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Protocols

Plate Reader Calibration Protocols

1. OD600 Reference point
You will use LUDOX-S30 as a single point reference to obtain a ratiometric conversion factor to transform your absorbance data into a standard OD600 measurement. This has two key objectives.With standard 1 cm pathlength spectrophotometers, the reading is still instrument dependent (see above). With plate readers the path length is less than 1 cm and is volume dependent. In this instance the ratiometric conversion can both transform Abs600 measurements (i.e. the basic output of the instrument and not standardised optical density with 1 cm pathlength) into OD600 measurements, whilst simultaneously accounting for instrument differences.
[IMPORTANT NOTE: many plate readers have an automatic path length correction, this is based on volume adjustment using a ratio of absorbance measurements at 900 and 950 nm. Because scattering increases with longer wavelengths, this adjustment is confounded by scattering solutions, such as dense cells. YOU MUST THEREFORE TURN OFF PATHLENGTH CORRECTION.]
To measure your standard LUDOX Abs600 you must use the same cuvettes, plates and volumes (suggestion: use 100 μl for plate reader measurement and 1 mL for spectrophotometer measurement) that you will use in your cell based assays. The LUDOX solution is only weakly scattering and so will give a low absorbance value.
If using plates prepare a column of 4 wells with 100 μl 100% LUDOX and 4 wells containing 100 μl H2O. Repeat the measurement in all relevant modes used in your experiments (e.g. settings for orbital averaging).
If using a cuvette, you will only have enough material for a single measurement, but repeat the reading multiple times. Use the same cuvette to measure the reference with H2O (this value will be subtracted by the instrument to give the OD600 reading).

Materials:
1ml LUDOX (provided in kit)
H20 (provided by team)
96 well plate or cuvettes (provided by team)
Method
Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
Add 100 μl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
Record the data in the table below or in your notebook
Import data into Excel (OD600 reference point tab) Sheet_1 provided

2. Protocol FITC fluorescence standard curve
You will prepare a dilution series of FITC in 4 replicates and measure the fluorescence in a 96 well plate in your plate reader or individually in cuvettes in a fluorimeter. By measuring these in all standard modes in your plate reader or fluorimeter, you will generate a standard curve of fluorescence for FITC concentration. You will be able to use this to correct your cell based readings to an equivalent fluorescein concentration. You will then be able to convert this into a concentration of GFP.
Before beginning this protocol ensure that you are familiar with the GFP settings and measurement modes of your instrument.

Materials:
187 μg FITC (provided in kit)
10ml 1xPBS (phosphate buffered saline; provided by team)
96 well plate or cuvettes (provided by team)

Method
Prepare the FITC stock solution:
Spin down FITC stock tube to make sure pellet is at the bottom of tube.
Prepare 10x FITC stock solution by resuspending FITC in 1 mL of 1xPBS
Incubate the solution at 42°C for 4 hours
Dilute the 10x FITC stock solution in half with 1xPBS to make a 5x FITC solution and resulting concentration of FITC stock solution 2.5 μM.
[Note: it is important that the FITC is properly dissolved. To check this after the incubation period pipetted up and down – if any particulates are visible in the pipette tip continue to incubate overnight.]
Prepare the serial dilutions of FITC:
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 MUST CONTAIN PBS BUFFER ONLY. Initially you will setup the plate with the FITC stock in column 1 and an equal volume of 1xPBS in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing.
Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
Add 200 μl of FITC 5x stock solution into A1, B1, C1, D1
Transfer 100 μl of FITC stock solution from A1 into A2.
Mix A2 by pipetting up and down 3x and transfer 100 μl into A3...
Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...
Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...
Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste
TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.
Repeat dilution series for rows B, C, D
Measure fluorescence of all samples in all standard measurement modes in instrument
Record the data in your notebook
Import data into Excel (FITC standard curve tab) Sheet_1 provide

Plate Reader Measurement protocol

Prior to performing the measurement on the cells you should perform the calibration measurements. This will ensure that you understand the measurement process and that you can take the cell measurements under the same conditions.

Materials:

Competent cells (Escherichia coli strain DH5α)
LB (Luria Bertani) media as an alternative
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
50 ml Falcon tube
Incubator at 37°C
1.5 ml eppendorf tubes for sample storage
2x 96-well plates:
1 Completely translucent - clear
1 Completely opaque - black
Ice bucket with ice
Pipettes

Devices (from InterLab Measurement Kit):

Positive control
Negative control
Device 1: J23101+I13504
Device 2: J23106+I13504
Device 3: J23117+I13504

Method
Day 1: transform Escherichia coli DH5α with these following plasmids:

Positive control
Negative control
Device 1: J23101+I13504
Device 2: J23106+I13504
Device 3: J23117+I13504

Day 2: Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol.

Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.

Day 3: Cell growth, sampling, and assay

Using 96-well plates and a plate reader:

Set your instrument to read OD600 (as OD calibration setting)
Measure OD600 of the overnight cultures
Record data in your notebook
Import data into Excel (normalisation tab) Sheet_1 provided
Dilute the cultures to a target OD600 of 0.02 (see the volume of preloading culture and media in Excel (normalisation tab) Sheet_1) in 20 ml LB medium + Chloramphenicol in 50 mL falcon tube.
Incubate the cultures at 37°C and 220 rpm.
Take 200 μL (1% of total volume) samples of the cultures at 0, 1, 2, 3, 4, 5, and 6 hours of incubation
Place samples on ice.
At the end of sampling point you need to measure your samples (OD and Fl measurement), see the below for details.
Record data in your notebook
Import data into Excel (cell measurement tab) Sheet_1 provided

Measurement

It is important that you use the same instrument settings that you used when measuring the FITC standard curve. This includes using the sample volume 100 ul. Samples should be laid out according to the figure below. Pipette 100 μl of each sample into each well of the clear plate and then 100 ul of sample into each well of the black plate. Set the instrument settings as those that gave the best results in your calibration curves (no measurements off scale). If necessary you can test more than one of the previously calibrated settings to get the best data (no measurements off scale).
Hint: No measurement off scale means the data you get does not out of range of your calibration curve.

Transformation Protocol

Materials:
Resuspended DNA
10pg/µl Control DNA
Competent Cells
2ml Microtubes
Floating Foam Tube Rack
Ice & ice bucket
Lab Timer
42°C water bath
SOC Media
37°C incubator
LB broth
Chloramphenicol
Petri plates w/ LB agar and chloramphenicol antibiotic
Sterile spreader or glass beads
Pipettes and Tips
Protocol

1. Thaw competent cells on ice
2. Pipette 25µl of competent cells into 2ml tube
3. Pipette 1µl of resuspended DNA into 2ml tube
4. Pipette 1µl of control DNA into 2ml tube
5. Close 2ml tubes, incubate on ice for 30min
6. Heat shock tubes at 42°C for 1 min
7. Incubate on ice for 5min
8. Pipette 200µl SOC media to each transformation
9. Incubate at 37°C for 2 hours, shaker or rotor recommended:
10. Pipette each transformation on two chlor petri plates for a 20µl and 200µl plating
11. Incubate transformations overnight (14-18hr) at 37°C
12. Pick single colonies and inoculate in 5ml of LB with chlor antibiotic to grow up cell cultures overnight (14-18hr) at 37°C

Continuous culture protocol from Execter iGEM

This is the protocol we are using for the sequential batch culture, we are also using a ministat to continuous culture our kill switch. This is the main part of our project and is quite a lot of work so adapt this as you like to fit it in.
Make LB broth and No Salt LB broth.
LB broth recipe for 1 litre is 10g Tryptone, 5g Yeast extract, 10g NaCl
No salt LB is as above but No NACl (we are doing this as we can’t find a reason for having salt in LB and are interested to see the growth characteristics)
Transform your killswitch into whichever strain is best for your killswitch and overnight a colony in 5 ml of LB broth.
Take the OD in triplicate of the overnight and get an average. Use whatever settings you have for your plate reader. Calculate the amount of overnight culture you need to add to get a starting OD of 0.05 in 50 ml of media
Use this equation (0.05*50)/starting average OD
Put 50 ml of the media (one of LB and one of No salt LB) into 250ml erlenmeyer flasks and add antibiotics for the control you are using to the recommended iGEM concentration. Amp- 100 micrograms/ml, Cm- 35 micrograms /ml, Kan- 50micrograms/ml, Tet- 5 microgram/ml
Innoculate with the required amount and incubate the flasks at 37 degrees and 220 rpm.
Each morning and evening, take the OD of the culture and add to a fresh flask as before to reach a starting OD of 0.05 again. We are not allowed in the lab before nine or after five thirty so these are the times we will be doing it but whatever suits you is fine. This will keep the culture going.
Take a sample from the culture and make a glycerol stock using 0.5ml of sample and 0.5ml of 50% glycerol every day in week 1 and every two days in week 2 (this is what we are doing if it’s too much then however is fine). Then test all of these in whichever way is appropriate for your killswitch.
If you could miniprep and send for sequencing DNA from each of your samples this would also be really interesting as we would like to know how much mutation happens, and how much needs to happen in order for a kill switch no longer be functional.

3A Assembly Protocol

This protocol is used to combine two parts into a plasmid backbone with higher efficiency than standard assembly

Materials

• RFC 10 compatible parts
• Linearlized plasmid backbone
• EcoRI
• XbaI
• SpeI
• PstI
• NEB Buffer 2
• BSA
• dH2O
• DNA Purification kit
• Transformation materials (with positive/negative agar plate controls)

Procedure

1. Digest the first Part with EcoRI and SpeI (may use EcoRI-HF instead of EcoRI)
1. Fill a PCR tube with the following reaction (see Discussion for example):
   1. 500 ng plasmid DNA (see Discussion for example calculation)
   2. Fill to 50μL with 1X NEB Buffer 2 (comes as 10X so you have to dilute)
   3. .5 μl of 100X BSA (see Discussion for calculation)
   4. 1 μl each enzyme (always add enzyme last!)
2. Incubate at 37°C for 60 min then at 80°C for 20 min to heat inactivate enzymes (return to 4°C forever at the end)
3. Place BSA solution in freezer after use
2. Digest the second Part with XbaI and PstI
3. Digest plasmid backbone with EcoRI and PstI
4. Purify restriction digests with DNA purification kit (optional)
5. Ligate the two constructs and the linearized plasmid together
   1. For a total reaction volume of 20 μl, add 2 μl from each digest, 1 μl T4 DNA ligase, and 1X T4 DNA ligase reaction buffer
   2. Incubate ligation at 16 degrees celsius overnight
   3. Heat shock at 65 for 20 minutes
   4. Store products at -20°C if not used immediately for transformation
6. Transform the ligation product

First, digest the first Part with EcoRI and SpeI (may use EcoRI-HF instead of EcoRI). Next, Fill a PCR tube with the following reaction: 500 ng plasmid DNA, fill to 50 μL with 1X NEB Buffer 2 (comes as 10X so you have to dilute), 0.5 μL of 100X BSA, and 1 μL of each enzyme. Incubate at 37°C for 30 min then at 80°C for 20 min to heat inactivate enzymes (return to 4°C forever at the end). Digest the second Part with XbaI and PstI and digest plasmid backbone with EcoRI and PstI. Purify restriction digests with DNA purification kit. Then, ligate the two constructs and the linearized plasmid together. For a total reaction volume of 20 μl, add 2 μl from each digest, 1 μl T4 DNA ligase, and 1X T4 DNA ligase reaction buffer. Incubate ligation at room temperature for 10 min. Store products at -20°C if not used immediately for transformation. Finally, transform the ligation product.

Discussion
DNA Calculation Example (with 25ng/μL DNA)
500ng DNA * (1μL/25ng) = 20 μl DNA solution

Making 100X BSA Solution

1. Cover a 100mL bottle with foil
2. Add 100mL of ddH20
3. Measure and add 1g of BSA
4. Swirl to dissolve

Total Digest Reaction Example
Say you have 74.7ng/μL DNA

1. Add 6.69μl DNA
2. Add .5 μl 100X BSA
3. Add 1 μl of first enzyme
4. Add 1 μl of second enzyme
5. Add 4.08 μl of 10X NEB Buffer 2
6. Add 36.73 μl of ddH20

Source: http://dspace.mit.edu/bitstream/handle/1721.1/65066/BioBrickAssemblyFinalAuthorsVersion.pdf?sequence=2

Acid Digestion for Phosphates

Purpose
This is a protocol for converting both polyphosphates and organic phosphates to the orthophosphate form.

Materials
Phenolphthalein
Sulfuric acid solution
Carefully add 300 mL concentrated Sulfuric acid to approximately 600 mL distilled water and dilute to 1 L with distilled water.
Ammonium persulfate, crystal
Sodium hydroxide, 1M

Equipment
Hot plate
125 mL Erlenmeyer flask (acid washed)
50 mL graduated cylinders (acid washed)

Protocol
Measure 50 mL or an appropriate amount of sample diluted to 50 mL with distilled water. Add to the Erlenmeyer flask.
Add 1 drop phenolphthalein indicator. If a red color develops, add sulfuric acid solution until color just disappears.
Add 1 mL of sulfuric acid solution and 0.4 g of ammonium persulfate.
Boil gently for 30 to 40 minutes or until the total volume is 10 mL.
Cool, add 1 drop of phenolphthalein and neutralize to a faint pink color with 1 N sodium hydroxide.
Make up to 50 mL with distilled water. The digested sample is then tested for total phosphate as outlined in Section 12.

Discussion
Provide any commentary or advice you or another person has for running this protocol

Source
Pennsylvania Department of Environmental Protection Document “Chapter 8: TOTAL PHOSPHORUS”

Paragraph form
Obtain a 50 mL sample of the phosphate solution and place in the Erlenmeyer flask. If necessary, obtain a smaller volume of sample and dilute to 50 mL. Add a drop of phenolphthalein to the sample and, if the sample turns red, add drops sulfuric acid until the red color disappears. Then, add one mL of the prepared sulfuric acid solution and 0.4 grams of ammonium persulfate to the solution. Using the hot plate, boil the solution gently for 30 to 40 minutes, when the total volume is 10 mL. Let the solution cool, and then add another drop of phenolphthalein. Add drops of the 1 M sodium hydroxide solution until the solution is a faint pink color. Add distilled water to bring the total volume back to 50 mL.

Ascorbic Acid Procedure

Purpose
This is a protocol for determining the concentration of phosphate-phosphorus in a solution.

Materials
Phenolphthalein
Sulfuric acid solution
Dilute 70 mL of concentrated Sulfuric acid to 500 mL with distilled water
Potassium antimonyl tartrate solution, 4.1 mM
Dissolve 1.3715 g of Potassium antimonyl tartrate in 400 mL of distilled water in a 500 mL volumetric flask and dilute to 500 mL. Store in a glass stoppered bottle
Ammonium molybdate Solution, 0.032 M
Dissolve 20 g of Ammonium molybdate in 500 mL of distilled water. Store in a glass stoppered bottle
Ascorbic acid solution, 0.1 M
Potassium dihydrogen phosphate solution, 5 mg/L

Equipment
Spectrophotometer with an infrared phototube for use at 880 nm with a light path of at least 2.5 cm (1 in)
An analytical balance capable of weighing to 0.1 mg accuracy
Acid washed glassware

Protocol
If the calibration plot has yet to be prepared, dilute the potassium dihydrogen phosphate to produce three samples of 50 mL for each of the following concentrations: 0.1 mg/L, 0.2 mg/L, 0.4 mg/L, 0.6 mg/L, 0.8 mg/L, and 1.0 mg/L. On each sample, perform the acid digestion and the ascorbic acid procedure. Use the average result of the three samples for each concentration when creating the calibration plot.
Prepare one sample of a known phosphorus concentration and perform the acid digestion and ascorbic acid procedure. Compare the known concentration to the results obtained via the calibration plot in order to check the accuracy of the calibration plot. Mix 50 mL of the 5 M sulfuric acid solution with 5 mL of of the potassium antimonyl tartrate solution to an acid cleaned, dry 125 mL Erlenmeyer flask and then mix thoroughly. Then,add 15 mL of the ammonium molybdate solution to the mixture and mix thoroughly again. Then, add 30 mL of the ascorbic acid solution and mix the solution again. If turbidity occurs, shake and then let stand until turbidity clears. This solution is only stable for 4 hours. Then, place 50 mL of sample into a fresh, acid cleaned, dry 135 mL Erlenmeyer flask. Add a drop of phenolphthalein, and, if necessary, add drops of sulfuric acid until a red color disappears. Take 8 mL of the combined reagent previously mixed and add to the flask with the sample. Let the sample sit for 10 to 30 minutes. Measure the absorbance of the solution using a spectrophotometer at a wavelength of 880 nm. The blank should be prepared by carrying out an acid digestion and ascorbic acid procedure for 50 mL of distilled water. Then, compare the results of the sample against a calibration curve

If this is the first run through of the procedure, start here to create a calibration curve. If not, skip to step 4

1. Dilute the 5 mg/L standard potassium dihydrogen phosphate to produce 3 samples for each concentration of 50 mL. each

	final conc.	volume of standard	final vol. in flask
a	0.1 mg/L	1.0 mL	50.0 mL
b	0.2 mg/L	2.0 mL	50.0 mL
c	0.4 mg/L	4.0 mL	50.0 mL
d	0.6 mg/L	6.0 mL	50.0 mL
e	0.8 mg/L	8.0 mL	50.0 mL
f	1.0 mg/L	10.0 mL	50.0 mL

2. Perform the acid digestion and the ascorbic acid procedure (starting at step 5) for each of the 18 samples.
3. Use the average of absorbance or transmittance for each concentration to preparing the curve.
   1. If the curve appears to differ from a straight line, the curve may be straightened to approximate a straight line by plotting the data on semi-logarithmic graph paper
4. Prepare 1 sample from step 1 and run through both the acid digestion and ascorbic acid procedure in order to verify the accuracy of the calibration curve.
5. Keep the sulfuric acid solution, potassium antimonyl tartrate solution, the ammonium molybdate solution, and the ascorbic acid solution at room temperature
6. Add 50.0 mL of 5 M sulfuric acid solution and 5 mL of the potassium antimonyl tartrate solution to an acid cleaned, dry 125 mL Erlenmeyer flask. Mix thoroughly.
7. Add 15 mL of ammonium molybdate solution. Mix thoroughly.
8. Add 30 mL of ascorbic acid solution. Mix thoroughly. If turbidity occurs, shake and let stand until turbidity clears. This solution is only stable for 4 hours.
9. Pipette 50.0 mL or an appropriate amount diluted to 50 mL of digested sample into an acid cleaned, dry 125 mL Erlenmeyer flask.
10. Add 1 drop of phenolphthalein indicator to the digested sample. If a red color develops, add 5 N sulfuric acid until the color disappears.
11. Add 8.0 mL of the combined reagent and mix thoroughly.
12. Allow at least 10 minutes (but not more than 30 minutes) for color development.
13. Measure absorbance at 880 nm using a reagent blank to zero the spectrophotometer.
    1. The reagent blank is made using 50 mL of distilled water carried through the digestion step and ascorbic acid procedure.
    2. In highly colored or turbid samples, prepare a sample blank by adding all the reagents to the sample except the ascorbic acid and potassium antimonyl tartrate. Subtract the absorbance of this blank from the absorbance of the sample.
14. Check the sample’s absorbance against the calibration curve and determine the concentration. Correct for dilution.

Source
Pennsylvania Department of Environmental Protection Document “Chapter 8: TOTAL PHOSPHORUS”

Bioreactor Flow Rate Protocol

Purpose
To consistently measure flow rate from the bioreactor system

Materials
Bioreactor
Dry 1L bottles
Graduated Cylinder (100ml)
Two other people
Water

Equipment
Stopwatch
Thermometer

Protocol
Prepare bioreactor by ensuring tubing is tightly sealed, filter canisters are sitting upright on the table, and the pump is on and refilling the bioreactor. Record the water temperature. Ensure the water level in the bioreactor is staying at the constant level around the overflow port before opening any outlet streams. Adjust the inlet valve accordingly. If measuring only one outlet stream, open desired outlet stream and adjust bioreactor inlet valve to maintain constant water level in bioreactor. Let the effluent run into the large beaker while adjusting valves. Prepare for sampling by obtaining dry bottles and someone to time. Place the bottle in the already running effluent stream and immediately begin timing for 10 seconds. Remove the bottle from the stream at 10 seconds, DO NOT close the valve at 10 seconds. If measuring all outlet streams, follow the above procedure identically at each outlet valve. Make sure to adjust the inlet stream to maintain a constant water level. Three people are recommended, one per outlet stream and one additional person to run the stopwatch. Measure the effluent liquid volume in a large graduated cylinder. Repeat the process to get an average volume for 10 seconds. Divide the average output volume by 10 seconds to get flow rate per second.

E. coli Transformation Protocol

Purpose
To transform parts from the registry into competent cells

Materials

1. Parts from Kit Plates
2. Plates with Antibiotic Resistance (Must be warmed up in 37℃ room)
   1. 2 plates per part (specific antibiotic type depends on part resistance)
   2. 1 plate for negative control (for each type of antibiotic used)
3. 1 LB Plate (for positive control)
4. 1 LB Plate w/ amp for blank plate
5. Eppendorf Tubes
1. 2 tubes for controls
2. 1 tube per part
6. PCR Tubes (1 tube per part)
7. 5 alpha Competent cells
   1. 50µL for controls
   2. 25µL per part
8. SOC Media
   1. 400µL for controls
   2. 200µL per part
9. Ice
10. Glass Beads
11. Pipettes and Tips (Filtered Tips if possible)
12. Deionized Water

Equipment

1. 42℃ Water Bath (BIND 134 balance room)
2. 37℃ Incubator (with shaker)

Expected Data

• Cell growth observed on the Positive Control (Ensures that cells are growing properly)
• No cell growth observed on the Negative Control (Ensures that the antibiotic is functioning)
• Observed growth and fluorescence on the RFP control plate

Paragraph Form
The purpose of this protocol is to transform parts from the registry into competent cells. Prepare the lab space by wiping the counter with ethanol and lighting a flame. Next, add 10µL of DI water to the kit plates and pipette up and down. Transferred this to a labeled PCR tube and repeat for each part. Warm the water bath to 42℃. Transfer competent cells to ice from the -80℃ freezer. Next, add 25µL of competent cells to each Eppendorf tube. The top of the cell containers should be marked to show use, and each Eppendorf tube should be labeled with the part name of control. Return the cells to the -80℃ freezer as quickly as possible. Next, add 4 µL of DNA were to the experimental Eppendorf tubes. Mix the DNA well and store leftover DNA was in PCR tubes in the -20℃ freezer. Incubate tubes on ice for 45 minutes. Next, heat shock cells at 42℃ for 60 seconds to open the cell walls for DNA insertion. Place Eppendorf tubes into Styrofoam holders and place into the water bath. Time very precisely and immediately place tubes back into ice after 60 seconds. After about a minute, move tubes around in ice because the nearby ice has probably melted. Put cells back on ice for 5 minutes. Add 200µL of SOC media to each tube. Incubate cells for 2 hours at 37℃ at 250 rpm. Next, plate 200µL of the cells on antibiotic plates for the experimental and negative control. Plate 200µL of cells on LB plates labeled positive control. Scatter the cell suspension in drops on the plate. Pour about five glass beads onto the plate. Swirl the plate to move the glass beads around and evenly coat the media with the liquid suspension of cells. Ensure that all plates are properly labeled. Incubate plates overnight from 12-18 hours at 37℃. There should be cell growth on the positive control and no cell growth on the negative control.

Extraction and Quantification of Intracellular Poly-P

Reagents

• Sodium phosphate glass type 45 (Sigma-Aldrich, S4379-100MG-089K5011)
• Toluidine blue dye (Loba Chemie, Cat. No. 52040-25GM)
• Glacial acetic acid (Rankem, Product Code A0030)
• Chloroform (HiMedia, Cat. No. AS039-2.5LT)
• Isoamyl alcohol (HiMedia, Cat. No. MB091-500ML)
• De-ionized water

Equipment

• Boiling water bath (OVFU)
• Sonicator (Sartorius Stedim Labsonic R M)
• Centrifuge (Remi C-24 Plus)
• Spectrophotometer (Intech Microprocessor Uv-Vis Spectrophotometer Single Beam 290)

Procedure Preparation of standard curve of sodium phosphate glass type 45

1. Weigh 0.3 mg of sodium phosphate glass type 45 and dissolve it in 150 µl de-ionized water to make a 2 µg/ µl standard stock solution.
2. Make 30 mg/ L toluidine blue stock solution with double distilled water.
3. Make 0.2 N acetic acid stock solution with glacial acetic acid and double distilled water.
4. Use 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 µl of the standard stock solution to obtain 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 µg polyphosphate in the experimental set up.
5. Make three replicates of each set up.
6. Set up the experiment in test tubes following Table 1. (I suggest changing amount of water to make volume constant)
   Vortex the contents of each test tube and incubate for 15 minutes at 25°C.
   Record the absorbance at 630 nm, by setting de-ionized water as blank.
   Prepare a standard curve of sodium polyphosphate glass type 45 in Microsoft Excel by plotting the amount of polyphosphate in the X-axis and the A630 in the Y-axis (Figure 1).

   Microbial cell lysis for extracting polyphosphate granule

   Take 5 ml of bacterial culture grown for 72 hours at 37°C in a medium with phosphorus source.
   Make 2 mM with K2PO4, as https://www.ncbi.nlm.nih.gov/pmc/articles/PMC124021/ says that is a sufficient
   Stock - add 45 ul of 0.22 M stock to 5 ml of culture
   Take 10 ml of microalgal and cyanobacterial culture grown for 25 days at 28°C in BG-11 (non-N2 fixer medium) and BG-0 (N2 fixer medium).
   Centrifuge the samples at 2,350 g for 5 minutes. Discard the supernatant.
   Dissolve the cell pellets in 500 µl autoclaved de-ionized water and centrifuge at 2350 g for 5 minutes. Discard the supernatant.
   Take the fresh weight of the samples.
   Add 600 µl of de-ionized water to the samples and mix by flickering.
   Sonicate the samples for 5 minutes at 30 Hz (Cycle 0.5, Amplitude 65%).
   Place the tubes containing the samples in boiling water bath at 100°C and boil for 2 hours.

   Recovery of polyphosphate granules and quantification by spectrophotometer

   After boiling, cool down the tubes at room temperature.
   Add 600 µl of 24:1 (v/v) chloroform: isoamyl alcohol solution to all the tubes. Mix by vigorous shaking.
   Centrifuge at 13,520 g for 15 minutes at room temperature.
   Collect the upper aqueous phase in separate tubes. CRITICAL STEP: Care should be taken to pipette the aqueous phase without disturbing the organic phase. The presence of even small amount of organic phase in the next steps may give ambiguous results.
   Take 300 µl of the aqueous phase in a fresh test tube. Add 3 ml each of toluidine blue solution (Stock conc. of 30 mg/ L) and 0.2 N acetic acid solution. Mix by gentle vortexing and incubate for 15 minutes at 25°C till the colour of the solution changes from blue to purple. CRITICAL STEP: The change in colour implies that polyphosphate is successfully extracted and is present in the aqueous phase. No colour change indicates that extraction is unsuccessful and has to be done again.
   Make a control in the same way with 300 µl de-ionized water.
   Record the absorbance at 630 nm, by setting de-ionized water as blank.
   Calculation of the amount of polyphosphate in the microbial cells
   Calculate the amount of polyphosphate present in the samples by trend analysis of its A630 on the standard curve (Figure 1).
   Calculate the amount of polyphosphate in µg present per gm of sample fresh weight by the following formula: (amount of polyphosphate in ug/ gm of sample fresh weight) = 1,000 x (ug of polyphosphate from standard curve) / (fresh weight of sample in mg)
   Source: http://www.nature.com/protocolexchange/protocols/4073#/anticipated_results
   

   Serial Number	Amount of standard polyphosphate (ug)	Volume of standard polyphosphate solution added (uL)	Volume of de-ionized water added (uL)	Volume of toluidine blue solution added (ml)	Volume of acetic acid solution added (ml)
   1	0	0	300	3	3
   2	2	1	300	3	3
   3	4	2	300	3	3
   4	6	3	300	3	3
   5	10	5	300	3	3
   6	12	6	300	3	3
   7	14	7	300	3	3
   8	16	8	300	3	3
   9	18	9	300	3	3
   10	20	10	300	3	3
   11	22	11	300	3	3
   12	24	12	300	3	3
   13	26	13	300	3	3
   14	28	14	300	3	3
   15	30	15	300	3	3