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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

Generating Chemically Competent Cells

Risk Assessment

The following protocol has been taken from [http://openwetware.org/index.php?title=TOP10_chemically_competent_cells&oldid=288222 Openwetware.org]

Overview

This protocol is a variant of the Hanahan protocol using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the Bloom05 patent as well. This protocol has been tested on TOP10, MachI and BL21(DE3) cells. See Bacterial Transformation for a more general discussion of other techniques. Jesse '464 patent describes using this buffer for DH5α cells. The Bloom04 patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells.

This is the chemical transformation protocol used by Tom Knight and the [http://partsregistry.org Registry of Standard Biological Parts].

Materials

Detergent-free, sterile glassware and plasticware (see procedure)
Table-top OD600nm spectrophotometer
SOB

SOB

0.5% (w/v) yeast extract
2% (w/v) tryptone
10 mM NaCl
2.5 mM KCl
20 mM MgSO₄

Per liter:

5 g yeast extract
20 g tryptone
0.584 g NaCl
0.186 g KCl
2.4 g MgSO₄

Some formulations of SOB use 10 mM MgCl₂ and 10 mM MgSO₄ instead of 20 mM MgSO₄.
Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.

CCMB80 buffer

10 mM KOAc pH 7.0 (10 ml of a 1M stock/L or 0.98g/L)
80 mM CaCl₂.2H₂O (11.8 g/L)
20 mM MnCl₂.4H₂O (4.0 g/L)
10 mM MgCl₂.6H₂O (2.0 g/L or 5ml of 2M stock/L)
10% glycerol (100 ml/L)
Fill up with DI water AFTER regulating pH

adjust pH DOWN to 6.4 with 0.1N HCl if necessary
- adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
sterile filter and store at 4°C
slight dark precipitate appears not to affect its function

Procedure

Preparing glassware and media

Eliminating detergent

Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent free for these protocols. The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.

Prechill plasticware and glassware

Prechill 250mL centrifuge tubes and screw cap tubes before use.

Preparing seed stocks

Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C
- room temperature works well
Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C
- room temperature works well
Add glycerol to 15%
Aliquot 1 ml samples to Nunc cryotubes
Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
- This step may not be necessary
Place in -80°C freezer indefinitely.

Preparing competent cells

Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3
- This takes approximately 16 hours.
- Controlling the temperature makes this a more reproducible process, but is not essential.
- Room temperature will work. You can adjust this temperature somewhat to fit your schedule
- Aim for lower, not higher OD if you can't hit this mark
Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
- Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
- It is often easier to resuspend pellets by mixing before adding large amounts of buffer
Gently resuspend in 80 ml of ice cold CCMB80 buffer
- sometimes this is less than completely gentle. It still works.
Incubate on ice 20 minutes
Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.
Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
Incubate on ice for 20 minutes
Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates
Store at -80°C indefinitely.
- Flash freezing does not appear to be necessary
Test competence (see below)
Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.

Measurement of competence

Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
- This is at 10 pg/μl or 10^-5 μg/μl
- This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 ml of TE
Hold on ice 0.5 hours
Heat shock 60 sec at 42C
Add 250 μl SOC
Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
- using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
- For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
- Ampicillin and kanamycin appear to do fine with 1 hour growth
Plate 20 μl on AMP plates using sterile 3.5 mm glass beads
- Good cells should yield around 100 - 400 colonies
- Transformation efficiency is (dilution factor=15) x colony count x 10⁵/µgDNA
- We expect that the transformation efficiency should be between 5x10⁸ and 5x10⁹ cfu/µgDNA

References

Hanahan91 pmid=1943786
Reusch86 pmid=3536850
Addison04 pmid=15470891
Bloom04 US Patent 6,709,852
Bloom05 US Patent 6,855,494
Jesse05 US Patent 6,960,464

</Biblio>

Back to Protocols

@@ Line 1: / Line 1: @@
-{{Template:Team:Cambridge/CAM_2012_TEMPLATE_HEAD_DIARY}}
+{{Template:Team:Cambridge/CAM_2012_TEMPLATE_HEADNEW}}
+<html>
+<div id="template_content_wide" style="position: absolute; top: 200px; left: 40px; height: 620px; width: 1110px; overflow: auto; overflow-y: scroll;">
+</html>
+=Generating Chemically Competent Cells=
+'''[[Team:Cambridge/RiskAssessments/ChemicallyCompetentCellGeneration|Risk Assessment]]'''
 The following protocol has been taken from [http://openwetware.org/index.php?title=TOP10_chemically_competent_cells&oldid=288222 Openwetware.org]
@@ Line 29: / Line 39: @@
 *Some formulations of SOB use 10 mM MgCl<sub>2</sub> and 10 mM MgSO<sub>4</sub> instead of 20 mM MgSO<sub>4</sub>.
 *Adjust to pH 7.5 prior to use.  This requires approximately 25 ml of 1M NaOH per liter.
 ===CCMB80 buffer===
-* 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
+* 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L or 0.98g/L)
 * 80 mM CaCl<sub>2</sub>.2H<sub>2</sub>O (11.8 g/L)
 * 20 mM MnCl<sub>2</sub>.4H<sub>2</sub>O (4.0 g/L)
-* 10 mM MgCl<sub>2</sub>.6H<sub>2</sub>O (2.0 g/L)
+* 10 mM MgCl<sub>2</sub>.6H<sub>2</sub>O (2.0 g/L or 5ml of 2M stock/L)
 * 10% glycerol (100 ml/L)
+* Fill up with DI water AFTER regulating pH
 * adjust pH DOWN to 6.4 with 0.1N HCl if necessary
@@ Line 55: / Line 65: @@
 ===Preparing seed stocks===
-* Streak TOP10 cells on an SOB plate and grow for single colonies at 23&deg;C
+# Streak TOP10 cells on an SOB plate and grow for single colonies at 23&deg;C
-** room temperature works well
+#* room temperature works well
-* Pick single colonies into 2 ml of SOB medium  and shake overnight at 23&deg;C
+# Pick single colonies into 2 ml of SOB medium  and shake overnight at 23&deg;C
-** room temperature works well
+#* room temperature works well
-* Add glycerol to 15%
+# Add glycerol to 15%
-* Aliquot 1 ml samples to Nunc cryotubes
+# Aliquot 1 ml samples to Nunc cryotubes
-* Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
+# Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
-** This step may not be necessary
+#* This step may not be necessary
-* Place in -80&deg;C freezer indefinitely.
+# Place in -80&deg;C freezer indefinitely.
 ===Preparing competent cells===
-* Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20&deg;C to an OD600nm of 0.3
+# Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20&deg;C to an OD600nm of 0.3
-** This takes approximately 16 hours.
+#* This takes approximately 16 hours.
-** Controlling the temperature makes this a more reproducible process, but is not essential.
+#* Controlling the temperature makes this a more reproducible process, but is not essential.
-** Room temperature will work.  You can adjust this temperature somewhat to fit your schedule
+#* Room temperature will work.  You can adjust this temperature somewhat to fit your schedule
-** Aim for lower, not higher OD if you can't hit this mark
+#* Aim for lower, not higher OD if you can't hit this mark
-* Centrifuge at 3000g at 4&deg;C for 10 minutes in a flat bottom centrifuge bottle.
+# Centrifuge at 3000g at 4&deg;C for 10 minutes in a flat bottom centrifuge bottle.
-** Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
+#* Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
-** It is often easier to resuspend pellets by mixing ''before'' adding large amounts of buffer
+#* It is often easier to resuspend pellets by mixing ''before'' adding large amounts of buffer
-* Gently resuspend in 80 ml of ice cold CCMB80 buffer
+# Gently resuspend in 80 ml of ice cold CCMB80 buffer
-** sometimes this is less than completely gentle.  It still works.
+#* sometimes this is less than completely gentle.  It still works.
-* Incubate on ice 20 minutes
+# Incubate on ice 20 minutes
-* Centrifuge again at 4&deg;C and resuspend in 10 ml of ice cold CCMB80 buffer.
+# Centrifuge again at 4&deg;C and resuspend in 10 ml of ice cold CCMB80 buffer.
-* Test OD of a mixture of 200 &mu;l SOC and 50 &mu;l of the resuspended cells.
+# Test OD of a mixture of 200 &mu;l SOC and 50 &mu;l of the resuspended cells.
-* Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
+# Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
-* Incubate on ice for 20 minutes
+# Incubate on ice for 20 minutes
-* Aliquot to chilled screw top 2 ml vials or 50 &mu;l into chilled microtiter plates
+# Aliquot to chilled screw top 2 ml vials or 50 &mu;l into chilled microtiter plates
-* Store at -80&deg;C indefinitely.
+# Store at -80&deg;C indefinitely.
-** Flash freezing does not appear to be necessary
+#* Flash freezing does not appear to be necessary
-* Test competence (see below)
+# Test competence (see below)
-* Thawing and  refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.
+# Thawing and  refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.
 ===Measurement of competence===
-* Transform 50 &mu;l of cells with 1 &mu;l of standard pUC19 plasmid (Invitrogen)
+# Transform 50 &mu;l of cells with 1 &mu;l of standard pUC19 plasmid (Invitrogen)
-** This is at 10 pg/&mu;l or 10<sup>-5</sup> &mu;g/&mu;l
+#* This is at 10 pg/&mu;l or 10<sup>-5</sup> &mu;g/&mu;l
-** This can be made by diluting 1 &mu;l of NEB pUC19 plasmid (1 &mu;g/&mu;l, NEB part number N3401S) into 100 ml of TE
+#* This can be made by diluting 1 &mu;l of NEB pUC19 plasmid (1 &mu;g/&mu;l, NEB part number N3401S) into 100 ml of TE
-* Hold on ice 0.5 hours
+# Hold on ice 0.5 hours
-* Heat shock 60 sec at 42C
+# Heat shock 60 sec at 42C
-* Add 250 &mu;l SOC
+# Add 250 &mu;l SOC
-* Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
+# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
-** using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
+#* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
-** For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
+#* For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
-** Ampicillin and kanamycin appear to do fine with 1  hour growth
+#* Ampicillin and kanamycin appear to do fine with 1  hour growth
-* Plate 20 &mu;l on AMP plates using sterile 3.5 mm glass beads
+# Plate 20 &mu;l on AMP plates using sterile 3.5 mm glass beads
-** Good cells should yield around 100 - 400 colonies
+#* Good cells should yield around 100 - 400 colonies
-** Transformation efficiency is (dilution factor=15) x colony count x 10<sup>5</sup>/µgDNA
+#* Transformation efficiency is (dilution factor=15) x colony count x 10<sup>5</sup>/µgDNA
-**We expect that the transformation efficiency should be between 5x10<sup>8</sup> and 5x10<sup>9</sup> cfu/µgDNA
+#*We expect that the transformation efficiency should be between 5x10<sup>8</sup> and 5x10<sup>9</sup> cfu/µgDNA
 ==References==
-<biblio>
+<Biblio>
 # Hanahan91 pmid=1943786
 # Reusch86 pmid=3536850
@@ Line 111: / Line 121: @@
 # Bloom05 US Patent 6,855,494
 # Jesse05 US Patent 6,960,464
-</biblio>
+</Biblio>
+<center>'''[[Team:Cambridge/Protocols|Back to Protocols]]'''</center>
+<html>
+</div>
+</html>
-{{Template:Team:Cambridge/CAM_2012_TEMPLATE_FOOT}}
+{{Template:Team:Cambridge/CAM_2012_TEMPLATE_FOOTNEW}}

Team:Cambridge/Protocols/Chemicallycompetentcells

From 2012.igem.org

Latest revision as of 23:47, 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

Generating Chemically Competent Cells

Overview

Materials

SOB

CCMB80 buffer

Procedure

Preparing glassware and media

Eliminating detergent

Prechill plasticware and glassware

Preparing seed stocks

Preparing competent cells

Measurement of competence

References