Introduction of cell measurement

The goal of cell measurement of the interlab study is to explore a major question: How close can the numbers be when fluorescence is measured all around the world? So we measure GFP fluorescence in our lab with plate reader (Tecan Infinite M200pro). This year, six devices and one positive control and one negative control were provided by the registry.

After the experiment, eight required devices were created:
Positive control: I20270 in pSB1C3;
Negative control: R0040 in pSB1C3;
Test Device 1: J23101.B0034.E0040.B0010.B0012 in pSB1C3;
Test Device 2: J23106.B0034.E0040.B0010.B0012 in pSB1C3;
Test Device 3: J23117.B0034.E0040.B0010.B0012 in pSB1C3;
Test Device 4: J23100.B0034.E0040.B0010.B0012 in pSB1C3;
Test Device 5: J23104.B0034.E0040.B0010.B0012 in pSB1C3;
Test Device 6: J23116.B0034.E0040.B0010.B0012 in pSB1C3.

Protocol of cell measurement

I. Materials

• Measurement Kit (provided with the iGEM distribution shipment)
• iGEM Parts Distribution Kit Plates (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
• Incubator at 37°C
• 1.5 ml eppendorf tubes
• Ice bucket with ice
• Micropipettes
• Micropipette tips
• 96 well plates, black with clear flat bottom preferred

II. Calibration Protocols (before Cell measurement!)

1. OD₆₀₀Reference point - LUDOX Protocol

1) Add 100 μl LUDOX into wells A1, B1, C1, D1.
2) Add 100 μl of ddH₂O into wells A2, B2, C2, D2.
3) Measure absorbance at 600 nm of all samples in the same measurement mode.

2. Particle Standard Curve - Microsphere Protocol

1) Obtain the tube labeled "Silica Beads" from the InterLab test kit and vortex vigorously for 30 seconds.
2) Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube.
3) Add 904 μL of ddH₂O to the microspheres.
4) Vortex well. Obtain Microsphere Stock Solution.
5) Add 100 μl of ddH₂Ointo wells A2, B2, C2, D2....A12, B12, C12, D12.
6) Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds.
7) Immediately add 200 μlof microspheres stock solution into A1.
8) Transfer 100 μl of microsphere stock solution from A1 into A2.
9) Mix A2 by pipetting up and down 3x and transfer 100 μl into A3.
10) And so on..................
11) Mix A10 by pipetting up and down 3x and transfer 100 μl into A11.
12) Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.
13) Repeat dilution series for rows B, C, D.
14) Re-Mix (Pipette up and down) each row of plate immediately before putting in the plate reader.
15) Measure Abs₆₀₀ of all samples .

3. Fluorescence standard curve - Fluorescein Protocol

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 1mL of 1xPBS.
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.
4) Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12.
5) Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1.
6) 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.
7) And so on..................
8) Mix A10 by pipetting up and down 3x and transfer 100 μl into A11.
9) Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.
10) Repeat dilution series for rows B, C, D.
11) Measure fluorescence of all samples.

III. Cell measurement protocol

1) Transform 8 plasmids into DH5α competent cells, grown in incubator for 12 hrs at 37℃.
2) Pick 2 colonies from each of the transformation plates and inoculate in 10 mL LB medium and Chloramphenicol. Grow the cells for 16hrs at 37°C and 220 rpm.
3) Cell growth, sampling, and assay.
4) Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor).
5) Measure Abs₆₀₀ of these cultures.
6) Dilute the cultures further to a target Abs₆₀₀ of 0.02 in a final volume of 12 ml LB medium and Chloramphenicol in 50 mL falcon tube.
7) Incubate the cultures at 37°C and 220rpm. Take 500µL samples of the cultures at 0 and 6 hours of incubation.Place the samples on ice.
8) Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
9)Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice.
10) At the end of sampling point measure samples (Abs₆₀₀ and fluorescence measurement).
11) Measurements of absorbance and fluorescence:
①OD₆₀₀
Device: Plate Reader (Tecan Infinite M200pro)
Wavelengths: 600 nm absorption
②Fluorescence
Device: Plate Reader (Tecan Infinite M200pro), 96-well plates.
Wavelengths: 485 nm excitation, 530 nm emission

Measurements of cell measurement

I. OD₆₀₀ Reference point

Table 1. Absorbance at 600nm for LUDOX and H₂O

II.Particle Standard Curve

Table 2. The Particle Standard Curve in plate reader with 485nm excitation, 530nm emission

Table 3. The Particle Standard Curve (log scale)

III. Fluorescence standard curve

Table 4. The Fluorescein Standard Curve in plate reader with 485nm excitation, 530nm emission

Table 5. The Fluorescein Standard Curve (log scale)

Ⅳ. Cell Measurements

1. OD₆₀₀–Background

Table 6. OD₆₀₀–Background

2. Fluorescence Background (485nm excitation, 530nm emission)

Table 7. Fluorescence Background

3. FI/ABS₆₀₀

Table 8. FI/ABS₆₀₀

4. MEFL / particle

Table 9. MEFL / particle

Introduction of colony forming units

The goal of this part of interlab study is 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 year, two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures were provided by the Registry. The results show increased number of colonies in the higher cell concentration of each culture.

There are three steps in this part: Starting Sample Preparation, Dilution Series, CFU/mL/OD Calculation. After the first step, duplicate cultures of BBa_I20270 (Positive Control) and BBa_R0040 (Negative Control) were diluted to twelve dilution replicate in total, six for Positive Controls (in triplicate per culture), the other six replicates for Negative Controls:
BBa_I20270 Culture 1, Dilution Replicate 1;
BBa_I20270 Culture 1, Dilution Replicate 2;
BBa_I20270 Culture 1, Dilution Replicate 3;
BBa_I20270 Culture 2, Dilution Replicate 1;
BBa_I20270 Culture 2, Dilution Replicate 2;
BBa_I20270 Culture 2, Dilution Replicate 3;
BBa_R0040 Culture 1, Dilution Replicate 1;
BBa_R0040 Culture 1, Dilution Replicate 2;
BBa_R0040 Culture 1, Dilution Replicate 3;
BBa_R0040 Culture 2, Dilution Replicate 1;
BBa_R0040 Culture 2, Dilution Replicate 2;
BBa_R0040 Culture 2, Dilution Replicate 3.

Protocol of Colony Forming Units

I. Materials

• Overnight Cell cultures (BBa_I20270 Culture 1/2, BBa_R0040 Culture 1/2)
• LB (Luria Bertani) media with Cam
• Microplate reader
• Agar + Cam plates (36 in total)
• 37 ℃ incubator

II. Protocol

1. Starting Sample Preparation
1) Diluting the overnight culture 1:8 (8-fold dilution) in LB + Cam before measuring the OD₆₀₀: Add 25 μL culture to 175 μL LB + Cam in a well in a black 96-well plate, with a clear, flat bottom.
2) Dilute overnight culture to OD₆₀₀ = 0.1 in 1mL of LB + Cam media.
3) Do this in triplicate for each culture.
4) Check the OD₆₀₀ and make sure it is 0.1 (minus the blank measurement).
2. Dilution Series
For each Starting Sample (total for all 12 samples)
1) You will need 3 LB Agar + Cam plates (36 total).
2) Prepare three 2.0 mL tubes (36 total) with 1900 μL of LB + Cam media for Dilutions 1, 2, and 3.
3) Prepare two 1.5 mL tubes (24 total) with 900 μL of LB + Cam media for Dilutions 4 and 5.
4) Label each tube according to the figure below (Dilution 1, etc.) for each Starting Sample.
5) Pipet 100 μL of Starting Culture into Dilution 1. Vortex tube for 5-10 secs.
6) Repeat Step 5 for each dilution through to Dilution 5 as shown below.
7) Aseptically spead plate 100 μL on LB + Cam plates for Dilutions 3, 4, and 5.
3. CFU/mL/OD Calculation
1) Count the colonies on each plate with fewer than 300 colonies.
2) Multiple the colony count by the Final Dilution Factor on each plate.

Measurements of cell measurement

I. Starting Sample Preparation

Table 10. Starting Sample Preparation

II. CFU/mL/OD Calculation

Table 11. CFU/mL/OD Calculation

We met some challenges and gained from them during the process of the whole Interlab, and we will make a brief discussion below:

Ⅰ. Part of Cell Measurement

Thanks to the detailed experimental protocols and materials provided to us by the igem office, we have completed our cell measurement department very successfully.

In the meantime, we had mastered molecular cloning experiments, as well as the use of Plate Reader. The only drawback was that we didn't know the expected results of the experiment, and we couldn't make sure that if our data were correct before submitting them to the igem office.

Ⅱ. Part of Flow Cytometry

We were not so sure about the apparatus used in this part when reading the protocols of flow cytometry. Therefore, we used conventional type of flow cytometer, which could only measure and analysize ONE sample at a time. It proved that using the traditional flow cytometer a big mistake because this part not only cost us much time (about 5 hours and more) but also damaged the instrument due to the long-time measurement.

After the deadline of data submission, we knew that what should be used in this part was the flow cytometer of high-throughput type that could read 96-well plates. It was a big regret that we failed to read out this information from the protocols.

Ⅲ. Colony Forming Units

We learned that the OD₆₀₀ detected by the Plate Reader was actually Abs₆₀₀, so we had to measure the calibration parameter before the formal experiment.

In this experiment, there were many samples need to be diluted, which may spend lots of time, and the bacteria were still growing with the OD value increasing. In addition, plate counting was also a difficult process, because the single colonies that grew on the higher concentration plates grew together. Therefore, we couldn't guarantee the accuracy of the results.

Q: Who did the actual work to acquire these measurements?
A: Ye Qiang, Yucheng Chen, Niangui Cai, Yunyun Hu, Qiupeng Wang, Junhong Chen and Ruofan Yang.
Q: What other people should be credited for these measurements?
A: Yousi Fu.
Q: On what dates were the protocols run and the measurements taken?
A: Required devices were transformed on 25th July, 2018. All samples were measured on 26th July, 2018.

[1]https://2017.igem.org/Team:XMU-China/InterLab
[2]https://2016.igem.org/Team:XMU-China/Interlab
[3]http://parts.igem.org/Help:Parts
[4]https://2018.igem.org/Measurement/InterLab/Plate_Reader