Team:Stockholm/InterLab

iGEM Stockholm 2018 Wiki

InterLab Study

Aim

In earlier InterLab studies, iGEM aimed to standardize Green Fluorescent Protein (GFP) measurement by determining how comparable the fluorescence measurements were when conducted in different laboratories around the world. However, they have found that when the population of cells were measured in bulk, there is still a great source of variability because the number of cells in the sample differs. For this year’s Fifth InterLab Study, we aimed to reduce lab-to-lab variability in the measurement of fluorescence by standardizing it to Colony-Forming Units (CFU) or absolute cell number. From here we hope to solve the inconsistency in measurements between different laboratories and increase the precision of fluorescence measurements.

Overview

Reproducibility of results between various different laboratories has proven difficult in scientific research. Often times, laboratory protocols need some adjustments to work in the same manner in different conditions and laboratories. The iGEM Measurement Committee has aimed throughout the years to develop and standardize a measurement procedure for GFP in hopes to have a reliable and repeatable measurement. For this year’s InterLab Study 2018, we are reducing this laboratory variability by standardizing Optical Density (OD) measurements to either CFU or with absolute cell number. The protocol to perform this analysis is provided by the measurement committee as well as the necessary parts and reagents to conduct our measurements.

Procedure and Materials

Members

Chrismar Garcia and Stamatina Rentouli performed all the planning, the experiments and the analysis. Hanna Norbäck assisted in preparing the plates for CFU and planning the experiment.

BioBricks used for the Study

Negative control BBa_R0040
Positive control BBa_I20270
Test Device 1 BBa_J364000
Test Device 2 BBa_J364001
Test Device 3 BBa_J364002
Test Device 4 BBa_J364007
Test Device 5 BBa_J364008
Test Device 6 BBa_J364009

Equipment

  • Plate reader: BMG Labtech CLARIO star
  • Flow Cytometer: Beckman Gallios

Materials

  • 1 ml LUDOX CL-X
  • ddH2O
  • 96 well plate

Methods

  1. 100μL of LUDOX from InterLab are added into wells A1, B1, C1, D1
  2. 100μL of ddH2O into wells A2, B2, C2, D2
  3. Absorbance at 600 nm are measured for all samples
  4. Record the data in the table provided

Materials

  • 300 μL Silica beads
  • ddH2O
  • 96 well plates

Methods

  1. 100μL of ddH2O are added into wells A2, B2, C2, D2...A12, B12, C12, D12
  2. 200μL of microspheres stock solutions are then added into A1
  3. Transfer 100μL of microsphere stock solution from A1 into A2
  4. Mix A2 by pipetting up and down 3x and transfer 100μL into A3
  5. Mix A3 by pipetting up and down 3x and transfer 100μL into A4
  6. Mix A4 by pipetting up and down 3x and transfer 100μL into A5
  7. Mix A5 by pipetting up and down 3x and transfer 100μL into A6
  8. Mix A6 by pipetting up and down 3x and transfer 100μL into A7
  9. Mix A7 by pipetting up and down 3x and transfer 100μL into A8
  10. Mix A9 by pipetting up and down 3x and transfer 100μL into A10
  11. Mix A10 by pipetting up and down 3x and transfer 100μL into A11
  12. Mix A11 by pipetting up and down 3x and transfer 100μL into liquid waste
  13. Repeat serial dilution for rows B, C, D
  14. Measure Abs600 of all samples

Materials

  • Fluorescein
  • 10mL 1x PBS pH7.4-7.6
  • 96 well plate

Methods

  1. 100μL of ddH22O are added into wells A2, B2, C2, D2...A12, B12, C12, D12
  2. 200μL of microspheres stock solutions are then added into A1
  3. Transfer 100μL of microsphere stock solution from A1 into A2
  4. Mix A2 by pipetting up and down 3x and transfer 100μL into A3
  5. Mix A3 by pipetting up and down 3x and transfer 100μL into A4
  6. Mix A4 by pipetting up and down 3x and transfer 100μL into A5
  7. Mix A5 by pipetting up and down 3x and transfer 100μL into A6
  8. Mix A6 by pipetting up and down 3x and transfer 100μL into A7
  9. Mix A7 by pipetting up and down 3x and transfer 100μL into A8
  10. Mix A9 by pipetting up and down 3x and transfer 100μL into A10
  11. Mix A10 by pipetting up and down 3x and transfer 100μL into A11
  12. Mix A11 by pipetting up and down 3x and transfer 100μL into liquid waste
  13. Repeat serial dilution for rows B, C, D
  14. Measure fluorescence of all samples in instrument
Cell Measurement & Transformation

Transformations

Materials

  • Competent cells (DH5 α)
  • LB media
  • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
  • 50mL Falcon tube covered in foil Incubator at 37°C
  • 1.5mL Eppendorf tubes for sample storage
  • Ice bucket with ice
  • Micropipettes and tips
  • 96 well plate

Methods

We made overnight cultures of two colonies of each transformation plate. The transformation was successful and we got colonies in the plates the following day. To further conclude that the transformation was successful we performed a colony PCR to make sure that the BioBricks had the right band size.

Cell Measurements

  • Day 1. Transformation of Escherichia coli DH5-alpha
  • Day 2. Inoculation of transformed cells in LB medium with antibiotic
  • Day 3. Cell growth, Sampling, and Assay
Flow Cytometry

For the Flow Cytometry part we used the SpheroTech Rainbow calibration beads, type RCP-30-5A.

During the cell measurement protocol, we also prepared a sample of SpheroTech beads and placed in well A10 of each plate. After measuring each plate with the plate reader, we collected 10,000 events per well, from all wells with our flow cytometer.

Results

A PDF with our data can be found here.

Calibration 1: OD Reference Point

Figure 1. Abs600 x 4 replicates of both Ludox vs H2O.

Calibration 2: Particle Standard Curve

Figure 2. Abs600 x particle count/100µL (top) and same figure on a logarithmic scale (bottom).

Calibration 3: Fluorescein Standard Curve

Figure 3. Fluorescence x fluorescein concentration (µM) (top) and same figure on a logarithmic scale (bottom).

Colony Forming Units

To be able to directly find the concentration of the culture, we calibrated the OD600 to colony forming units (CFU) in which an assumption of 1 bacterial cell will give rise to 1 colony. For this protocol, we used two Positive control (BBa_I20270) and two Negative Control (BBa_R0040).

This part of the InterLab study is divided into three parts:

Part 1. Preparation of our Starting Sample:

We incubated our culture for 18 hours overnight and measured its OD600 the following day. Since our initial measurement of our culture’s OD600 was high, we diluted this overnight culture into a value of 0.1 with 1mL of LB + Cam media and made triplicate for each control that we had.

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Part 2. Dilution Series:

From the triplicate samples we have made from the previous steps, we then made a serial dilution for each individual replicate giving us a total of 12 Starting sample (6 Positives and 6 for Negative Controls). From our serial dilution, we plated only Dilution 3 (Final Dilution Factor 8 x 104), Dilution 4 (Final Dilution Factor 8 x 105) & Dilution 5 (Final Dilution Factor 8 x 106) for each of the replicate samples.

Part 3. CFU/mL/OD Calculation:

For the last part of this study, we counted the colony on each plate with fewer than 300 colonies and we calculated the colony count by the Final Dilution Factor on each plate:

Example: using Dilution 4

# colonies x Final Dilution Factor = CFU/mL

125 x (8 x 105) = 1 x 108 CFU/mL in Starting Sample