Safety

As The Lung Ranger project has resulted in the construction of a device (the L.A.S.S.O.) that would eventually be used by non-scientists, the team felt that we should implement some well-considered safety protocols to ensure that:

1. Users do not come into direct contact with microorganisms, genetically modified or otherwise
2.Genetically modified microorganisms do not come into contact with wild-type microorganisms which could result in horizontal gene-transfer
3.Genetically engineered organisms are disposed of safely

This has resulted in the design and prototyping of a set of components and practices to be utilised in the context of home-visits to patients undertaken by trained medical staff.

Storage of Genetically Modified Microorganisms

Cultures of our modified E. coli would be freeze-dried before being stored in sealed LB-Agar plates containing selective antibiotics at 4^oC.

Rehydration of Freeze-Dried Cultures

On the day prior to a home-visit, water would be added to cultures with a sterile syringe via a one-way, filter-sterilizing valve and the plates incubated at 37^oC overnight.

Addition of Sputum to Rehydrated Cultures

Sputum samples would be collected in a sterile syringe which would then be fitted the plate (Figure 3) and the plunger depressed to add the sputum to the culture. It may be necessary, due to the variability in sputum viscosity, to aspirate Sputolysin® - a reducing agent - along with the sputum sample to thin the sample for use. The filter sterilizing valve will prevent wild-type bacterial cells from contacting the genetically modified bacteria inside the plate and thus preventing any uptake of transgenes by wild-type microorganisms.

Processing of Sample

The plate containing a sputum sample would be agitated by hand to ensure adequate coverage of the medium by the sputum before being placed in the L.A.S.S.O. (figure 4). After light detection, the plate would be transferred to a waste compartment by depressing the plunger-arm (figure 5). The plunger-arm would be reset and the process can be repeated until the waste compartment is filled.

Removal of Used Culture Plates

When the waste compartment is filled, the L.A.S.S.O. can be separated into two sections (figure 6), allowing the waste compartment to be either replaced with an empty compartment or for the waste compartment to be emptied into a sealable bag which would be autoclaved to dispose of the genetically modified microorganisms

Protocols

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Competent Cells

Day 1

1. Streak out frozen glycerol stock of bacterial cells onto an LB plate (no antibiotics). Work sterile. Grow cells overnight at 37^oC.

Day 2

2. Select a single colony of E. coli from fresh LB plate and inoculate a 5ml starter culture of LB (with no antibiotics). Grow at 37^oC in shaker overnight.

Day 3

3. Inoculate 50ml fresh growth medium with 500μl starter culture and grow at 37^oC for 2 hours.
4. When OD₆₀₀ reaches 0.35-0.4, immediately put the cells on ice, chill the culture for 20-30 minutes, swirling occasionally to ensure even cooling. Chill centrifuge bottles on ice.
5. Harvest the cells by centrifugation at 3000g (around 4000rpm in the Beckman Ja-10 rotor) for 15 minutes at 4^OC.
6. Decant the supernatant and gently resuspend each pellet in about 5ml of ice-cold transformation buffer¹.
7. Aliquot in 200μl batches into sterile 1.5ml microcentrifuge tubes and snap freeze with liquid nitrogen.
Store frozen cells at -80^O.
¹. Sambrook, J., and Russell, D. W. (2001) Molecular cloning : a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

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Polymerase Chain Reaction

This protocol can be used to amplify the target genes required for the biosensors from genomic DNA and add appropriate restriction sites required for ligation into the vector pUniprom. Primers were supplied by Sigma-Aldrich, the DNA polymerase used was Herculase II Fusion DNA Polymerase which was supplied by Aligent Technologies.

Set up Standard PCR reaction (final volume = 50μl):

Component	Volume (μl)
5x Herculase buffer	10
100μM forward primer	0.5
100μM reverse primer	0.5
10mM dNTP's	0.5
DMSO	2.5
H20	34.5
gDNA	1
Herculase II	0.5

Thermocycler protocol

Cycle(s)	Time/Temp	Process
1	3 mins at 95oC	Denature DNA
30-40	1 min at 95oC	DNA denatures into single strands
	0.5 min at 75oC	Primers bind to DNA template
	1 min/kb at 68oC	Primers extended 5’-3’ (1 min/kb)-elongation phase
1 cycle	5 min at 72oC	Final elongation
	10oC	Final hold

Agarose Gel Electrophoresis

DNA products were run on a 1% agarose gel for to separate charged nucleic acids by size for analysis/purification.

GelRed was used to stain nucleic acids and gels were visualised on a BioRad GelDoc.

Standard 1% agarose gel

1. Measure 1g of agarose
2. Pour agarose powder to 100ml of 1xTAE¹ (tris-acetate EDTA buffer) in a microwavable flask
3. Microwave until agarose has dissolved (1-3mins) and there is a rolling boil - Caution HOT!
4. Allow solution to cool down
5. Add Gel Red (6ul per 100ml 1% agarose solution)
6. Pour cooled agarose solution into a gel tray and place required combs in place (prevent any air bubbles)
7. Allow poured gel to sit on bench top for 20-30 minutes to solidify
8. Once gel has solidified, remove the comb and place gel into the electrophoresis unit.
9. Fill the gel tank with 1xTAE
10. Carefully load DNA weight ladder into the first lane of the gel
11. Carefully load samples into the additional wells of the gel (be careful not to disturb the wells)
12. Run the gel at 100V for 30 mins until the dye line is approximately 75-80% of the way down the gel.
13. Turn off the power, disconnect the electrodes from the power source and then carefully remove the gel from the gel box
14. UV light used to visualize the DNA fragments.

1. Sambrook, J., and Russell, D. W. (2001) Molecular cloning : a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

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Gel Extraction

QIAquick gel Extraction Kit Protocol designed to extract and purify DNA of 70bp to 10kb from standard or low-melt agarose gels in TAE buffer.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100mg=100μl).
3. Incubate at 50^oC for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2-3 min during the incubation.
4. After the gel slice has dissolved completely, check that the colour of the mixture is yellow
5. 5. Add 1 gel volume of isopropanol to the sample and mix
6. if the DNA will be subsequently used for sequencing, in vitro transcription, or microinjection, add 500μl Buffer QG to the QIAquick column and centrifuge for 1 min. Discard flow-through and place and place the QIAquick column back into the same tube.
7. To wash, add 750μl Buffer PE to QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube. Centrifuge the QIAquick column in the provided 2ml collection tube for 1 min to remove residual wash buffer.
8. Place QIAquick into a clean 1.5ml microcentrifuge tube.
9. To elute DNA, add 50μl of water to the centre of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30ul water to the centre of the QIAquick membrane, let stand for 1min, and then centrifuge for 1 min.
10. If the purified DNA is to be analysed on a gel, add 1 volume of loading dye to 5 volumes of purified DNA.

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Restriction Digests

Restriction Digests were performed to prepare DNA for ligation. Digests were carried out using enzymes supplied by Roche and New England Biolabs. Digests were performed at 37^oC for 3 hours.

An aliguot of alkaline phosphatase was added to restriction digests of vectors at 2hrs and a second aliquot at 2.5hrs Reactions were stopped after three hours. Digested PCR products were separated from restriction enzymes and buffers using Strataclean resin (Aligent Technologies). Digested vectors were purified on a 1% agarose gel and then extracted.

60μl reactions following PCR:

1. 50μl PCR product
2. 1.5μl restriction enzyme 1
3. 1.5μl restriction enzyme 2
4. 6μl cognate digestion Buffer for enzyme(s)
5. 1μl H₂0


Heatblock for 3 hours at 37^oC

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Strataclean

√RD sample= volume of Strataclean
√60= 7.745μl

1. Ensure Strataclean is vortexed before use.
2. Add 7.75μl to each restriction digest.
3. Vortex for 15 sec and then centrifuge for 1 min at 13,000rpm in a microfuge.
4. Pipette supernatant into a clean microcentrifuge tube.

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Ligations

For ligation of genes into vectors, the digested the gene of interest and the appropriate vector were incubated with T4 DNA ligase at room temperature for 3hrs.

Total reaction volume=10μl

Cycles	Vector Only	2:1	3:1
10x T4 ligase buffer	1μl		1μl		1μl
Insert	-	2μl		2μl
Vector	1μl	1μl		1μl
T4 ligase	1μl	1μl		1μl
H2o	7μl	5μl		4μl

N.b. T4 ligase added last to the reaction.

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Heat Shock Transformation of competent cells

Plasmids or ligation products containing the desired genes were transformed into chemocompetent E.coli cells.

1. Thaw competent cells on ice. Usually 10-20 minutes.
2. Pipette 100μl of cells into a pre-chilled eppendorf tube.
3. Add 10μl of ligation reaction or 2μl of miniprepped plasmid
4. Mix by gently flicking the tube
5. Chill on ice for 30 minutes
6. Heat shock at 42^oC for 90 seconds.
7. Return to ice for 2 minutes
8. Add 1ml of LB medium to cells and recover by shaking at 37^oC (ampicillin selection requires less recovery time than chloramphenicol selection)
9. Centrifuge for 3 minutes
10. Remove supernatant and resuspend in 100μl of LB
11. Plate out the cells on selective LB agar plates containing appropriate antibiotics and spread using sterile glass spreader
12. Incubate overnight at 37^oC.

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Colony PCR

PCR was used to identify the desired clones on the plate of transformed cells. PCR was performed on the samples using Taq polymerase supplied by Aligent Technologies.

DNA preparation

1. Pick a single colony and twizzle into 30μl of nuclease-free H₂o
2. Boil for 10 minutes at 100^oC
3. Centrifuge for 1 minute at 13000rpm in a microfuge 4. 5μl of DNA was then used as part of the reaction mixture outlined below


Colony PCR reaction:

Prepare the following PCR reaction on ice (N.B. adding the enzyme last):

Thermocycler Protocol

Component	Volume (μl)
10x PCR reaction Mg (Roche)	2
100μM forward primer	0.2
100μM reverse primer	0.2
10mM dNTP's	0.2
DMSO	2
DNA	5
H20	11.2
Taq Polymerase	0.5

Cycle(s)	Time/Temp	Process
1	3 mins at 95oC	Denature DNA
30-40	1 min at 95oC	DNA denatures into single strands
	0.5 min at 75oC	Primers bind to DNA template
	1 min/kb at 68oC	Primers extended 5’-3’ (1 min/kb)-elongation phase
1 cycle	5 min at 72oC	Final elongation
	10oC	Final hold

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Plasmid Miniprep

Plasmids were isolated using a QIAprep Spin Miniprep Kit Protocol.

A 5mL overnight culture of cells was centrifuged at 4000rpm for 10min. The resulting pellet was lysed in an alkaline lysis buffer and centrifuged. Supernatant is passed through a silica membrane to isolate plasmid DNA.

1. Resuspend pelleted bacterial cells in 250μl Buffer P1 and transfer to a microcentrifuge tube.
N.b. Ensure that RNase A has been added to Buffer P1.
2. Add 250μl Buffer P2 and gently invert the tube 4-6 times to mix.
Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA.
N.b. Do not allow the lysis reaction to proceed for more than 5 min.
3. Add 350μl Buffer N3 and invert the tube immediately but gently 4-6 times.
4. Centrifuge for 10 min at 13,000 rpm in a microfuge.
5. Apply the supernatants form step 4 to the QIAprep Spin column by decanting or pipetting.
6. Centrifuge for 30-60 seconds. Discard the flow-through.
7. (Optional): wash the QIAPrep Spin Column by adding 0.5ml Buffer PB and centrifuging for 30-60 seconds. Discard the flow-through.
8. Wash QIAPrep Spin column by adding 0.7ml of Buffer PE and centrifuging for 30-60s.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
10. Place the QIAprep column in a clean 1.5ml microcentrifuge tube. To elute DNA, add 50μl water to the centre of each QIAprep Spin column, let stand for 1 min, and centrifuge for 1 min.

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Site directed mutagenesis (Quick-change)

Site directed mutagenesis was used to remove internal restriction sites from genes. The method used relies on PCR using primers containing a single nucleotide mutation, which maintains the amino acid sequence and has specificity 15bp up- and downstream of the nucleotide that is to be changed. Herculase II polymerase was used in PCR reactions to amplify the target plasmid containing the mutation. PCR products were digested with the restriction enzyme DpnI to remove any methylated DNA leaving only the PCR product.

PCR reaction:

thermocycler process

Component	0.5x DNA template mixture (μl)	3x DNA template mixture (μl)
5x Herculase buffer	10		10
100μM forward primer	1	1
100μM reverse primer	1	1
10mM dNTP	1	1
H20	36.5	34
DNA template	0.5	3
Herculase II	1	10

Cycle(s)	Time/Temp	Process
1	3 mins at 95oC	Denature DNA
30	1 min at 95oC	DNA denatures into single strands
	0.5 min at 75oC	Primers bind to DNA template
	1 min/kb at 68oC	Primers extended 5’-3’ (1 min/kb)-elongation phase
1 cycle	5 min at 72oC	Final elongation
	10oC	Final Hold

PCR reaction then held at 10^oC until further analysis.

Run a 5ul aliquot of each PCR product on a 1% agarose gel to check for amplified target gene. Digest PCR products with restriction enzyme DpnI for 3 hours at 37^oC to remove any methylated DNA leaving only the PCR product. 5μl or 10μl of PCR product then transformed into competent E. coli cells and plated onto appropriate LB selective media.

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Glycerol Stock

1. Pick single colonies from agar plate
2. Inoculate 5ml LB broth overnight
3. Add 1ml of overnight culture to 1ml of 60% glycerol in a cryotube
4. Freeze at -80^oC

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Testing the biosensors

In order to test our complete biosensors the following protocol was followed:

• Prepare 5ml O/N cultures in LB medium containing the completed constructs supplemented with appropriate antibiotics
• Following 16hr of growth at 37^oC, subculture each sample into fresh growth medium (1:100)
• Spike cultures with saturating concentrations of synthetic signalling molecules
• Grow cultures for a further 2 hours to allow sufficient time for GFP /mCherry production
• Take 1 ml aliquots and proceed with western blot protocol

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Cell Fractionation

In order to test our complete biosensors the following protocol was followed:

• Inoculate 5ml of LB growth medium supplemented with appropriate antibiotic
• Allow 16hr growth at 37^oC and then subculture into 100ml of fresh growth medium (1:100 dilution)
• Grow cells for a further 8 hours and pellet
• Wash pellet once in 50mM Tris-HCl, pH 7.5.
• Repellet the cells and then resuspend in 2ml of the same buffer
• Lyse by sonication
• Centrifuge sonicated samples to remove unbroken cells
• Ultracentrifuge the supernatant to pellet the cell membranes
• Remove the supernatant (corresponds to the soluble fraction of the cell)
• Resuspend membrane pellet in 1ml of 50mM Tris-HCl, pH 7.5
• Separate 10μl of the samples by SDS PAGE (12% acrylamide), transfer to PVDF and probe with specific antibody (detailed western blot protocol below)

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Western blot

Western blotting was used to detect the expression of proteins in the E. coli chassis. 1mL of an overnight culture of cells were pelleted by centrifugation.
The pellets were resuspended in Laemmli buffer and boiled for 2mins to lyse the cells. Samples were then loaded on freshly prepared 4-12% polyacrylamide gels. Gels were run in SDS buffer at 180V for 45-60mins to separate proteins by size.
Proteins were transferred onto PVDF membrane by semi-dry transfer at 175mA for 20min for western blot analysis, following the transfer membranes were blocked in 5% milk/TBS overnight. The target proteins contained a hemagglutinin tag for detection by monoclonal Anti-HA−Peroxidase antibodies supplied by Sigma-Aldrich. Membranes were washed in Tris buffered saline containing 0.1% Tween 201 and incubated with the antibody at 1:10000 concentration in TBST for 1 hour at room temperature.
Photo-detection of the cross-reacting bands used ECL reagent (luminol + HRP) and exposure on photographic film.

1. Sambrook, J., and Russell, D. W. (2001) Molecular cloning : a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.