Team:KCL UK/InterLab
KCL_UK InterLab
Introduction
Through iGEM Interlab, teams from all over the world participating in iGEM collaborate to help reduce lab to lab variability in measurements.
This year the question we are trying to answer is: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
Below shows the protocol we followed in order to help answer this question.
Materials
- Measurement Kit containing:
- 1ml LUDOX CL-X
- 150 μL Silica Bead (microsphere suspension)
- Fluorescein (powder, in amber tube)
- iGEM Parts Distribution Kit Plates (you will obtain the test devices from the parts kit plates)
- 1x PBS (phosphate buffered saline, pH 7.4 - 7.6)
- ddH2O (ultrapure filtered or double distilled water)
- Competent cells (Escherichia coli strain DH5α)
- LB (Luria Bertani) media
- Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
- 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
- Incubator at 37°C
- 1.5 ml eppendorf tubes
- Ice bucket with ice
- Micropipettes (capable of pipetting a range of volumes between 1 μL and 1000 μL)
- Micropipette tips
- 96 well plates, black with clear flat bottom preferred, at least 3-4 plates
Calibration Protocols
Calibration 1: OD600 Reference point - LUDOX Protocol
We used LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform our absorbance (Abs600) data from our plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer. We turned off pathlength correction when taking measurements from our plate reader.
Method
We used LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform our absorbance (Abs600) data from our plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer. We turned off pathlength correction when taking measurements from our plate reader.
- Add 100 μl LUDOX into wells A1, B1, C1, D1
- Add 100 μl of dd H2O into wells A2, B2, C2, D2
- Measure absorbance at 600 nm.
We recorded the data as shown below.
Calibration 2: Particle Standard Curve - Microsphere Protocol
We prepared a dilution series of monodisperse silica microspheres and measured the Abs600 in our plate reader. The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement allowed us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.
Method
Prepare the Microsphere Stock Solution:
- Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex vigorously for 30 seconds.
- Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
- Add 904 μL of ddH O to the microspheres
- Vortex well. This is our Microsphere Stock Solution.
Prepare the serial dilution of Microspheres:
- Add 100 μl of ddH2O into wells A2, B2, C2, D2. A12, B12, C12, D12
- Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds
- Immediately add 200 μl of microspheres stock solution into A1
- Transfer 100 μl of microsphere 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…
- Continue this until A11
- Mix A11 by pipetting up and down 3x and transfer 100μl into liquid waste
- Repeat dilution series for rows B, C, D
- Measure Abs of all samples in instrument
- We then recorded our data, as shown below.
Calibration 3:Fluorescence standard curve - Fluorescein Protocol
We prepared a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in our plate reader. By measuring these in our plate reader, we were able to generate a standard curve of fluorescence for fluorescein concentration. Were were able to use this to convert our cell based readings to an equivalent fluorescein concentration.
Method
Prepare the fluorescein stock solution:
- Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
- Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM: 100 μL of 10x fluorescein stock into 900 μL 1x PBS
Prepare the serial dilutions of fluorescein:
- Add 100 μl of PBS into wells A2, B2, C2, D2...A12, B12, C12, D12
- Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1
- Transfer 100 μl of fluorescein 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…
- Continue this until A11
- Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste
- Repeat dilution series for rows B, C, D
- Measure fluorescence of all samples in instrument
- We then recorded the data as shown below
Cell measurement Protocol
Method
Day 1: transform Escherichia coli DH5α; with these following plasmids (all in pSB1C3):
Day 2: Pick 2 colonies from each of the transformation plates and inoculate in 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
- Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
- Measure Abs600 of these 1:10 diluted cultures
- Record the data
- Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube covered with foil to block light.
- Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. Place the samples on ice.
- Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
- Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice.
- Measure Abs600 and fluorescence of the samples from the 0 hour and 6 hour time point.
- Record data as shown below.
Protocol: Colony Forming Units per 0.1 OD600E. coli cultures
This procedure can be used to calibrate OD600 to colony forming unit (CFU) counts, which are directly relatable to the cell concentration of the culture, i.e. viable cell counts per mL. This protocol assumes that 1 bacterial cell will give rise to 1 colony.
For the CFU protocol, we needed to count colonies for our two Positive Control cultures and our two Negative Control cultures.
Step 1: Starting Sample Preparation
This protocol will result in CFU/mL for 0.1 OD600.
1.Measure the OD600 of our cell cultures. Include blank media (LB + Cam) as well.
Preparation: Add 25 µL culture to 175 µL LB + Cam in a well in a black 96-well plate, with a clear, flat bottom. Each well should have 200 µL .
2.Dilute overnight culture to OD600 = 0.1 in 1mL of LB + Cam media. Do this in triplicate for each culture.
Use (Cı)(Vı) = (C2)(V2) to calculate your dilutions
Step 2: Dilution Series Instructions
- Prepare three 2.0 mL tubes (36 total) with 1900 µL of LB + Cam media for Dilutions 1, 2, and 3
- Prepare two 1.5 mL tubes (24 total) with 900 µL of LB + Cam media for Dilutions 4 and 5
- Pipet 100 µL of Starting Culture into Dilution 1. Discard tip. Vortex tube for 5-10 secs.
- Repeat Step 3 for each dilution through to Dilution 5 as shown below.
- Aseptically spread plate 100 µL on LB + Cam plates for Dilutions 3, 4, and 5.
- Incubate at 37°C overnight and count colonies after 18-20 hours of growth.
Step 3: CFU/mL/OD Calculation Instructions
Based on the assumption that 1 bacterial cell gives rise to 1 colony, we calculated colony forming units (CFU) per 1mL of an OD600 = 0.1 culture, as follows:
- Count the colonies on each plate with fewer than 300 colonies.
- Multiple the colony count by Final Dilution Factor of 8 X 10^5 for Dilution 4.
- We recorded the data as shown below