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Latest revision as of 23:50, 26 October 2012

Parts for a reliable and field ready biosensing platform

Implementation of biosensors in real world situations has been made difficult by the unpredictable and non-quantified outputs of existing solutions, as well as a lack of appropriate storage, distribution and utilization systems. This leaves a large gap between a simple, functional sensing mechanism and a fully realised product that can be used in the field. We aim to bridge this gap at all points by developing a standardised ratiometric luciferase output in a Bacillus chassis. This output can be linked up with prototyped instrumentation and software for obtaining reliable quantified results. Additionally, we have reduced the specialized requirements for the storage and distribution of our bacteria by using Bacillus' sporulation system. To improve the performance of our biosensing platform we have genetically modified Bacillus’ germination speed. Lastly, we demonstrated the robustness of our system by testing it with a new fluoride riboswitch, providing the opportunity to tackle real life problems.

One minute tour! :)

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Judging Form

Please help the judges by filling out this form. Tell them what medal you think you deserve and why. Tell them which special prizes you should win. Help them find your best parts. Show them how you thought about the safety of your project. Helping the judges will help you too.

Team: Cambridge
Region: Europe
iGEM Year:2012
Track:Foundational Advance
Project Name:Parts for a reliable and field ready biosensing platform
Project Abstract: Implementation of biosensors in real world situations has been made difficult by the unpredictable and non-quantified outputs of existing solutions, as well as a lack of appropriate storage, distribution and utilization systems. This leaves a large gap between a simple, functional sensing mechanism and a fully realised product that can be used in the field.

We aim to bridge this gap at all points by developing a standardised ratiometric luciferase output in a Bacillus chassis. This output can be linked up with prototyped instrumentation and software for obtaining reliable quantified results. Additionally, we have reduced the specialized requirements for the storage and distribution of our bacteria by using Bacillus' sporulation system. To improve the performance of our biosensing platform we have genetically modified Bacillus’ germination speed. Lastly, we demonstrated the robustness of our system by testing it with a new fluoride riboswitch, providing the opportunity to tackle real life problems.

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iGEM Medals for non-software teams

We believe our team deserves the following medal:
- Bronze
- Silver
- √Gold

Because we met the following criteria (check all that apply and provide details where needed)

Requirements for a Bronze Medal

√Register the team, have a great summer, and plan to have fun at the Regional Jamboree.
√Successfully complete and submit this iGEM 2012 Judging form.
√Create and share a Description of the team's project using the iGEM wiki and the team's parts using the Registry of Standard Biological Parts.
√Plan to present a Poster and Talk at the iGEM Jamboree.
√Enter information detailing at least one new standard BioBrick Part or Device in the Registry of Standard Biological Parts. Including:
- √Primary nucleaic acid sequence
- √Description of function
- √Authorship
- Safety notes, if relevant.
- √Acknowedgment of sources and references
√Submit DNA for at least one new BioBrick Part or Device to the Registry.

Additional Requirements for a Silver Medal

√Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device.
√Enter this information and other documentation on the part's 'Main Page' section of the Registry
Part Number(s): [http://partsregistry.org/Part:BBa_K911004 BBa_K911004]

Additional Requirements for a Gold Medal: (one OR more)

Improve an existing BioBrick Part or Device and enter this information back on the Experience Page of the Registry.
Part Number(s): None
√Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.
Link to this information on your wiki. Page name: Team:Cambridge/Outreach/Collaboration
√Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.
Link to this information on your wiki.
Page name: Team:Cambridge/HumanPractices/Overview,Team:Cambridge/HumanPractices/MarketResearch,Team:Cambridge/HumanPractices/FutureDirections

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iGEM Prizes

All teams are eligible for special prizes at the Jamborees. more... To help the judges, please indicate if you feel you should be evaluated for any of the following special prizes:

√Best Human Practice Advance
√Best Experimental Measurement
Best Model

Please explain briefly why you should receive any of these special prizes:

Best Human Practice Advance:

We feel that we deserve this prize for three reasons:

We explored the impacts, *both positive and negative*, of synthetic biology as a solution to real world problems, through interviewing professionals working in a relevant field, namely the impact of arsenic water contamination in Bangladesh.
We recognized existing problems with the way the current direction of synthetic. On going through the registry we found that most of the characterization data for biosensing parts is often neither comparable nor replicable. We have worked to solve this issue, for example with our ratiometric dual channel output.
*Our project doesn’t stop here*, in Chanel number 6 (Team:Cambridge/HumanPractices/FutureDirections) we considered the future implications and technological applications of our project, as well as the means by which it could be improved by subsequent users. We feel that the end to an iGEM project should not be the conclusion of an idea, but the start of it.

Best BioBrick Measurement Approach:

It is absolutely vital that a quantitative, numerical, robust, and flexible measurement approach exists to relay information to a user that is an accurate representation of the input processed by a biological device. Working from these principles, the following was done:

We designed and built Biologger, a *cheap, arduino-based, fully functional automatic rotary device* that has an incorporated ratiolumnometer
Our project is entirely open-sourced and open-platform. We have published source code for the two applications which serve to operate the device, one for PCs and the other for Android devices, as well as the open source circuit design that provides this ratiometric reading. Furthermore, the Android app is able to receive its data wirelessly, which we feel is a great advance in BioBrick measurement.
Our dual-channel luciferase reporter was successfully tested with a dilution series of E.coli transformed with the Lux Operon (under pBAD) biobrick (Part BBa_K325909) of the Cambridge iGEM 2010 team. It can detect, with good accuracy, both different light intensities, as well as the percentages of blue or orange frequencies in a sample.
Our device was successfully tested using artificial light to detect different frequencies (colours) as well.

Having done all the above, we believe that this fully open-sourced instrumentation kit (mechanical) chassis, electronics, software code), estimated at *$35.00* (or $85.00 if a Bluetooth modem is required), is a complete BioBrick measurement solution for any and all BioBricks with a light output.

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Team_Parts

To help the judges evaluate your parts, please identify 3 of your parts that you feel are best documented and are of the highest quality.

Best new BioBrick part (natural)
[http://partsregistry.org/Part:BBa_K911003 BBa_K911003]
Best new BioBrick part (engineered)
[http://partsregistry.org/Part:BBa_K911004 BBa_K911004]
Best improved part(s): None

List any other parts you would like the judges to examine:[http://partsregistry.org/Part:BBa_K911001 BBa_K911001], [http://partsregistry.org/Part:BBa_K911008 BBa_K911009], [http://partsregistry.org/Part:BBa_K911008 BBa_K911008]

Please explain briefly why the judges should examine these other parts:

Magnesium Sensitive Riboswitch [http://partsregistry.org/Part:BBa_K911001 BBa_K911001]
As a riboswitch sensing construct, this part is an entirely new type of biosensor (along with the fluoride construct) that could potentially change the way we think about designing input genetic circuits. Unlike the fluoride riboswitch, it is a derepression system and therefore serves to demonstrate the principle that riboswitches can be used regardless of whether they turn on or off their reporter.
Fluorescent ratiometric construct for standardizing promoter output [http://partsregistry.org/Part:BBa_K911009 BBa_K911009]
Fluorescence is a major cornerstone for biosensors in the registry, however, most parts do not involve the use of a ratiometric output, which has been shown in the literature to provide much more reliable and meaningful data. This part not only furthers the development of ratiometric measurements in molecular biology but due to the choice of promoters and terminators it can be used to characterize the difference in activity between E. coli and B. Subtilis
Fast Germination (B. subtilis) [http://partsregistry.org/Part:BBa_K911008 BBa_K911008]
This part is entirely novel for the registry and fully utilizes the recombination machinery inherent in the Bacillus chassis. Have spores that can germinate at a faster rate is certainly a worthy achievement and could help with experiments with B. Subtilis that any future iGEM teams may wish to perform.

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iGEM Safety

For iGEM 2012 teams are asked to detail how they approached any issues of biological safety associated with their projects.

The iGEM judges expect that you have answered the four safety questions Safety page on your iGEM 2012 wiki.

Please provide the link to that page: Page name: Team:Cambridge/Safety

Attribution and Contributions

For iGEM 2012 the description of each project must clearly attribute work done by the team and distinguish it from work done by others, including the host labs, advisors, and instructors.

Please provide the link to that page, or comments in the box below: Page name: Team:Cambridge/Attributions

Comments

If there is any other information about your project you would like to highlight for the judges, please provide a link to your wiki page here: Team:Cambridge/Overview/DesignProcess

Transformation of Bacillus subtilis

A technique used to introduce foreign DNA into competent Bacillus cells.

Before you start

Mediums needed:

Protocols

**Making Bacillus competent**

Grow one blank plate of Bacillus subtilis (or several if you want to transform different strains) for 20 hours at 37^oC (plate kept on the bench for several days would be better)
Inoculate about 12ml of Medium A with several colonies. Mix the contents of the tube. Check with OD₆₅₀. Start OD should be between 0.1 and 0.2. Be careful to pipette 0.8ml of this mixture into the cuvette to measure and dispose of it after measurement to avoid contamination in the main mixture.
Incubate at 37^oC with vigorous shaking. Read the OD₆₅₀ every 20 minutes (always dispose of the sample used for measuring)
Plot log(OD₆₅₀) as a function of time. After a brief lag, you should observe an exponential increase. After a while, it will leave the exponential growth; the moment at which it leaves the exponential path is denoted as t₀. This should take about 100 minutes and the OD should be between 0.7 and 1.0.
At t₀ incubate for 90 minutes at 37^oC with vigorous shaking.
Transfer 0.05ml of this culture into 0.45ml of pre-warmed Medium B in an Eppendorf tube. One tube needs to be prepared for each transformation, plus one extra for a DNA-less control.
Incubate the diluted cultures at 37^oC with shaking for 90 minutes. At this moment, the cells are HIGHLY COMPETENT.
To check for competency, you can look at cells under the microscope; competent cells are very mobile.

Transforming

Spin Eppendorf tubes containing cells. Discard 400µl of the liquid medium (leaving 100µl - this will concentrate the cells). Resuspend the pellet in the remaining culture.
To transform from competent glycerol stocks, spin the tube at about 11000rpm for 20 minutes, remove the supernatent (glycerol), and add 100µl of pre-warmed medium B
Mix the cells thoroughly
Add 0.6µg of DNA to the competent cells
Incubate for 30min at 37^oC with shaking
Plate 100µl of transformed cells onto selective agar

Glycerol stocks

To freeze competent Bacillus cells, spin down the fresh competent cells to obtain a pellet
Remove all supernatant
Re-suspend cells in 500µl 60% glycerol
Freeze tubes at -80^oC

Risk Assessment Back to Protocols

@@ Line 1: / Line 1: @@
-{| style="color:#1b2c8a;background-color:#3ae2e8;" cellpadding="3" cellspacing="0" border="1" bordercolor="#fff" width="62%" align="center"
+{{Template:Team:Cambridge/CAM_2012_TEMPLATE_HEADNEW}}
-!align="center"|[[Team:Cambridge|Home]]
-!align="center"|[[Team:Cambridge/Team|Team]]
-!align="center"|[https://igem.org/Team.cgi?year=2012&team_name=Cambridge Official Team Profile]
-!align="center"|[[Team:Cambridge/Project|Project]]
-!align="center"|[[Team:Cambridge/Parts|Parts Submitted to the Registry]]
-!align="center"|[[Team:Cambridge/Modeling|Modeling]]
-!align="center"|[[Team:Cambridge/Notebook|Notebook]]
-!align="center"|[[Team:Cambridge/Safety|Safety]]
-!align="center"|[[Team:Cambridge/Attributions|Attributions]]
-!align="center"|[[Team:Cambridge/Sponsors|Sponsors]]
-|}
+<html>
+<div id="template_content_wide" style="position: absolute; top: 200px; left: 40px; height: 620px; width: 1110px; overflow: auto; overflow-y: scroll;">
+</html>
 ==Transformation of ''Bacillus subtilis''==
 A technique used to introduce foreign DNA into competent Bacillus cells.
-===Theory===
+===Before you start===
-''Bacillus subtilis'' is a rod-shaped, soil-dwelling bacterium . It is widely used as a model organism for studying biology of Gram-positive bacteria which, in contrast to Gram-negative ''E.coli'', do not possess the outer membrane over the peptidoglycan cell wall. Additionally, ''Bacillus'' is naturally competent, which means that no sophisticated pre-treatment is needed for transformation with foreign DNA.
-===Practice===
+Mediums needed:
-* Add to a clean Eppendorf tube:
+*[[Team:Cambridge/Protocols/MediumB|Medium A]]
-<center>
+*[[Team:Cambridge/Protocols/MediumB|Medium B]]
-:{| border="1px" style="text-align:center;"
-|'''Reagent'''
-|scope="col" width="80" | '''Quantity'''
-|-
-|scope="col" width="200" | competent ''Bacillus'' cells
-|400&mu;l
-|-
-|DNA
-|3&mu;l
-|-
-|}
-</center>
-* Incubate at 37ºC for 1 hour.
+==='''Protocols'''===
-* Plate on an agar plate containing antibiotic, which allows for selection of successful transformants.
-===Safety===
+===='''Making ''Bacillus'' competent'''====
-All equipment (including gloves) that may have come into contact with the bacteria must be autoclaved for decontamination purposes.
+#Grow one blank plate of ''Bacillus subtilis'' (or several if you want to transform different strains) for 20 hours at 37<sup>o</sup>C (plate kept on the bench for several days would be better)
+#Inoculate about 12ml of Medium A with several colonies. Mix the contents of the tube. Check with OD<sub>650</sub>. Start OD should be between 0.1 and 0.2. Be careful to pipette 0.8ml of this mixture into the cuvette to measure and dispose of it after measurement to avoid contamination in the main mixture.
+#Incubate at 37<sup>o</sup>C with vigorous shaking. Read the OD<sub>650</sub> every 20 minutes (always dispose of the sample used for measuring)
+#Plot log(OD<sub>650</sub>) as a function of time. After a brief lag, you should observe an exponential increase. After a while, it will leave the exponential growth; the moment at which it leaves the exponential path is denoted as t<sub>0</sub>. This should take about 100 minutes and the OD should be between 0.7 and 1.0.
+#At t<sub>0</sub> incubate for 90 minutes at 37<sup>o</sup>C with vigorous shaking.
+#Transfer 0.05ml of this culture into 0.45ml of pre-warmed Medium B in an Eppendorf tube. One tube needs to be prepared for each transformation, plus one extra for a DNA-less control.
+#Incubate the diluted cultures at 37<sup>o</sup>C with shaking for 90 minutes. At this moment, the cells are HIGHLY COMPETENT.
+#To check for competency, you can look at cells under the microscope; competent cells are very mobile.
+===='''Transforming'''====
+#Spin Eppendorf tubes containing cells. Discard 400µl of the liquid medium (leaving 100µl - this will concentrate the cells). Resuspend the pellet in the remaining culture.
+#To transform from competent glycerol stocks, spin the tube at about 11000rpm for 20 minutes, remove the supernatent (glycerol), and add 100µl of pre-warmed medium B
+#Mix the cells thoroughly
+#Add 0.6µg of DNA to the competent cells
+#Incubate for 30min at 37<sup>o</sup>C with shaking
+#Plate 100µl of transformed cells onto selective agar
+===='''Glycerol stocks'''====
+#To freeze competent ''Bacillus'' cells, spin down the fresh competent cells to obtain a pellet
+#Remove all supernatant
+#Re-suspend cells in 500µl 60% glycerol
+#Freeze tubes at -80<sup>o</sup>C
+<center>'''[[Team:Cambridge/RiskAssessments/TransformationofBsubtilis|Risk Assessment]]'''</center>
 <center>'''[[Team:Cambridge/Protocols|Back to Protocols]]'''</center>
+<html>
+</div>
+</html>
+{{Template:Team:Cambridge/CAM_2012_TEMPLATE_FOOTNEW}}

Team:Cambridge/Protocols/TransformationofB.subtilis

From 2012.igem.org

Latest revision as of 23:50, 26 October 2012

Contents

Judging Form

iGEM Medals for non-software teams

Requirements for a Bronze Medal

Additional Requirements for a Silver Medal

Additional Requirements for a Gold Medal: (one OR more)

iGEM Prizes

Please explain briefly why you should receive any of these special prizes:

Team_Parts

iGEM Safety

Attribution and Contributions

Comments

Transformation of Bacillus subtilis

Before you start

Protocols

**Making Bacillus competent**

Transforming

Glycerol stocks