Purdue Biomakers

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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.