SPORDUINOParts for a Reliable and Field Ready Biosensing Platform

CAMBRIDGE iGEM 2012: 2012.igem.org/Team:Cambridge

A novel � uoride sensor based on a riboswitch construct• We have characterised a recently isolated fl uoride riboswitch in both E. coli and B. subtilis. Th is riboswitch de-represses transcription in the presence of F- ions, allowing a suitable reporter to be used to quantify the levels

of fl uoride in the assay.

• We have found that our riboswitch has 2 sensitivity ranges; when chloroform treated, which puts pores in the cell membrane and allows free entry of fl uoride ions, the cells become more sensitive compared to when assayed intact. Th is has great potential as the linear range of the response of the porous cells in-cludes the WHO guidelines for safe levels, whilst the intact cells have a linear response in the range of the current levels of fl uoride groundwater contamination in Nepal and Bangladesh.• We chose to work with riboswitches as we felt that they are an underused resource by the iGEM community as their specifi city, and improved response times compared to inducible promoters, makes them ideal biosensing tools.• In the boxplot on the left , the whiskers show regions where the response to the riboswitch appears to be linear, but where the data was not as consistent as the solid blocks. Th e red line indicates the reported toxic threshold of many bacteria to fl uoride ions, 30mM. Th e green line represents the WHO safe limit for fl uoridated drinking water, 79μM

Le� : Our Fluoride construct, as kindly provided by the Breaker lab at Yale

Middle: A representative assay of the riboswitch in porous cells, the linear, and therefore easiest to calibrate, region appears to lie between 0 and 100μM

Right: A representative assay of the riboswitch in non-porous B.subtilis, the linear region of the response appears to be in the range of 1 to 20mM, all values written on the tubes represent the � uoride concentration in mM

• We decided to investigate the potential of ratiometric reporter constructs for accurate and reproducible characterisation and quantifi cation. We designed fl uorescence and lumines-cence-based constructs that would function in both E.coli and B.subtilis.

• Th e principle of ratiometric reporters is the normalisation of an inducible signal to a constitutive one, to take variations in culture density, size and productivity into account internal-ly. Th is requires two reporter channels. In this case, the inducible signal is eCFP and the constitutive signal is eYFP.

• We tested pSpac, an inducible promoter in B. subtilis and E.coli, with our construct. Results show that while there is a strong relationship between eYFP, expressed under the constitutive promoter pVeg, and OD600, increasing concen-tration of IPTG increases the CFP/YFP gradient (see boxplot on the left ), which is what we expected.• We wanted to use the bacterial lux operon for our light output, as unlike fi refl y luciferase it doesn’t need to be supplied with luciferin. Th is presented a challenge to fi nd the second channel, as there are no mutagenised colour variants. We investigated BRET (bioluminescence resonance en-ergy transfer) to provide wavelength variants. Th e vibrio fi sheri Y-1 strain YFP shift s the spectrum signifi cantly, but is unstable above 18°C.

• We fused mOrange to the LuxA subunit as described by Dachuan Ke and Shiao-Chun Tu (photochem + photobio, 2011). Th ey showed that this results in an additional emission at 540 nm.

• We designed a synthetic reporter construct including this second channel, which was generously synthesised by DNA 2.0 and codon-optimised for bacillus

• Characterisation has been obstructed by time, toxicity and stability issues. Th e construct glows. Overlaying fi lters used in instrumentation over our construct and an unaltered lux operon suggests a possible spectral shift . However this is not

an adequately controlled or quantifi ed experiment and we do not consider it as confi rmation. We hope to have a com-plete spectrum to present.

Daubner SC, Astorga AD, Leisman GB and Baldwin TO (1987)

Above: Ratiometric reporter principleLe� : Spectrum shi� by vischeri Y-1 strain YFP

Right top: mOrange-LuxA spectrum shi� , by Dachuan Ke and Shiao-Chun TuRight bottom: Ratiometric luciferase constructLe� : From le� to right, the � rst photo shows the glowing construct; second and third photo shows a possible spectral shi�

It is absolutely vital that a quantitative, numerical, robust, and � exible measurement ap-proach exists to relay easily interpretable information to a user that is an accurate represen-tation of the input processed by a biological device. Working from these principles, Biolog-ger was developped:

1. A cheap, arduino-based, fully functional automatic rotary device that has an incorpo-rated ratioluminometer of our own design.2. Open-sourced and open-platform. 3. Two applications: one for PCs and the one for Android devices.

4. Circuit design that provides this ratiometric reading uses two Light-Dependent resistors and colored fi lter gels.5. Wireless data transmission ability with Android application - advances BioBrick meas-urement.6. Dual-channel luciferase reporter - 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.

7. Di� erent light intensities are detectable, as well as the percentages of blue or orange frequencies in a sample.

8. Di� erent frequencies (colours) of light are also detectable.9. Estimated at $35.00 (or $85.00 if Bluetooth modem is required).10. Complete BioBrick measurement solution for BioBricks with light output.

Using a Bacillus subtilis chassis allows longer term, more user-friendly storage. • B.subtilis forms dormant spores which are stable and can be stored long term

without needing freezing or nutrients• Th e tube of bacteria simply sits on the user’s shelf until needed• When required, a simple protocol and germination medium supplied in the

kit is applied, aft er a short incubation period the sensor can be loaded into the arduino device

• Overexpression of certain B.subtilis operons (we used spoVA) reduces the ger-mination period so that the cells are ready quicker

DIC microscopy image of B.subtilis spores, phase bright (yellow) spots represent spores, phase dark (red-orange) objects represent cells.

Packaged cuvette

• Fluoride contamination is a problem that faces people in many countries. Current estimates based upon geographical surveys (rock being the primary source of fl uoride contamination in groundwater) indicate that much of North Africa, India and China are heavily aff ected.

• Considerable fl uoride contamination of groundwater from these areas has been directly reported, as well as many of the health problems associated with endemic fl uorosis.

• Th is is a particularly big problem for poorly developed countries, where the primary water source is oft en untreated well water – taken directly from contaminated aquifers.

• Cheap sensors of � uoride are urgently needed so high resolution maps of a� ected areas can be taken. Th ese maps will allow appro-priate action to be taken on the problem. For example, methods such as well switching have been shown to be of high effi cacy in areas where contamination is heterogeneous over the area.

• Possible problem with our kit (1): � e law. Th e law on GM products varies from country to coun-try and this may limit the available markets for a product.

• Possible problem with our kit (2): Public perception. It is very hard to fi nd unbiased sources when looking at public preception. More in depth research into public opinion, in particular market specifi c research, will be required before the launching of the product into the particular market. We predict that the public will be concerned with possible infections and disposal of our prod-uct.

Above: From Amini M., Abbaspour K.C., Berg M., Winkel L., Hug S.J., Hoehn E., Yang H., Johnson C.A (2008): Statistical modeling of global geogenic arsenic contamination in groundwater. Environmental Science and Technology 42, 3669 –3675

Right: patient with Fluorosis

A Case Study into the Suitability of our system for use in the � eld: Fluoride Contamination of Ground Water in Rural India