] week, before any laboratory work began. This included carrying out the following risk assessments to determine what control measures would be required.<br />
<br />
(i)'''Chemical Hazards''':<br />
At the beginning of the project written risk assessments were already available in the host laboratories for all procedures that involved potentially hazardous chemicals. These risk assessments were reviewed and the recommended control measures were strictly followed throughout. No additional chemical hazards specific to this project were identified.<br />
<br />
(ii)'''Radioisotopes and carcinogens''':<br />
None of them were used in this project.<br />
<br />
(iii)'''Biological hazards''':<br />
Throughout the project, we used the ''Escherichia coli'' strain DH5α, ''Bacillus subtilis'' strain 3610, ''Bacillus subtilis'' strain 168 and ''Bacillus sphaericus'' strain LMG 22257. Wild-type ''E. coli'' is classified as a hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP). However, ''E. coli'' strain DH5α is derived from a laboratory strain ''E. coli'' K12 strain, which is recognised as disabled and equivalent to an ACDP hazard group 1 organism (i.e. unlikely to cause disease). ''E. coli'' K12 and its derivatives such as strain DH5α are unable to colonise in humans or animals and consequently pose negligible risk to human or animal health. Wild-type ''Bacillus subtilis'' (i.e. strain 3610) is classified as an ACDP hazard group 1 organism and its derivative ''B. subtilis'' strain 168 has disabling auxotrophs mutations (e.g. conferring a requirement for tryptophan, Zeigler ''et al'', 2008) that makes it even less likely to colonise or cause harm to human or animal health. Also ''Bacillus sphaericus'' LMG 22257 is classified as an ADCP hazard group 1 organism making it unlikely to cause any harm to either human or animal health. The potential of any sequences cloned into these bacterial hosts during the project to pose additional hazards was also assessed. None of these sequences were associated with pathogenic traits or traits that might significantly enhance the survival outside the lab. Therefore, no specific safety issues, other than those associated with use of any non-pathogenic microorganism, were identified. It was concluded that containment level 1(CL1) would be sufficient to ensure researcher safety. Nonetheless, all work was carried out in strict compliance with the host laboratory's standard safety procedures, which were more stringent that those required for CL1.<br />
<br />
(iv)'''Other hazards''':<br />
The project also involved conducting some work in the Engineering structures laboratory, where we made and broke concrete 'blocls'. Appropriate safety regulations for the type of work in this laboratory were followed. When we were in the structures lab, we wore safety goggles, steel toe cap boots and gloves, which will be able to protect us from lab based accidents.<br />
<br />
* '''Public Safety''':<br />
Our project concerns repairing cracks on concrete surfaces by spraying these surfaces with spores of engineered derivatives of ''Bacillus subtilis'' 168. It is recommended that workers carrying out the spraying should wear an appropriate face mask to minimize inhaling of spores. During the spraying procedure spores could escape into the surrounding environment, but their concentration will diluted very rapidly with distance, greatly reducing potential hazards away from the immediate area of spraying. As outlined above, ''B. subtilis'' 168 is non-pathogenic and therefore very unlikely to pose a risk to public safety, particularly since any escaping spores will be unable to germinate in the absence of culture media.<br />
<br />
*'''Environmental Safety''':<br />
For reasons outlined above, the ''E. coli'' strain DH5α has very limited ability to survive outside the laboratory so taht in the very unlikely event of escape, it will be unable to survive, disseminate with and/or displace other organisms. Therefore no specific environmental hazards associated with the ''E. coli'' strain were identified.<br />
<br />
GM derivatives of ''Bacillus subtilis'' strain 168 will be released deliberately. This is a strain that has been maintained in the lab for 52 years, during which it has accumulated disabling mutations, such as tryptophan auxotrophy mentioned above, which greatly diminishes its ability to survive and disseminate outside the laboratory (Zeigler ''et al.'', 2008). It seems very unlikely that it could compete effectively and replace wild-type ''B. subtilis''. Nonetheless, the release of any GMO into the environment has been a concern since early days of genetic engineering and one of our instructors (Prof. Anil Wipat) has previously studied such issues (Wipat, 1990). To minimise concerns about environmental safety, in this project we designed the [[Team:Newcastle/Non-target-environment kill switch|Non-target-environment kill switch]] genetic part to prevent dissemination after release into the environment.<br />
<br />
'''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
We do not see any safety issues for the new Biobricks parts that we made this year. <br />
<br />
'''3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
Yes there is a biosafety group at the Centre for Bacterial Cell Biology and Institute of Cell and Molecular Biosciences, which includes the Institute Safety Officer (SSO), Biological Safety Supervisor (BSS), Genetic Modification Chairperson (GMC), Radiation Protection Supervisor (RPS), Laser Protection Officer and Lab Heads. They have reviewed the safety of the lab from time to time during the duration of the project.<br />
<br />
* If yes, what does your local biosafety group think about your project?<br />
<br />
They are aware about the whole project and they reviewed it thoroughly with the whole team. They discussed about each and every Biobrick part in detail and found no safety issues with it.<br />
<br />
'''4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A full risk assessment should be carried out before the work begins. This should consider: <br />
(i) Consequences of any identifiable hazard and<br />
(ii) The likelihood of the hazard arising.<br />
The risk can then be defined and appropriate control measures can be introduced to minimise the risk. <br />
<br />
The inclusion of a safety kill switch, such as that detailed in our project, could help reduce the risk of accidental release. In addition, strains should be marked with unique barcodes to allow them to be tracked, should they accidently escape.<br />
<br />
==Ethics==<br />
<br />
===Synthetic Biology: Background===<br />
<br />
Synthetic biology is a new research field that has a big potential in coming up with solutions for our everyday problems. However, ethical issues have been raised since the start of development in this field. Here we will discuss the ethical issues that our project will bring.<br />
<br />
====Biohackers====<br />
<br />
In this field, there are always people that view synthetic biology's negative aspects with concern, like worrying about the production of pathogens to be used as weapons. Their fears are that people will hack into systems to obtain data that might be used to do this. Therefore, we have to be very careful with the products of our research so that they remain safe in our hands.<br />
<br />
====Playing God!!!====<br />
<br />
<br />
In synthetic biology, we control the lives of bacteria. We insert proteins into bacteria to make them do what we want them to do. We make them kill themselves at the end of their job because they might be released into the environment and be harmful to other living things. It shows that we do not really treat the single-celled organism as life. However, we are coming up with a novel solution for the environment by healing concrete cracks.<br />
<br />
====Biosecurity====<br />
<br />
Bacteria might be harmful to the environment and living things around. Considering the fact that they are such small organisms and could not be spotted with the naked-eye if they are released in air, we make them kill themselves with the ‘kill switch’ biobrick. This can be very inconsiderate standing in a bacteria’s point of view, but it is the novel thing to do to avoid them hurting other living organisms.<br />
<br />
===Construction: Background===<br />
<br />
Cracks form in concrete structures as soon as they set. These cracks continue to grow in size if they are not repaired due to the weight of the structure, imposed load, freeze-thaw effect and wind loading. The bigger the cracks are, the higher the rate of water seeping into the steel reinforcements, causing them to corrode and thus weakening the structure. Therefore, we would like to use Bacilla Filla to fill up these cracks before things get worse.<br />
<br />
====Environmental Disaster====<br />
<br />
Concrete is a very widely used material in construction. In fact, cement that is used to make concrete is the second most widely used substance in the world after water. Some building structures have to be demolished because the cracks formed appear to be threatening the strength of the structures. These buildings have to be rebuilt in order to reinforce its tensile strength. This is an unsustainable method of recovery because cement is a material that requires a lot of energy to be produced. In the production of cement, the rotating kiln requires the temperature to be between 1350°C-1400°C, which not only uses up a lot of energy resources, but also produces a lot of carbon dioxide. These carbon dioxide contributes to the green house effect which then leads to global warming. Therefore, our project can help to reduce renovation of buildings which in turn lowers the amount of cement that has to be produced each year.<br />
<br />
====Post-Chernobyl Disaster====<br />
<br />
The nuclear power plant combustion that occurred in Chernobyl on 26 April 1986 has allowed radioactive elements to be released into the environment, threatening the health of people living within close proximity of the plant. Therefore, a shelter, the Shelter Implementation Plan (SIP) which will be completed in 2012 is currently being built to cover up the area that where the accident occurred so as to make it environmentally friendly. However, if cracks were to form, something that inevitably occurs for building structures, those radioactive elements will still be dispersed into the environment. With the help of our project, those cracks will be able to be filled-up and the area will be safe again.<br />
<br />
<br />
<br />
[1] Wipat, A. (1990). "''Release and detection of geneticaly engineered streptomycetes in soil''". PhD thesis, Microbiology Department, John Moores University.<br />
<br />
[2] Zeigler DR, Prágai Z, Rodriguez S, Chevreux B, Muffler A, Albert T et al. (2008). "''The origins of 168, W23, and other Bacillus subtilis legacy strains''". Journal of bacteriology, 190(21), 6983-95.<br />
<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/26_July_2010Team:Newcastle/26 July 20102010-10-27T21:04:12Z<p>RachelBoyd: /* Overnight cultures of B. subtilis 168 for chromosomal DNA extraction */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
='''Preparation for cloning of the ''rocF'' BioBrick'''=<br />
<br />
==Aim==<br />
In preparation for Gibson cloning of the ''rocF'' BioBrick we started work on mini preps of plasmid DNA, and on ''B. subtilis'' 168 chromosomal DNA extraction.<br />
<br />
==Re-hydration of registry parts==<br />
[[Image:Newcastlehydration.jpg|200px|thumb|right|Re-hydration of dried parts registry DNA]]<br />
We re-hydrated using sterile distill water: <br />
#[http://partsregistry.org/Part:pSB1C3 pSB1C3] (the plasmid we will be submitting our BioBricks to the registry in) and<br />
#[http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] (the double terminator we will be using for the ''rocF'' BioBrick) from the parts distribution.<br />
<br />
==Transformation of ''E. coli''==<br />
We transformed and plated separate tubes of ''E. coli'' DH5α with:<br />
<br />
# The above two re-hydrated plasmids<br />
# [http://partsregistry.org/Part:BBa_K143062 BBa_K143062], a LacI BioBrick sent to us by Imperial College, London, UK which we will use to help characterise many of our BioBricks, including ''rocF''.<br />
# A positive control which we had already prepared during our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with ''rfp'' insert.<br />
# A negative control (no vector), to verify the antibiotic plates are working (no growth should be observed on this plate).<br />
<br />
<br />
Please refer to the transformation protocol for ''E. coli'' DH5α here: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']].<br />
<br />
==Overnight cultures of ''B. subtilis'' 168 for chromosomal DNA extraction==<br />
The ''rocF'' coding sequence is to be amplified from the ''B. subtilis'' 168 genome by PCR. Before we can do this we need to extract 168 chromosomal DNA.<br />
<br />
Today we plated up overnight cultures of ''B. subtilis'' 168 so that we can do chromosome extraction tomorrow.<br />
<br />
='''PCR of Genomic DNA'''=<br />
<br />
==Aim:==<br />
<br />
To determine whether the genomic DNA has been extracted from ''B. subtilis'' strain 3610.<br />
<br />
==Materials:==<br />
<br />
* Pipette<br />
* Microfuge<br />
* Microtubes<br />
* Distilled H2O<br />
* Nucleotide DNTP<br />
* 5x GoTaq buffer<br />
* Template DNA (B. Subtilis ATCC 6633, 1:1 and 1:2)<br />
* Forward and reverse primers<br />
<br />
==Protocol:==<br />
<br />
* For the full protocol, please refer to [[Team:Newcastle/PCR|PCR]].<br />
<br />
===Conditions in ThermoCycler:===<br />
* Melting temperature, Tm used for Anneal step is 59°C.<br />
<br />
==Results:==<br />
Gel electrophoresis will be run tomorrow to determine the results.<br />
<br />
==Conclusion:==<br />
<br />
Please refer to Lab book dated [[Team:Newcastle/27_July_2010|27th July 2010]].<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/safetyTeam:Newcastle/safety2010-10-27T21:03:36Z<p>RachelBoyd: /* Safety Issues */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
==Safety Issues==<br />
<br />
These are the safety questions for the judging form:<br />
<br />
'''1. Would any of your project ideas raise safety issues in terms of:'''<br />
<br />
*'''Researcher Safety''':<br />
<br />
We worked in the Centre for Bacterial Cell Biology (CBCB) at Newcastle University for the entire project, where there are clearly defined safety rules and regulations that all laboratory workers must follow. One of our advisors, Dr Wendy Smith, guided us through the first [[Team:Newcastle/14_June_2010|introductory]] week, before any laboratory work began. This included carrying out the following risk assessments to determine what control measures would be required.<br />
<br />
(i)'''Chemical Hazards''':<br />
At the beginning of the project written risk assessments were already available in the host laboratories for all procedures that involved potentially hazardous chemicals. These risk assessments were reviewed and the recommended control measures were strictly followed throughout. No additional chemical hazards specific to this project were identified.<br />
<br />
(ii)'''Radioisotopes and carcinogens''':<br />
None of them were used in this project.<br />
<br />
(iii)'''Biological hazards''':<br />
Throughout the project, we used the ''Escherichia coli'' strain DH5α, ''Bacillus subtilis'' strain 3610, ''Bacillus subtilis'' strain 168 and ''Bacillus sphaericus'' strain LMG 22257. Wild-type ''E. coli'' is classified as a hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP). However, ''E. coli'' strain DH5α is derived from a laboratory strain ''E. coli'' K12 strain, which is recognised as disabled and equivalent to an ACDP hazard group 1 organism (i.e. unlikely to cause disease). ''E. coli'' K12 and its derivatives such as strain DH5α are unable to colonise in humans or animals and consequently pose negligible risk to human or animal health. Wild-type ''Bacillus subtilis'' (i.e. strain 3610) is classified as an ACDP hazard group 1 organism and its derivative ''B. subtilis'' strain 168 has disabling auxotrophs mutations (e.g. conferring a requirement for tryptophan, Zeigler ''et al'', 2008) that makes it even less likely to colonise or cause harm to human or animal health. Also ''Bacillus sphaericus'' LMG 22257 is classified as an ADCP hazard group 1 organism making it unlikely to cause any harm to either human or animal health. The potential of any sequences cloned into these bacterial hosts during the project to pose additional hazards was also assessed. None of these sequences were associated with pathogenic traits or traits that might significantly enhance the survival outside the lab. Therefore, no specific safety issues, other than those associated with use of any non-pathogenic microorganism, were identified. It was concluded that containment level 1(CL1) would be sufficient to ensure researcher safety. Nonetheless, all work was carried out in strict compliance with the host laboratory's standard safety procedures, which were more stringent that those required for CL1.<br />
<br />
(iv)'''Other hazards''':<br />
The project also involved conducting some work in the Engineering structures laboratory, where we made and broke concrete 'blocls'. Appropriate safety regulations for the type of work in this laboratory were followed. When we were in the structures lab, we wore safety goggles, steel toe cap boots and gloves, which will be able to protect us from lab based accidents.<br />
<br />
* '''Public Safety''':<br />
Our project concerns repairing cracks on concrete surfaces by spraying these surfaces with spores of engineered derivatives of ''Bacillus subtilis'' 168. It is recommended that workers carrying out the spraying should wear an appropriate face mask to minimize inhaling of spores. During the spraying procedure spores could escape into the surrounding environment, but their concentration will diluted very rapidly with distance, greatly reducing potential hazards away from the immediate area of spraying. As outlined above, ''B. subtilis'' 168 is non-pathogenic and therefore very unlikely to pose a risk to public safety, particularly since any escaping spores will be unable to germinate in the absence of culture media.<br />
<br />
*'''Environmental Safety''':<br />
For reasons outlined above, the ''E. coli'' strain DH5α has very limited ability to survive outside the laboratory so taht in the very unlikely event of escape, it will be unable to survive, disseminate with and/or displace other organisms. Therefore no specific environmental hazards associated with the ''E. coli'' strain were identified.<br />
<br />
GM derivatives of ''Bacillus subtilis'' strain 168 will be released deliberately. This is a strain that has been maintained in the lab for 52 years, during which it has accumulated disabling mutations, such as tryptophan auxotrophy mentioned above, which greatly diminishes its ability to survive and disseminate outside the laboratory (Zeigler ''et al.'', 2008). It seems very unlikely that it could compete effectively and replace wild-type ''B. subtilis''. Nonetheless, the release of any GMO into the environment has been a concern since early days of genetic engineering and one of our instructors (Prof. Anil Wipat) has previously studied such issues (Wipat, 1990). To minimise concerns about environmental safety, in this project we designed the [[Team:Newcastle/Non-target-environment kill switch|Non-target-environment kill switch]] genetic part to prevent dissemination after release into the environment.<br />
<br />
'''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
We do not see any safety issues for the new Biobricks parts that we made this year. <br />
<br />
'''3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
Yes there is a biosafety group at the Centre for Bacterial Cell Biology and Institute of Cell and Molecular Biosciences, which includes the Institute Safety Officer (SSO), Biological Safety Supervisor (BSS), Genetic Modification Chairperson (GMC), Radiation Protection Supervisor (RPS), Laser Protection Officer and Lab Heads. They have reviewed the safety of the lab from time to time during the duration of the project.<br />
<br />
* If yes, what does your local biosafety group think about your project?<br />
<br />
They are aware about the whole project and they reviewed it thoroughly with the whole team. They discussed about each and every Biobrick part in detail and found no safety issues with it.<br />
<br />
'''4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A full risk assessment should be carried out before the work begins. This should consider: <br />
(i) Consequences of any identifiable hazard and<br />
(ii) The likelihood of the hazard arising.<br />
The risk can then be defined and appropriate control measures can be introduced to minimise the risk. <br />
<br />
The inclusion of a safety kill switch, such as that detailed in our project, could help reduce the risk of accidental release. In addition, strains should be marked with unique barcodes to allow them to be tracked, should they accidently escape.<br />
<br />
==Ethics==<br />
<br />
===Synthetic Biology: Background===<br />
<br />
Synthetic biology is a new research field that has a big potential in coming up with solutions for our everyday problems. However, ethical issues have been raised since the start of development in this field. Here we will discuss the ethical issues that our project will bring.<br />
<br />
====Biohackers====<br />
<br />
In this field, there are always people that view synthetic biology's negative aspects with concern, like worrying about the production of pathogens to be used as weapons. Their fears are that people will hack into systems to obtain data that might be used to do this. Therefore, we have to be very careful with the products of our research so that they remain safe in our hands.<br />
<br />
====Playing God!!!====<br />
<br />
<br />
In synthetic biology, we control the lives of bacteria. We insert proteins into bacteria to make them do what we want them to do. We make them kill themselves at the end of their job because they might be released into the environment and be harmful to other living things. It shows that we do not really treat the single-celled organism as life. However, we are coming up with a novel solution for the environment by healing concrete cracks.<br />
<br />
====Biosecurity====<br />
<br />
Bacteria might be harmful to the environment and living things around. Considering the fact that they are such small organisms and could not be spotted with the naked-eye if they are released in air, we make them kill themselves with the ‘kill switch’ biobrick. This can be very inconsiderate standing in a bacteria’s point of view, but it is the novel thing to do to avoid them hurting other living organisms.<br />
<br />
===Construction: Background===<br />
<br />
Cracks form in concrete structures as soon as they set. These cracks continue to grow in size if they are not repaired due to the weight of the structure, imposed load, freeze-thaw effect and wind loading. The bigger the cracks are, the higher the rate of water seeping into the steel reinforcements, causing them to corrode and thus weakening the structure. Therefore, we would like to use Bacilla Filla to fill up these cracks before things get worse.<br />
<br />
====Environmental Disaster====<br />
<br />
Concrete is a very widely used material in construction. In fact, cement that is used to make concrete is the second most widely used substance in the world after water. Some building structures have to be demolished because the cracks formed appear to be threatening the strength of the structures. These buildings have to be rebuilt in order to reinforce its tensile strength. This is an unsustainable method of recovery because cement is a material that requires a lot of energy to be produced. In the production of cement, the rotating kiln requires the temperature to be between 1350°C-1400°C, which not only uses up a lot of energy resources, but also produces a lot of carbon dioxide. These carbon dioxide contributes to the green house effect which then leads to global warming. Therefore, our project can help to reduce renovation of buildings which in turn lowers the amount of cement that has to be produced each year.<br />
<br />
====Post-Chernobyl Disaster====<br />
<br />
The nuclear power plant combustion that occurred in Chernobyl on 26 April 1986 has allowed radioactive elements to be released into the environment, threatening the health of people living within close proximity of the plant. Therefore, a shelter, the Shelter Implementation Plan (SIP) which will be completed in 2012 is currently being built to cover up the area that where the accident occurred so as to make it environmentally friendly. However, if cracks were to form, something that inevitably occurs for building structures, those radioactive elements will still be dispersed into the environment. With the help of our project, those cracks will be able to be filled-up and the area will be safe again.<br />
<br />
<br />
<br />
[1] Wipat, A. (1990). "''Release and detection of geneticaly engineered streptomycetes in soil''". Unpublished PhD thesis, Microbiology Department, John Moores University.<br />
<br />
[2] Zeigler DR, Prágai Z, Rodriguez S, Chevreux B, Muffler A, Albert T et al. (2008). "''The origins of 168, W23, and other Bacillus subtilis legacy strains''". Journal of bacteriology, 190(21), 6983-95.<br />
<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/safetyTeam:Newcastle/safety2010-10-27T21:02:59Z<p>RachelBoyd: /* Safety Issues */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
==Safety Issues==<br />
<br />
These are the safety questions for the judging form:<br />
<br />
'''1. Would any of your project ideas raise safety issues in terms of:'''<br />
<br />
*'''Researcher Safety''':<br />
<br />
We worked in the Centre for Bacterial Cell Biology (CBCB) at Newcastle University for the entire project, where there are clearly defined safety rules and regulations that all laboratory workers must follow. One of our advisors, Dr Wendy Smith, guided us through the first [[Team:Newcastle/14_June_2010|introductory]] week, before any laboratory work began. This included carrying out the following risk assessments to determine what control measures would be required.<br />
<br />
(i)'''Chemical Hazards''':<br />
At the beginning of the project written risk assessments were already available in the host laboratories for all procedures that involved potentially hazardous chemicals. These risk assessments were reviewed and the recommended control measures were strictly followed throughout. No additional chemical hazards specific to this project were identified.<br />
<br />
(ii)'''Radioisotopes and carcinogens''':<br />
None of them were used in this project.<br />
<br />
(iii)'''Biological hazards''':<br />
Throughout the project, we used the ''Escherichia coli'' strain DH5α, ''Bacillus subtilis'' strain 3610, ''Bacillus subtilis'' strain 168 and ''Bacillus sphaericus'' strain LMG 22257. Wild-type ''E. coli'' is classified as a hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP). However, ''E. coli'' strain DH5α is derived from a laboratory strain ''E. coli'' K12 strain, which is recognised as disabled and equivalent to an ACDP hazard group 1 organism (i.e. unlikely to cause disease). ''E. coli'' K12 and its derivatives such as strain DH5α are unable to colonise in humans or animals and consequently pose negligible risk to human or animal health. Wild-type ''Bacillus subtilis'' (i.e. strain 3610) is classified as an ACDP hazard group 1 organism and its derivative ''B. subtilis'' strain 168 has disabling auxotrophs mutations (e.g. conferring a requirement for tryptophan, Zeigler ''et al'', 2008) that makes it even less likely to colonise or cause harm to human or animal health. Also ''Bacillus sphaericus'' LMG 22257 is classified as an ADCP hazard group 1 organism making it unlikely to cause any harm to either human or animal health. The potential of any sequences cloned into these bacterial hosts during the project to pose additional hazards was also assessed. None of these sequences were associated with pathogenic traits or traits that might significantly enhance the survival outside the lab. Therefore, no specific safety issues, other than those associated with use of any non-pathogenic microorganism, were identified. It was concluded that containment level 1(CL1) would be sufficient to ensure researcher safety. Nonetheless, all work was carried out in strict compliance with the host laboratory's standard safety procedures, which were more stringent that those required for CL1.<br />
<br />
(iv)'''Other hazards''':<br />
The project also involved conducting some work in the Engineering structures laboratory, where we made and broke concrete 'blocls'. Appropriate safety regulations for the type of work in this laboratory were followed. When we were in the structures lab, we wore safety goggles, steel toe cap boots and gloves, which will be able to protect us from lab based accidents.<br />
<br />
* '''Public Safety''':<br />
Our project concerns repairing cracks on concrete surfaces by spraying these surfaces with spores of engineered derivatives of ''Bacillus subtilis'' 168. It is recommended that workers carrying out the spraying should wear an appropriate face mask to minimize inhaling of spores. During the spraying procedure spores could escape into the surrounding environment, but their concentration will diluted very rapidly with distance, greatly reducing potential hazards away from the immediate area of spraying. As outlined above, ''B. subtilis'' 168 is non-pathogenic and therefore very unlikely to pose a risk to public safety, particularly since any escaping spores will be unable to germinate in the absence of culture media.<br />
<br />
*'''Environmental Safety''':<br />
For reasons outlined above, the ''E. coli'' strain DH5α has very limited ability to survive outside the laboratory so taht in the very unlikely event of escape, it will be unable to survive, disseminate with and/or displace other organisms. Therefore no specific environmental hazards associated with the ''E. coli'' strain were identified.<br />
<br />
GM derivatives of ''Bacillus subtilis'' strain 168 will be released deliberately. This is a strain that has been maintained in the lab for 52 years, during which it has accumulated disabling mutations, such as tryptophan auxotrophy mentioned above, which greatly diminishes its ability to survive and disseminate outside the laboratory (Zeigler ''et al.'', 2008). It seems very unlikely that it could compete effectively and replace wild-type ''B. subtilis''. Nonetheless, the release of any GMO into the environment has been a concern since early days of genetic engineering and one of our instructors (Prof. Anil Wipat) has previously studied such issues (Wipat, 1990). To minimise concerns about environmental safety, in this project we designed the [[Team:Newcastle/Non-target-environment kill switch|Non-target-environment kill switch]] genetic part to prevent dissemination after release into the environment.<br />
<br />
'''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
We do not see any safety issues for the new Biobricks parts that we made this year. <br />
<br />
'''3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
Yes there is a biosafety group at the Centre for Bacterial Cell Biology and Institute of Cell and Molecular Biosciences, which includes the Institute Safety Officer (SSO), Biological Safety Supervisor (BSS), Genetic Modification Chairperson (GMC), Radiation Protection Supervisor (RPS), Laser Protection Officer and Lab Heads. They have reviewed the safety of the lab from time to time during the duration of the project.<br />
<br />
* If yes, what does your local biosafety group think about your project?<br />
<br />
They are aware about the whole project and they reviewed it thoroughly with the whole team. They discussed about each and every Biobrick part in detail and found no safety issues with it.<br />
<br />
'''4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A full risk assessment should be carried out before the work begins. This should consider: <br />
(i) Consequences of any identifiable hazard and<br />
(ii) The likelihood of the hazard arising.<br />
The risk can then be defined and appropriate control measures can be introduced to minimise the risk. <br />
<br />
The inclusion of a safety kill switch, such as that detailed above, could help reduce the risk of accidental release. In addition, strains should be marked with unique barcodes to allow them to be tracked, should they accidently escape.<br />
<br />
==Ethics==<br />
<br />
===Synthetic Biology: Background===<br />
<br />
Synthetic biology is a new research field that has a big potential in coming up with solutions for our everyday problems. However, ethical issues have been raised since the start of development in this field. Here we will discuss the ethical issues that our project will bring.<br />
<br />
====Biohackers====<br />
<br />
In this field, there are always people that view synthetic biology's negative aspects with concern, like worrying about the production of pathogens to be used as weapons. Their fears are that people will hack into systems to obtain data that might be used to do this. Therefore, we have to be very careful with the products of our research so that they remain safe in our hands.<br />
<br />
====Playing God!!!====<br />
<br />
<br />
In synthetic biology, we control the lives of bacteria. We insert proteins into bacteria to make them do what we want them to do. We make them kill themselves at the end of their job because they might be released into the environment and be harmful to other living things. It shows that we do not really treat the single-celled organism as life. However, we are coming up with a novel solution for the environment by healing concrete cracks.<br />
<br />
====Biosecurity====<br />
<br />
Bacteria might be harmful to the environment and living things around. Considering the fact that they are such small organisms and could not be spotted with the naked-eye if they are released in air, we make them kill themselves with the ‘kill switch’ biobrick. This can be very inconsiderate standing in a bacteria’s point of view, but it is the novel thing to do to avoid them hurting other living organisms.<br />
<br />
===Construction: Background===<br />
<br />
Cracks form in concrete structures as soon as they set. These cracks continue to grow in size if they are not repaired due to the weight of the structure, imposed load, freeze-thaw effect and wind loading. The bigger the cracks are, the higher the rate of water seeping into the steel reinforcements, causing them to corrode and thus weakening the structure. Therefore, we would like to use Bacilla Filla to fill up these cracks before things get worse.<br />
<br />
====Environmental Disaster====<br />
<br />
Concrete is a very widely used material in construction. In fact, cement that is used to make concrete is the second most widely used substance in the world after water. Some building structures have to be demolished because the cracks formed appear to be threatening the strength of the structures. These buildings have to be rebuilt in order to reinforce its tensile strength. This is an unsustainable method of recovery because cement is a material that requires a lot of energy to be produced. In the production of cement, the rotating kiln requires the temperature to be between 1350°C-1400°C, which not only uses up a lot of energy resources, but also produces a lot of carbon dioxide. These carbon dioxide contributes to the green house effect which then leads to global warming. Therefore, our project can help to reduce renovation of buildings which in turn lowers the amount of cement that has to be produced each year.<br />
<br />
====Post-Chernobyl Disaster====<br />
<br />
The nuclear power plant combustion that occurred in Chernobyl on 26 April 1986 has allowed radioactive elements to be released into the environment, threatening the health of people living within close proximity of the plant. Therefore, a shelter, the Shelter Implementation Plan (SIP) which will be completed in 2012 is currently being built to cover up the area that where the accident occurred so as to make it environmentally friendly. However, if cracks were to form, something that inevitably occurs for building structures, those radioactive elements will still be dispersed into the environment. With the help of our project, those cracks will be able to be filled-up and the area will be safe again.<br />
<br />
<br />
<br />
[1] Wipat, A. (1990). "''Release and detection of geneticaly engineered streptomycetes in soil''". Unpublished PhD thesis, Microbiology Department, John Moores University.<br />
<br />
[2] Zeigler DR, Prágai Z, Rodriguez S, Chevreux B, Muffler A, Albert T et al. (2008). "''The origins of 168, W23, and other Bacillus subtilis legacy strains''". Journal of bacteriology, 190(21), 6983-95.<br />
<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/26_July_2010Team:Newcastle/26 July 20102010-10-27T21:01:35Z<p>RachelBoyd: /* PCR of Genomic DNA */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
='''Preparation for cloning of the ''rocF'' BioBrick'''=<br />
<br />
==Aim==<br />
In preparation for Gibson cloning of the ''rocF'' BioBrick we started work on mini preps of plasmid DNA, and on ''B. subtilis'' 168 chromosomal DNA extraction.<br />
<br />
==Re-hydration of registry parts==<br />
[[Image:Newcastlehydration.jpg|200px|thumb|right|Re-hydration of dried parts registry DNA]]<br />
We re-hydrated using sterile distill water: <br />
#[http://partsregistry.org/Part:pSB1C3 pSB1C3] (the plasmid we will be submitting our BioBricks to the registry in) and<br />
#[http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] (the double terminator we will be using for the ''rocF'' BioBrick) from the parts distribution.<br />
<br />
==Transformation of ''E. coli''==<br />
We transformed and plated separate tubes of ''E. coli'' DH5α with:<br />
<br />
# The above two re-hydrated plasmids<br />
# [http://partsregistry.org/Part:BBa_K143062 BBa_K143062], a LacI BioBrick sent to us by Imperial College, London, UK which we will use to help characterise many of our BioBricks, including ''rocF''.<br />
# A positive control which we had already prepared during our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with ''rfp'' insert.<br />
# A negative control (no vector), to verify the antibiotic plates are working (no growth should be observed on this plate).<br />
<br />
<br />
Please refer to the transformation protocol for ''E. coli'' DH5α here: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']].<br />
<br />
==Overnight cultures of ''B. subtilis'' 168 for chromosomal DNA extraction==<br />
The ''rocF'' coding sequence is to be taken from the ''B. subtilis'' 168 genome by PCR. Before we can do this we need to extract 168 chromosomal DNA.<br />
<br />
Today we plated up overnight cultures of ''B. subtilis'' 168 so that we can do chromosome extraction tomorrow.<br />
<br />
='''PCR of Genomic DNA'''=<br />
<br />
==Aim:==<br />
<br />
To determine whether the genomic DNA has been extracted from ''B. subtilis'' strain 3610.<br />
<br />
==Materials:==<br />
<br />
* Pipette<br />
* Microfuge<br />
* Microtubes<br />
* Distilled H2O<br />
* Nucleotide DNTP<br />
* 5x GoTaq buffer<br />
* Template DNA (B. Subtilis ATCC 6633, 1:1 and 1:2)<br />
* Forward and reverse primers<br />
<br />
==Protocol:==<br />
<br />
* For the full protocol, please refer to [[Team:Newcastle/PCR|PCR]].<br />
<br />
===Conditions in ThermoCycler:===<br />
* Melting temperature, Tm used for Anneal step is 59°C.<br />
<br />
==Results:==<br />
Gel electrophoresis will be run tomorrow to determine the results.<br />
<br />
==Conclusion:==<br />
<br />
Please refer to Lab book dated [[Team:Newcastle/27_July_2010|27th July 2010]].<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/26_July_2010Team:Newcastle/26 July 20102010-10-27T21:01:21Z<p>RachelBoyd: /* Transformation of E. coli */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
='''Preparation for cloning of the ''rocF'' BioBrick'''=<br />
<br />
==Aim==<br />
In preparation for Gibson cloning of the ''rocF'' BioBrick we started work on mini preps of plasmid DNA, and on ''B. subtilis'' 168 chromosomal DNA extraction.<br />
<br />
==Re-hydration of registry parts==<br />
[[Image:Newcastlehydration.jpg|200px|thumb|right|Re-hydration of dried parts registry DNA]]<br />
We re-hydrated using sterile distill water: <br />
#[http://partsregistry.org/Part:pSB1C3 pSB1C3] (the plasmid we will be submitting our BioBricks to the registry in) and<br />
#[http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] (the double terminator we will be using for the ''rocF'' BioBrick) from the parts distribution.<br />
<br />
==Transformation of ''E. coli''==<br />
We transformed and plated separate tubes of ''E. coli'' DH5α with:<br />
<br />
# The above two re-hydrated plasmids<br />
# [http://partsregistry.org/Part:BBa_K143062 BBa_K143062], a LacI BioBrick sent to us by Imperial College, London, UK which we will use to help characterise many of our BioBricks, including ''rocF''.<br />
# A positive control which we had already prepared during our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with ''rfp'' insert.<br />
# A negative control (no vector), to verify the antibiotic plates are working (no growth should be observed on this plate).<br />
<br />
<br />
Please refer to the transformation protocol for ''E. coli'' DH5α here: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']].<br />
<br />
==Overnight cultures of ''B. subtilis'' 168 for chromosomal DNA extraction==<br />
The ''rocF'' coding sequence is to be taken from the ''B. subtilis'' 168 genome by PCR. Before we can do this we need to extract 168 chromosomal DNA.<br />
<br />
Today we plated up overnight cultures of ''B. subtilis'' 168 so that we can do chromosome extraction tomorrow.<br />
<br />
='''PCR of Genomic DNA'''=<br />
<br />
==Aim:==<br />
<br />
To determine whether the genomic DNA has been extracted from ''B. Subtilis'' Strain 3610.<br />
<br />
==Materials:==<br />
<br />
* Pipette<br />
* Microfuge<br />
* Microtubes<br />
* Distilled H2O<br />
* Nucleotide DNTP<br />
* 5x GoTaq buffer<br />
* Template DNA (B. Subtilis ATCC 6633, 1:1 and 1:2)<br />
* Forward and reverse primers<br />
<br />
==Protocol:==<br />
<br />
* For the full protocol, please refer to [[Team:Newcastle/PCR|PCR]].<br />
<br />
===Conditions in ThermoCycler:===<br />
* Melting temperature, Tm used for Anneal step is 59°C.<br />
<br />
==Results:==<br />
Gel electrophoresis will be run tomorrow to determine the results.<br />
<br />
==Conclusion:==<br />
<br />
Please refer to Lab book dated [[Team:Newcastle/27_July_2010|27th July 2010]].<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/23_July_2010Team:Newcastle/23 July 20102010-10-27T21:00:01Z<p>RachelBoyd: /* Conclusion */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
=Arginine experiment=<br />
<br />
==Aim==<br />
The aim of this experiment is to determine whether ''B. subtilis'' 168 is able to take up external arginine.<br />
<br />
==Procedure==<br />
* Please refer to [[Team:Newcastle/Arginine test| Arginine test]]<br />
<br />
==Results==<br />
Arginine is an amino acid that is positively charged. Therefore if ''B. subtilis'' 168 is able to take up arginine, it will cause a pH change in the media. This would result in an increase in pH.<br />
<br />
|[[Image:Newcastle_arginine_test_230710.png|500px ]]<br />
<br />
'''Figure 1:''' Arginine test using pH indicator stick to measure pH changes of the media. <br />
<br />
{|border=1<br />
|-<br />
!Time (in minutes)<br />
!Control (1)<br />
!Control (2)<br />
!Control (3)<br />
!Test (1)<br />
!Test (2)<br />
|-<br />
|0<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|-<br />
|30<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|-<br />
|60<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|-<br />
|90<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|-<br />
|120<br />
|pH 7 <br />
|pH 7<br />
|pH 7<br />
|pH 8<br />
|pH 8<br />
|-<br />
|150<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 8<br />
|pH 8<br />
|-<br />
|180<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 9<br />
|pH 9<br />
|-<br />
|210<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 9<br />
|pH 9<br />
|-<br />
|240<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 9<br />
|pH 9<br />
|-<br />
|270<br />
|pH 7<br />
|pH 7<br />
|pH 8<br />
|pH 9<br />
|pH 9<br />
|-<br />
|300<br />
|pH 7<br />
|pH 7<br />
|pH 8<br />
|pH 9<br />
|pH 9<br />
|}<br />
<br />
'''Table 1:''' Arginine test using pH indicator stick to measure pH changes of the media. Table represents the change in pH over the time span of 300 minutes i.e. 5 hours. <br />
<br />
Here,<br />
#Control (1) - LB media <br />
#Control (2) - LB media with 10 mM of arginine<br />
#Control (3) - LB media plus ''B. subtilis'' 168<br />
#Test (1) - LB media with 10 mM of arginine plus ''B. subtilis'' 168<br />
#Test (2) - LB media with 10 mM of arginine plus ''B. subtilis'' 168<br />
<br />
==Conclusion==<br />
''B. subtilis'' 168 breaks down arginine to urea by producing arginase. The urea is then further broken down to ammonia and carbonate ions by urease. This will lead to an increase in pH. Both the test 1 and test 2, which contain ''B. subtilis'' 168 and 10 mM of arginine show an increase in pH from 7 to 9. While the control 1 and control 2, which contain no ''B. subtilis'' 168 remains at pH 7. The control 3 which contain ''B. subtilis'' 168 but without addition of arginine show an increase in pH from 7 to 8. This could be due to unidentified products that are secreted by the bacteria.<br />
<br />
Therefore this experiment indicates that ''B. subtilis'' 168 is able to utilise arginine, and thus increase the overall pH of the media. The production of carbonate ion will lead to its binding with calcium ions provided in the media leading to formation of calcium carbonate.<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/23_July_2010Team:Newcastle/23 July 20102010-10-27T20:59:24Z<p>RachelBoyd: /* Conclusion */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
=Arginine experiment=<br />
<br />
==Aim==<br />
The aim of this experiment is to determine whether ''B. subtilis'' 168 is able to take up external arginine.<br />
<br />
==Procedure==<br />
* Please refer to [[Team:Newcastle/Arginine test| Arginine test]]<br />
<br />
==Results==<br />
Arginine is an amino acid that is positively charged. Therefore if ''B. subtilis'' 168 is able to take up arginine, it will cause a pH change in the media. This would result in an increase in pH.<br />
<br />
|[[Image:Newcastle_arginine_test_230710.png|500px ]]<br />
<br />
'''Figure 1:''' Arginine test using pH indicator stick to measure pH changes of the media. <br />
<br />
{|border=1<br />
|-<br />
!Time (in minutes)<br />
!Control (1)<br />
!Control (2)<br />
!Control (3)<br />
!Test (1)<br />
!Test (2)<br />
|-<br />
|0<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|-<br />
|30<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|-<br />
|60<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|-<br />
|90<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|-<br />
|120<br />
|pH 7 <br />
|pH 7<br />
|pH 7<br />
|pH 8<br />
|pH 8<br />
|-<br />
|150<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 8<br />
|pH 8<br />
|-<br />
|180<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 9<br />
|pH 9<br />
|-<br />
|210<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 9<br />
|pH 9<br />
|-<br />
|240<br />
|pH 7<br />
|pH 7<br />
|pH 7<br />
|pH 9<br />
|pH 9<br />
|-<br />
|270<br />
|pH 7<br />
|pH 7<br />
|pH 8<br />
|pH 9<br />
|pH 9<br />
|-<br />
|300<br />
|pH 7<br />
|pH 7<br />
|pH 8<br />
|pH 9<br />
|pH 9<br />
|}<br />
<br />
'''Table 1:''' Arginine test using pH indicator stick to measure pH changes of the media. Table represents the change in pH over the time span of 300 minutes i.e. 5 hours. <br />
<br />
Here,<br />
#Control (1) - LB media <br />
#Control (2) - LB media with 10 mM of arginine<br />
#Control (3) - LB media plus ''B. subtilis'' 168<br />
#Test (1) - LB media with 10 mM of arginine plus ''B. subtilis'' 168<br />
#Test (2) - LB media with 10 mM of arginine plus ''B. subtilis'' 168<br />
<br />
==Conclusion==<br />
''B. subtilis'' 168 breaks down arginine to urea by producing arginase. The urea is then further broken down to ammonia and carbonate ions by urease. This will lead to an increase in pH. Both the test 1 and test 2, which contain ''B. subtilis'' 168 and 10 mM of arginine show an increase in pH from 7 to 9. While the control 1 and control 2, which contain no ''B. subtilis'' 168 remains at pH 7. The control 3 which contain ''B. subtilis'' 168 but without addition of arginine show an increase in pH from 7 to 8. This could be due to unidentified products that are secreted by the bacteria.<br />
<br />
Therefore this experiment have shown that ''B. subtilis'' 168 is able to utilise arginine, and thus increase the overall pH of the media. The production of carbonate ion will lead to its binding with calcium ions provided in the media leading to formation of calcium carbonate.<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/Arginine_testTeam:Newcastle/Arginine test2010-10-27T20:58:51Z<p>RachelBoyd: /* Materials Required */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
=Arginine Test=<br />
<br />
==Materials Required==<br />
* Plate containing ''Bacillus subtilis'' 168 colonies.<br />
* Flame (streaking) loop<br />
* LB media consisting of arginine<br />
* Auto pipette<br />
* Bursen Burner<br />
* Universal Tube<br />
* pH indicator paper<br />
<br />
==Procedures==<br />
* Perform the experiment using aseptic technique.<br />
* Transfer ''B. subtilis'' 168 colonies into universal tubes containing 5 ml of LB media and allowed to grow overnight at 37° C.<br />
* Transfer 1 ml of the overnight culture to another universal tube containing 4 ml of the following media:<br />
#Control (1) - LB media <br />
#Control (2) - LB media with 10 mM of arginine<br />
#Control (3) - LB media plus ''B. subtilis'' 168<br />
#Test (1) - LB media with 10 mM of arginine plus ''B. subtilis'' 168<br />
#Test (2) - LB media with 10 mM of arginine plus ''B. subtilis'' 168<br />
* Incubate the culture at 37° C with shaking.<br />
* Record the pH at every 30 min interval.Use 20 ul of the culture and measure the pH using the pH indicator paper.<br />
<br />
<br />
'''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]''' <br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/Arginine_testTeam:Newcastle/Arginine test2010-10-27T20:58:29Z<p>RachelBoyd: /* Procedures */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
=Arginine Test=<br />
<br />
==Materials Required==<br />
* Plate containing ''Bacillus subtilis'' 168 colonies.<br />
* Flame (streaking) loop<br />
* LB media consisting of arginine<br />
* Auto pipette<br />
* Bursen Burner<br />
* Universal Tube<br />
* pH measuring stick<br />
<br />
==Procedures==<br />
* Perform the experiment using aseptic technique.<br />
* Transfer ''B. subtilis'' 168 colonies into universal tubes containing 5 ml of LB media and allowed to grow overnight at 37° C.<br />
* Transfer 1 ml of the overnight culture to another universal tube containing 4 ml of the following media:<br />
#Control (1) - LB media <br />
#Control (2) - LB media with 10 mM of arginine<br />
#Control (3) - LB media plus ''B. subtilis'' 168<br />
#Test (1) - LB media with 10 mM of arginine plus ''B. subtilis'' 168<br />
#Test (2) - LB media with 10 mM of arginine plus ''B. subtilis'' 168<br />
* Incubate the culture at 37° C with shaking.<br />
* Record the pH at every 30 min interval.Use 20 ul of the culture and measure the pH using the pH indicator paper.<br />
<br />
<br />
'''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]''' <br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/Arginine_testTeam:Newcastle/Arginine test2010-10-27T20:56:38Z<p>RachelBoyd: /* Arginine Test */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
=Arginine Test=<br />
<br />
==Materials Required==<br />
* Plate containing ''Bacillus subtilis'' 168 colonies.<br />
* Flame (streaking) loop<br />
* LB media consisting of arginine<br />
* Auto pipette<br />
* Bursen Burner<br />
* Universal Tube<br />
* pH measuring stick<br />
<br />
==Procedures==<br />
* Perform the experiment using aseptic technique.<br />
* Transfer ''B. subtilis'' 168 colonies into universal tubes containing 5 ml of LB media and allowed to grow overnight at 37° C.<br />
* Transfer 1 ml of the overnight culture to another universal tube containing 4 ml of the following media:<br />
#Control (1) - LB media <br />
#Control (2) - LB media with 10 mM of arginine<br />
#Control (3) - LB media plus ''B. subtilis'' 168<br />
#Test (1) - LB media with 10 mM of arginine plus ''B. subtilis'' 168<br />
#Test (2) - LB media with 10 mM of arginine plus ''B. subtilis'' 168<br />
* Incubate the culture at 37° C with shaking.<br />
* Record the pH at every 30 min interval.Use 20 ul of the culture and measure the pH using the pH indicator strip.<br />
<br />
<br />
'''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]''' <br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/Filamentous_CellsTeam:Newcastle/Filamentous Cells2010-10-27T20:46:01Z<p>RachelBoyd: /* Cloning strategy */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
<br />
=Filamentous cell formation by overexpression of ''yneA''=<br />
<br />
''Bacillus subtilis'' cell division is dependent on FtsZ. FtsZ forms a 30 subunit ring at the midpoint of the cell and contracts.<br />
<br />
YneA indirectly stops the formation of the FtsZ ring. In nature, ''yneA'' is expressed during SOS response, allowing the cell to repair DNA damage before continuing with the division cycle.<br />
<br />
It is hypothesized that YneA acts through unknown transmembrane proteins to inhibit FtsZ ring formation; we call these unknown components "Blackbox proteins".<br />
<br />
By expressing YneA and therefore inhibiting FtsZ ring formation, cells will grow filamentous.<br />
<br />
<br />
==Part==<br />
<br />
[[Image:yneA_brick2.png]]<br />
<br />
Our ''IPTG-inducible filamentous cell formation part'' puts ''yneA'' under the control of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302003 strongly LacI-repressible promoter that we designed, hyperspankoid]. In the presence of LacI, induction with IPTG will result in a filamentous cell phenotype. <br />
<br />
The part has no terminator, allowing for transcriptional fusion with ''gfp'' and visualisation under the microscope.<br />
<br />
This is part [http://partsregistry.org/Part:BBa_K302012 BBa_K302012] on the [http://partsregistry.org parts registry]. <br />
<br />
<br />
<br />
[[Image:biochemical_pathway_filamentous.png|700px]]<br />
<br />
==Computational model==<br />
<br />
{|<br />
|<br />
|-<br />
|[[Image:Newcastle_ModelFilamentous.png|600px]]<br />
|We wrote a computational model of our filamentous cell system in SBML and simulated it in COPASI to help us verify our part's behaviour before we built it. The graph on the left shows that FtsZ ring formation is low when ''yneA'' is highly expressed.<br />
|}<br />
<br />
{|<br />
|<br />
|-<br />
|[[Image:Newcastle CellDesigner Filamentous.png|600px]]<br />
|Visualisation of the model's biochemical network in CellDesigner.<br />
|}<br />
<br />
Downloads:<br />
*[[Media:Newcastle_filamentous.mod.txt|SBML-shorthand]]<br />
*[[Media:Newcastle_filamentous.xml.txt|SBML]]<br />
<br />
<br />
==Cloning strategy==<br />
<br />
[[Media:yneA cloning strategy.pdf|yneA cloning strategy]]<br />
<br />
==Characterisation==<br />
<br />
We integrated our part into the ''Bacillus subtilis'' 168 chromosome at ''amyE'' (using the integration vector pGFP-rrnB) and selected for integration by testing for the ability to hydrolyse starch. Homologous recombination at ''amyE'' destroys endogenous expression of amylase. Colonies that are not able to break down starch on agar plate do not have a white halo when exposed to iodine.<br />
<br />
The part was co-transcribed with ''gfp'' fluorescent marker by transcriptional fusion after the ''yneA'' coding sequence.<br />
<br />
We characterised the part first without, and then with, LacI repression (using the integration vector pMutin4 to integrate ''lacI'' into the ''Bacillus subtilis'' 168 chromosome). When testing the part under LacI repression cells were induced with IPTG for two hours.<br />
<br />
<br />
<br />
{|<br />
|[[Image:Newcastle_filamentous_control_pc_expt1.jpg|thumb|Normal ''Bacillus subtilis ''168|280px|centre]]<br />
|[[Image:Newcastle_filamentous_pc_expt1.jpg|thumb|Filamentous cells|280px|centre]]<br />
|[[Image:Newcastle_filamentous_gfp_expt1.jpg|thumb|Filamentous cells showing GFP signal |280px|centre]]<br />
|} <br />
<br />
<center><br />
{|<br />
|[[Image:Newcastle_filamentous_pc_expt2.jpg|thumb|Filamentous cells (integrated at ''amyE'')|300px|centre]]<br />
|<br />
|[[Image:Newcastle_filamentous_gfp_expt2.jpg|thumb|Filamentous cells showing GFP signal(integrated at ''amyE'') | 300px|centre]]<br />
|}<br />
</center><br />
<br />
===Graphs===<br />
<br />
====Table1:====<br />
{| border="1"<br />
|-<br />
!Stats:<br />
!168<br />
!''yneA''<br />
!pMutin4 0μM IPTG<br />
!pMutin4 1μM IPTG<br />
|-<br />
|Average:<br />
|1.34μm<br />
|3.53μm<br />
|1.74μm<br />
|3.19μm<br />
|-<br />
|Max:<br />
|2.30μm<br />
|6.00μm<br />
|3.62μm<br />
|9.77μm<br />
<br />
|-<br />
|Min:<br />
|0.55μm<br />
|1.31μm<br />
|0.88μm<br />
|1.14μm<br />
|-<br />
|Median:<br />
|1.33μm<br />
|3.27μm<br />
|1.62μm<br />
|2.66μm<br />
|-<br />
|Standard Deviation:<br />
|0.32μm<br />
|1.01μm<br />
|0.80μm<br />
|1.56μm<br />
|}<br />
<br />
<br />
====Figure1:====<br />
{|<br />
|-<br />
|Distribution of cell lengths is not normal, so the mean is misleading; we are reporting the median instead.<br />
|-<br />
|[[Image:Teamnewcastle_yneA168.png|600px]]<br />
|-<br />
|Figure1: shows statistics for populations of cells<br />
*overexpression of the ''yneA'' construct (Δ''amyE'':pSpac(hy)-oid::''yneA''(cells with YneA construct but no inhibitory regulation) ) leads to a longer cell length compared with our control ''Bacillus subtilis 168''.<br />
*pMT4_0.0: YneA construct in pMutin4 vector with inhibition and no IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4) <br />
*pMT4_1.0: YneA construct in pMutin4 vector with inhibition and 1.0 μM IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4) <br />
|-<br />
|with inhibition cell lengths are comparable to ''Bacillus subtilis 168'' at 0μM IPTG and longer with IPTG induction.<br />
|}<br />
<br />
<br />
====Figure2:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_yneA1.jpg|300px]][[Image:Teamnewcastle_yneA.jpg|300px]] <br />
|-<br />
|'''Figure2''': ''Bacillus subtilis 168'' cells (left),''Bacillus subtilis'' expressing ''yneA''(centre) and ''Bacillus subtilis'' overexpressing ''yneA''(right)<br />
|-<br />
|The images we have taken this data from had very different numbers of cells, so the cells counts are misleading therefore we are reporting the proportions of cells at a given length. <br />
|}<br />
<br />
<br />
====Figure 3:====<br />
{|<br />
|-<br />
|[[Image:newcastle_no induction.jpg|600px]]<br />
|-<br />
|Figure 3 shows the percentage of cells at different lengths (μm) uninduced<br />
|}<br />
<br />
<br />
====Figure 4:====<br />
{|<br />
|-<br />
|Figure 4:''Bacillus subtilis'' 168 cells (left) and non-induced cells (right)<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_noindBS.jpg|300px]] <br />
|-<br />
|}<br />
<br />
<br />
=====Figure 5:=====<br />
{|<br />
|-<br />
|[[Image:newcastle_0.2 induction.jpg|600px]]<br />
|-<br />
|Figure 5: shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG <br />
|}<br />
<br />
====Figure 6:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_0.2indBS.jpg|300px]]<br />
|-<br />
|Figure 6: ''Bacillus subtilis 168'' cells (left) and cells induced at 0.2mM IPTG (right)<br />
|}<br />
<br />
<br />
====Figure 7:====<br />
{|<br />
|-<br />
|[[Image:newcastle_1IPTG.jpg|600px]]<br />
|-<br />
|Figure 7: shows the percentage of cells at different lengths (μm) induced at 1mM IPTG <br />
|}<br />
<br />
<br />
====Figure 8:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_1indBS2.jpg|300px]] <br />
|-<br />
|Figure 8: ''Bacillus subtilis'' 168 cells (left) and cells induced at 1mM IPTG(right)<br />
|}<br />
<br />
==Research==<br />
<br />
[[Team:Newcastle/Initial_filamentous|Initial Research]]<br />
<br />
==References==<br />
<br />
Kawai, Y., Moriya, S., & Ogasawara, N. (2003). ''"Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis"''. Molecular microbiology, 47(4), 1113-22.<br />
<br />
Mo, A.H. & Burkholder, W.F., (2010). ''"YneA , an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis , Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity"'' ᰔ †. Society, 192(12), 3159-3173.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/Filamentous_CellsTeam:Newcastle/Filamentous Cells2010-10-27T20:45:18Z<p>RachelBoyd: /* Cloning strategy */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
<br />
=Filamentous cell formation by overexpression of ''yneA''=<br />
<br />
''Bacillus subtilis'' cell division is dependent on FtsZ. FtsZ forms a 30 subunit ring at the midpoint of the cell and contracts.<br />
<br />
YneA indirectly stops the formation of the FtsZ ring. In nature, ''yneA'' is expressed during SOS response, allowing the cell to repair DNA damage before continuing with the division cycle.<br />
<br />
It is hypothesized that YneA acts through unknown transmembrane proteins to inhibit FtsZ ring formation; we call these unknown components "Blackbox proteins".<br />
<br />
By expressing YneA and therefore inhibiting FtsZ ring formation, cells will grow filamentous.<br />
<br />
<br />
==Part==<br />
<br />
[[Image:yneA_brick2.png]]<br />
<br />
Our ''IPTG-inducible filamentous cell formation part'' puts ''yneA'' under the control of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302003 strongly LacI-repressible promoter that we designed, hyperspankoid]. In the presence of LacI, induction with IPTG will result in a filamentous cell phenotype. <br />
<br />
The part has no terminator, allowing for transcriptional fusion with ''gfp'' and visualisation under the microscope.<br />
<br />
This is part [http://partsregistry.org/Part:BBa_K302012 BBa_K302012] on the [http://partsregistry.org parts registry]. <br />
<br />
<br />
<br />
[[Image:biochemical_pathway_filamentous.png|700px]]<br />
<br />
==Computational model==<br />
<br />
{|<br />
|<br />
|-<br />
|[[Image:Newcastle_ModelFilamentous.png|600px]]<br />
|We wrote a computational model of our filamentous cell system in SBML and simulated it in COPASI to help us verify our part's behaviour before we built it. The graph on the left shows that FtsZ ring formation is low when ''yneA'' is highly expressed.<br />
|}<br />
<br />
{|<br />
|<br />
|-<br />
|[[Image:Newcastle CellDesigner Filamentous.png|600px]]<br />
|Visualisation of the model's biochemical network in CellDesigner.<br />
|}<br />
<br />
Downloads:<br />
*[[Media:Newcastle_filamentous.mod.txt|SBML-shorthand]]<br />
*[[Media:Newcastle_filamentous.xml.txt|SBML]]<br />
<br />
<br />
==Cloning strategy==<br />
<br />
[[Media:''yneA'' cloning strategy.pdf|yneA cloning strategy]]<br />
<br />
==Characterisation==<br />
<br />
We integrated our part into the ''Bacillus subtilis'' 168 chromosome at ''amyE'' (using the integration vector pGFP-rrnB) and selected for integration by testing for the ability to hydrolyse starch. Homologous recombination at ''amyE'' destroys endogenous expression of amylase. Colonies that are not able to break down starch on agar plate do not have a white halo when exposed to iodine.<br />
<br />
The part was co-transcribed with ''gfp'' fluorescent marker by transcriptional fusion after the ''yneA'' coding sequence.<br />
<br />
We characterised the part first without, and then with, LacI repression (using the integration vector pMutin4 to integrate ''lacI'' into the ''Bacillus subtilis'' 168 chromosome). When testing the part under LacI repression cells were induced with IPTG for two hours.<br />
<br />
<br />
<br />
{|<br />
|[[Image:Newcastle_filamentous_control_pc_expt1.jpg|thumb|Normal ''Bacillus subtilis ''168|280px|centre]]<br />
|[[Image:Newcastle_filamentous_pc_expt1.jpg|thumb|Filamentous cells|280px|centre]]<br />
|[[Image:Newcastle_filamentous_gfp_expt1.jpg|thumb|Filamentous cells showing GFP signal |280px|centre]]<br />
|} <br />
<br />
<center><br />
{|<br />
|[[Image:Newcastle_filamentous_pc_expt2.jpg|thumb|Filamentous cells (integrated at ''amyE'')|300px|centre]]<br />
|<br />
|[[Image:Newcastle_filamentous_gfp_expt2.jpg|thumb|Filamentous cells showing GFP signal(integrated at ''amyE'') | 300px|centre]]<br />
|}<br />
</center><br />
<br />
===Graphs===<br />
<br />
====Table1:====<br />
{| border="1"<br />
|-<br />
!Stats:<br />
!168<br />
!''yneA''<br />
!pMutin4 0μM IPTG<br />
!pMutin4 1μM IPTG<br />
|-<br />
|Average:<br />
|1.34μm<br />
|3.53μm<br />
|1.74μm<br />
|3.19μm<br />
|-<br />
|Max:<br />
|2.30μm<br />
|6.00μm<br />
|3.62μm<br />
|9.77μm<br />
<br />
|-<br />
|Min:<br />
|0.55μm<br />
|1.31μm<br />
|0.88μm<br />
|1.14μm<br />
|-<br />
|Median:<br />
|1.33μm<br />
|3.27μm<br />
|1.62μm<br />
|2.66μm<br />
|-<br />
|Standard Deviation:<br />
|0.32μm<br />
|1.01μm<br />
|0.80μm<br />
|1.56μm<br />
|}<br />
<br />
<br />
====Figure1:====<br />
{|<br />
|-<br />
|Distribution of cell lengths is not normal, so the mean is misleading; we are reporting the median instead.<br />
|-<br />
|[[Image:Teamnewcastle_yneA168.png|600px]]<br />
|-<br />
|Figure1: shows statistics for populations of cells<br />
*overexpression of the ''yneA'' construct (Δ''amyE'':pSpac(hy)-oid::''yneA''(cells with YneA construct but no inhibitory regulation) ) leads to a longer cell length compared with our control ''Bacillus subtilis 168''.<br />
*pMT4_0.0: YneA construct in pMutin4 vector with inhibition and no IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4) <br />
*pMT4_1.0: YneA construct in pMutin4 vector with inhibition and 1.0 μM IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4) <br />
|-<br />
|with inhibition cell lengths are comparable to ''Bacillus subtilis 168'' at 0μM IPTG and longer with IPTG induction.<br />
|}<br />
<br />
<br />
====Figure2:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_yneA1.jpg|300px]][[Image:Teamnewcastle_yneA.jpg|300px]] <br />
|-<br />
|'''Figure2''': ''Bacillus subtilis 168'' cells (left),''Bacillus subtilis'' expressing ''yneA''(centre) and ''Bacillus subtilis'' overexpressing ''yneA''(right)<br />
|-<br />
|The images we have taken this data from had very different numbers of cells, so the cells counts are misleading therefore we are reporting the proportions of cells at a given length. <br />
|}<br />
<br />
<br />
====Figure 3:====<br />
{|<br />
|-<br />
|[[Image:newcastle_no induction.jpg|600px]]<br />
|-<br />
|Figure 3 shows the percentage of cells at different lengths (μm) uninduced<br />
|}<br />
<br />
<br />
====Figure 4:====<br />
{|<br />
|-<br />
|Figure 4:''Bacillus subtilis'' 168 cells (left) and non-induced cells (right)<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_noindBS.jpg|300px]] <br />
|-<br />
|}<br />
<br />
<br />
=====Figure 5:=====<br />
{|<br />
|-<br />
|[[Image:newcastle_0.2 induction.jpg|600px]]<br />
|-<br />
|Figure 5: shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG <br />
|}<br />
<br />
====Figure 6:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_0.2indBS.jpg|300px]]<br />
|-<br />
|Figure 6: ''Bacillus subtilis 168'' cells (left) and cells induced at 0.2mM IPTG (right)<br />
|}<br />
<br />
<br />
====Figure 7:====<br />
{|<br />
|-<br />
|[[Image:newcastle_1IPTG.jpg|600px]]<br />
|-<br />
|Figure 7: shows the percentage of cells at different lengths (μm) induced at 1mM IPTG <br />
|}<br />
<br />
<br />
====Figure 8:====<br />
{|<br />
|-<br />
|[[Image:Teamnewcastle_yneA168BS.jpg|300px]][[Image:Teamnewcastle_1indBS2.jpg|300px]] <br />
|-<br />
|Figure 8: ''Bacillus subtilis'' 168 cells (left) and cells induced at 1mM IPTG(right)<br />
|}<br />
<br />
==Research==<br />
<br />
[[Team:Newcastle/Initial_filamentous|Initial Research]]<br />
<br />
==References==<br />
<br />
Kawai, Y., Moriya, S., & Ogasawara, N. (2003). ''"Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis"''. Molecular microbiology, 47(4), 1113-22.<br />
<br />
Mo, A.H. & Burkholder, W.F., (2010). ''"YneA , an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis , Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity"'' ᰔ †. Society, 192(12), 3159-3173.<br />
<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/UreaseTeam:Newcastle/Urease2010-10-27T20:39:02Z<p>RachelBoyd: /* Flux balance analysis */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
=Calcium carbonate precipitation via urease expression=<br />
<br />
''Bacillus subtilis'' produce urease, which catalyses the hydrolysis of urea into ammonium and carbonate (CO<sub>3</sub><sup>2-</sup>). Since the cell walls of the bacteria are negatively charged, they draw cations from the environment, including Ca<sup>2+</sup>, to deposit on their cell surface. The Ca<sup>2+</sup> ions subsequently react with the CO<sub>3</sub><sup>2-</sup> ions, leading to the precipitation of CaCO<sub>3</sub> at the cell surface.<br />
<br />
In order for ''B. subtilis'' to fill up cracks in concrete, enhanced production of calcium carbonate must be achieved: we need to up-regulate urease production.<br />
<br />
Previous experiments involving up-regulating ''ureA'', ''ureB'' and ''ureC'' in ''B. subtilis'' have not lead to an increase in urease production. This could be due to yet unidentified genes that are involved in the process. Therefore, we looked for another strategy.<br />
<br />
==Flux balance analysis==<br />
In order to identify pathways which indirectly lead to urea hydrolysis we performed flux balance analysis using the [http://gcrg.ucsd.edu/Downloads/Cobra_Toolbox COBRA Matlab Toolbox] and [http://systemsbiology.ucsd.edu/In_Silico_Organisms/Other_Organisms a model of the core ''B. subtilis'' 168 metabolic network].<br />
<br />
To simplify the process of devising SBML models we used [http://www.staff.ncl.ac.uk/d.j.wilkinson/software/sbml-sh/ SBML Shorthand].<br />
<br />
Flux balance analysis (FBA) is a widely used approach for studying biochemical networks. FBA calculates the flow of metabolites through a metabolic network, thereby making it possible to predict the growth rate of an organism or the rate of production of a biotechnologically important metabolite under some set conditions. [http://www.nature.com/nbt/journal/v28/n3/abs/nbt.1614.html]<br />
<br />
By using FBA to calculate the flow of metabolites through the ''B. subtilis'' 168 biochemical network during maximum urease activity, we were able to identify the arginine biosynthesis and catabolism pathways as potential targets.<br />
<br />
[[Image:Newcastle_Arginine_and_Ornithine_Degradation.png|600px]]<br />
<br />
Taken from [http://seed-viewer.theseed.org/seedviewer.cgi?page=Subsystems&subsystem=Arginine_and_Ornithine_Degradation&organism=224308.1 SEED]<br />
<br />
<br />
<br />
Downloads:<br />
*[[Media:Newcastle_FBA_growth.txt|Metabolic flux in standard conditions (maximal growth)]]<br />
*[[Media:Newcastle_FBA_urease.txt|Metabolic flux during maximum urease activity]] (reaction rxn00101, Urea amidohydrolase), showing large flux through the L-Arginine amidinohydrolase reaction (rxn00394).<br />
*[[Media:Newcastle_flux_balance_analysis.m.txt|Matlab code]]<br />
<br />
==Parts==<br />
<br />
By increasing arginine and arginase production we can increase urea hydrolysis indirectly. Arginase breaks down arginine to urea and ornithine, leading to an increase of urea inside the cell. We believe that in turn the urea itself will increase urease production.<br />
<br />
We plan to produce two parts, one which will enhance arginine production, and one which will enhance arginase production. These will be combined into a composite urea/urease part.<br />
<br />
[[Image: rocFalan.jpeg|600px|Alan showing urease pathway]]<br />
<br />
<br />
===Arginine Part===<br />
[[Image:Newcastle IPTG-inducible L-arginine.png]]<br />
<br />
''SR1'' is a small untranslated regulatory RNA from the ''Bacillus subtilis'' genome. It acts as an antisense RNA to ''ahrC'' mRNA thereby inhibiting its translation. ''ahrC'' mRNA encodes the AhrC protein, which represses arginine biosynthesis and positively regulates arginine catabolism.[http://www.ncbi.nlm.nih.gov/pubmed/17020585]<br />
<br />
Transcription of ''SR1'' results in an increase in arginine biosynthesis and a decrease in arginine catabolism, and therefore an overall increase in the level of arginine.<br />
<br />
This is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302013 part BBa_K302013] on the [http://partsregistry.org parts registry].<br />
<br />
===Arginase Part===<br />
<br />
[[Image:IPTG-inducible arginase.png]]<br />
<br />
The ''rocF'' gene codes for the enzyme arginase, which breaks arginine into ornithine and urea. This is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302014 part BBa_K302014] on the [http://partsregistry.org parts registry].<br />
<br />
===Composite urea/urease Part===<br />
<br />
[[Image:IPTG-inducible urea urease.png]]<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K302015 Part BBa_K302015] on the [http://partsregistry.org parts registry] combines the above two parts. The part increases urea hydrolysis indirectly, by increasing arginine and arginase production. Arginase breaks down arginine to urea and ornithine, leading to an increase of urea inside the cell. In turn the urea itself leads to urease production. Urease breaks urea into ammonia and carbonate ions and the carbonate ions are then transported to the extracellular face of the cell membrane.<br />
<br />
==Computational model==<br />
We composed a computational model of our system in SBML and simulated it in Copasi to help us verify our parts had the expected behaviour before we built them. The graph below shows that carbonate increases over time, as desired.<br />
<br />
[[Image:ModelrocFsr1.png|400px]]<br />
<br />
Downloads:<br />
*[[Media:Newcastle_urease.mod.txt|SBML-shorthand]]<br />
*[[Media:Newcastle_urease.xml.txt|SBML]]<br />
<br />
==References==<br />
#Jeffrey D Orth, Ines Thiele and Bernhard Ø Palsson. 2010. "''What is flux balance analysis?''" Nature Biotechnology. 28, p.245–248.<br />
#Heidrich N, Chinali A, Gerth U, Brantl S. 2006. "''The small untranslated RNA SR1 from the Bacillus subtilis genome is involved in the regulation of arginine catabolism''" Mol Microbiol. 2006 Oct;62(2):520-36.<br />
#Kim JK, Mulrooney SB, and Hausinger RP. 2005. "''Biosynthesis of Active Bacillus subtilis Urease in the Absence of Known Urease Accessory Proteins''". Journal of Bacteriology.p.7150–7154.<br />
#Tittelboom KV, Belie ND, Muynck WD, Verstraete W. 2010. "''Use of bacteria to repair cracks in concrete''". Cement and Concrete Research. 40. p.157–166.<br />
#Gardan R, Rapoport G and Debarbouille M. 1995. "''Expression of the rocDEF Operon Involved in Arginine Catabolism in Bacillus subtilis''". Journal of Molecular Biology. 249, p.843–856.<br />
#Canton B, Labno A,and Endy D. 2008. "''Refinement and standardization of synthetic biological parts and devices''". Nature Biotechnology. 26, p.787-793.<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoydhttp://2010.igem.org/Team:Newcastle/UreaseTeam:Newcastle/Urease2010-10-27T20:38:46Z<p>RachelBoyd: /* Flux balance analysis */</p>
<hr />
<div>{{Team:Newcastle/mainbanner}}<br />
<br />
=Calcium carbonate precipitation via urease expression=<br />
<br />
''Bacillus subtilis'' produce urease, which catalyses the hydrolysis of urea into ammonium and carbonate (CO<sub>3</sub><sup>2-</sup>). Since the cell walls of the bacteria are negatively charged, they draw cations from the environment, including Ca<sup>2+</sup>, to deposit on their cell surface. The Ca<sup>2+</sup> ions subsequently react with the CO<sub>3</sub><sup>2-</sup> ions, leading to the precipitation of CaCO<sub>3</sub> at the cell surface.<br />
<br />
In order for ''B. subtilis'' to fill up cracks in concrete, enhanced production of calcium carbonate must be achieved: we need to up-regulate urease production.<br />
<br />
Previous experiments involving up-regulating ''ureA'', ''ureB'' and ''ureC'' in ''B. subtilis'' have not lead to an increase in urease production. This could be due to yet unidentified genes that are involved in the process. Therefore, we looked for another strategy.<br />
<br />
==Flux balance analysis==<br />
In order to identify pathways which indirectly lead to urea hydrolysis we performed flux balance analysis using the [http://gcrg.ucsd.edu/Downloads/Cobra_Toolbox COBRA Matlab Toolbox] and [http://systemsbiology.ucsd.edu/In_Silico_Organisms/Other_Organisms a model of the core ''B. subtilis'' 168 metabolic network].<br />
<br />
To simplify the process of devising SBML models we used [http://www.staff.ncl.ac.uk/d.j.wilkinson/software/sbml-sh/ SBML Shorthand]<br />
<br />
Flux balance analysis (FBA) is a widely used approach for studying biochemical networks. FBA calculates the flow of metabolites through a metabolic network, thereby making it possible to predict the growth rate of an organism or the rate of production of a biotechnologically important metabolite under some set conditions. [http://www.nature.com/nbt/journal/v28/n3/abs/nbt.1614.html]<br />
<br />
By using FBA to calculate the flow of metabolites through the ''B. subtilis'' 168 biochemical network during maximum urease activity, we were able to identify the arginine biosynthesis and catabolism pathways as potential targets.<br />
<br />
[[Image:Newcastle_Arginine_and_Ornithine_Degradation.png|600px]]<br />
<br />
Taken from [http://seed-viewer.theseed.org/seedviewer.cgi?page=Subsystems&subsystem=Arginine_and_Ornithine_Degradation&organism=224308.1 SEED]<br />
<br />
<br />
<br />
Downloads:<br />
*[[Media:Newcastle_FBA_growth.txt|Metabolic flux in standard conditions (maximal growth)]]<br />
*[[Media:Newcastle_FBA_urease.txt|Metabolic flux during maximum urease activity]] (reaction rxn00101, Urea amidohydrolase), showing large flux through the L-Arginine amidinohydrolase reaction (rxn00394).<br />
*[[Media:Newcastle_flux_balance_analysis.m.txt|Matlab code]]<br />
<br />
==Parts==<br />
<br />
By increasing arginine and arginase production we can increase urea hydrolysis indirectly. Arginase breaks down arginine to urea and ornithine, leading to an increase of urea inside the cell. We believe that in turn the urea itself will increase urease production.<br />
<br />
We plan to produce two parts, one which will enhance arginine production, and one which will enhance arginase production. These will be combined into a composite urea/urease part.<br />
<br />
[[Image: rocFalan.jpeg|600px|Alan showing urease pathway]]<br />
<br />
<br />
===Arginine Part===<br />
[[Image:Newcastle IPTG-inducible L-arginine.png]]<br />
<br />
''SR1'' is a small untranslated regulatory RNA from the ''Bacillus subtilis'' genome. It acts as an antisense RNA to ''ahrC'' mRNA thereby inhibiting its translation. ''ahrC'' mRNA encodes the AhrC protein, which represses arginine biosynthesis and positively regulates arginine catabolism.[http://www.ncbi.nlm.nih.gov/pubmed/17020585]<br />
<br />
Transcription of ''SR1'' results in an increase in arginine biosynthesis and a decrease in arginine catabolism, and therefore an overall increase in the level of arginine.<br />
<br />
This is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302013 part BBa_K302013] on the [http://partsregistry.org parts registry].<br />
<br />
===Arginase Part===<br />
<br />
[[Image:IPTG-inducible arginase.png]]<br />
<br />
The ''rocF'' gene codes for the enzyme arginase, which breaks arginine into ornithine and urea. This is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302014 part BBa_K302014] on the [http://partsregistry.org parts registry].<br />
<br />
===Composite urea/urease Part===<br />
<br />
[[Image:IPTG-inducible urea urease.png]]<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K302015 Part BBa_K302015] on the [http://partsregistry.org parts registry] combines the above two parts. The part increases urea hydrolysis indirectly, by increasing arginine and arginase production. Arginase breaks down arginine to urea and ornithine, leading to an increase of urea inside the cell. In turn the urea itself leads to urease production. Urease breaks urea into ammonia and carbonate ions and the carbonate ions are then transported to the extracellular face of the cell membrane.<br />
<br />
==Computational model==<br />
We composed a computational model of our system in SBML and simulated it in Copasi to help us verify our parts had the expected behaviour before we built them. The graph below shows that carbonate increases over time, as desired.<br />
<br />
[[Image:ModelrocFsr1.png|400px]]<br />
<br />
Downloads:<br />
*[[Media:Newcastle_urease.mod.txt|SBML-shorthand]]<br />
*[[Media:Newcastle_urease.xml.txt|SBML]]<br />
<br />
==References==<br />
#Jeffrey D Orth, Ines Thiele and Bernhard Ø Palsson. 2010. "''What is flux balance analysis?''" Nature Biotechnology. 28, p.245–248.<br />
#Heidrich N, Chinali A, Gerth U, Brantl S. 2006. "''The small untranslated RNA SR1 from the Bacillus subtilis genome is involved in the regulation of arginine catabolism''" Mol Microbiol. 2006 Oct;62(2):520-36.<br />
#Kim JK, Mulrooney SB, and Hausinger RP. 2005. "''Biosynthesis of Active Bacillus subtilis Urease in the Absence of Known Urease Accessory Proteins''". Journal of Bacteriology.p.7150–7154.<br />
#Tittelboom KV, Belie ND, Muynck WD, Verstraete W. 2010. "''Use of bacteria to repair cracks in concrete''". Cement and Concrete Research. 40. p.157–166.<br />
#Gardan R, Rapoport G and Debarbouille M. 1995. "''Expression of the rocDEF Operon Involved in Arginine Catabolism in Bacillus subtilis''". Journal of Molecular Biology. 249, p.843–856.<br />
#Canton B, Labno A,and Endy D. 2008. "''Refinement and standardization of synthetic biological parts and devices''". Nature Biotechnology. 26, p.787-793.<br />
<br />
{{Team:Newcastle/footer}}</div>RachelBoyd