Team:Mingdao/InterLab

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Interlab Study

Note

Description: the goal and main contents were quoted from iGEM International InterLab Measurement Study

Methods: the protocol was provided by iGEM InterLab Committee and described briefly in here

Results: the experiment and data presented here were all made by members of team Mingdao

Reference: Fifth International InterLab Measurement Study@iGEM

Instrument

The machine in the Biolab of Mingdao High School: Synergy H1 Hybrid Multi-Mode Microplate Reader



Introduction

"Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology. However, the ability to repeat measurements in different labs has been difficult. The Measurement Committee, through the InterLab study, has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. We chose GFP as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein."

Goal for the Fifth InterLab

"The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. Until we reach this point, synthetic biology will not be able to achieve its full potential as an engineering discipline, as labs will not be able to reliably build upon others’ work."

"This year, teams participating in the interlab study helped iGEM to answer the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?"

Calibration Reference

Calibration 1:OD600 Reference point - LUDOX Protocol

Materials

1ml LUDOX CL-X

ddH2O

96 well Black Clear Bottom Plate

Method

↓ Add 100 μl LUDOX into wells A1, B1, C1, D1

↓ Add 100 μl of ddH2 O into wells A2,B2,C2,D2

↓ Measure absorbance at 600 nm

↓ Record the data

Result

The table shows the OD600 measured by a spectrophotometer (see table above) and plate reader data for H2O and LUDOX corresponding to the expected results. The corrected Abs600 is calculated by subtracting the mean H2O reading. The reference OD600 is defined as that measured by the reference spectrophotometer. The correction factor to convert measured Abs600 to OD600 is thus the reference OD600 divided by Abs600. All cell density readings using this instrument with the same settings and volume can be converted to OD600 by multiplying by 4.200.

Calibration 2: Particle Standard Curve - Microsphere Protocol

Materials

300 μL silica beads Microsphere suspension

ddH2O

96 well Black Clear Bottom Plate

Method

Preparation of the Microsphere stock solution:

↓ Obtain Silica Beads

↓ Pipet 96 μL beads into an eppendorf

↓ Add 904 μL of ddH2O to the microspheres

↓ Vortex well to obtain stock Microsphere Solution.

↓ Preparation of microsphere serial dilutions as follows

↓ Measure Abs 600

↓ Record the data

Result

Raw Data

Particle Standard Curve

Particle Standard Curve(log scale)

Calibration 3: Fluorescence standard curve - Fluorescein Protocol

Plate readers report fluorescence values in arbitrary units that vary widely from instrument to instrument. Therefore absolute fluorescence values cannot be directly compared from one instrument to another. In order to compare fluorescence output of test devices between teams, it is necessary for each team to create a standard fluorescence curve. Although distribution of a known concentration of GFP protein would be an ideal way to standardize the amount of GFP fluorescence in E. coli cells, the stability of the protein and the high cost of its purification are problematic. The Interlab Study therefore uses the small molecule fluorescein, which has similar excitation and emission properties to GFP, but is cost-effective and easy to prepare. (The version of GFP used in the devices, GFP mut3b, has an excitation maximum at 501 nm and an emission maximum at 511 nm; fluorescein has an excitation maximum at 494 nm and an emission maximum at 525nm).

Teams will prepare a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in your plate reader. By measuring these in the plate reader, a standard curve of fluorescence for fluorescein concentration will be generated. THus, different teams will be able to use this to convert their cell based readings to an equivalent fluorescein concentration. Before beginning this protocol, teams should ensure that they are familiar with the GFP settings and measurement modes of their instrument. Each team needs to know what filters your instrument has for measuring GFP, including information about the bandpass width (530 nm / 30 nm bandpass, 25-30nm width is recommended), excitation (485 nm is recommended) and emission (520-530 nm is recommended) of this filter.

Materials

Fluorescein (provided in kit)

10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)

96 well Black Clear Bottom Plate

Method

Prepare the fluorescein stock solution

1. Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.

2. Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. [ Note : it is important that the fluorescein is properly dissolved. To check this, after the resuspension you should pipette up and down and examine the solution in the pipette tip – if any particulates are visible in the pipette tip continue to mix the solution until they disappear.]

3. 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 1xPBS

Prepare the serial dilutions of fluorescein

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 fluorescein 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.

1. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12

2. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1

3. Transfer 100 μl of fluorescein stock solution from A1 into A2.

4. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3

5. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...

6.Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...

7.Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...

8.Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...

9. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...

10. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...

11. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...

12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...

13. 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.

14. Repeat dilution series for rows B, C, D

15. Measure fluorescence of all samples in instrument

16. Record the data in your notebook

17. Import data into Excel sheet provided ( fluorescein standard curve tab )

Result

Raw Data

Fluorescein Standard Curves

Fluorescein Standard Curves(log scale)

Cell Measurement

Materials

 Competent cells ( Escherichia coli strain DH5 )

 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 for sample storage

 Ice bucket with ice

 Micropipettes and tips

 96 well Black Clear Bottom Plate

Workflow

Method

Day1

↓ Transform Escherichia coli DH5 with these plasmids

Day2

↓ Pick 2 colonies from each group

↓ Inoculate in 5-10 mL LB medium + Cm

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

Day 3

↓ Make a 1:10 dilution of each overnight culture in LB + Cm by putting 0.5mL of culture into 4.5mL of LB + Cm

↓ Measure Abs 600 of these 1:10 diluted cultures

↓ Record the data

↓ Dilute the cultures further to a target Abs6 00 of 0.02 in a final volume of 12 ml LB medium + Cm in 50 mL tube

↓ Incubate the cultures at 37°C and shake at 220 rpm for 6 hours.

↓ Measure your samples for Abs600 and fluorescence

↓ Record data in your notebook

Layout for Abs 600 and fluorescence measurement

Result

Fluorescence Raw Reading

Abs600 Raw Reading

Colony Forming Units per E. coli cultures at OD600=0.1

↓ Measure the OD600 of your cell cultures

↓ Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cm media. Do this in triplicate.

↓ Make the following serial dilutions for your triplicates

↓ Aseptically spread plate with 100 μL of the dilutions

↓ Incubate at 37°C overnight

↓ Count colonies after 18-20 hours of growth.

Result

Colony Forming Units per o.1 OD600 E.coli cultures

    Interlab Study

  • - Introduction
  • - Goal
  • - Calibration 1
  • - Calibration 2
  • - Calibration 3
  • - Cell Measurement
  • - Protocol