OriginALS

InterLab

Our team chose to participate in the Fifth International InterLab Measurement Study in synthetic biology.
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.
In this study we want to reduce lab-to-lab variability in fluorescence measurements by normalizing it to absolute cell count or colony-forming units (CFUs) instead of OD.
The fluorescence in the bacteria in our experiments occurs due to the presence of a gene that encodes a green fluorescent protein: GFP.
In cell and molecular biology, the GFP gene is frequently used as an expression reporter protein. Reporter genes are often used as an indication that a certain gene has been internalized or expressed in cells or organisms.

Equipment

All our measurements were taken in a plate reader that can read both absorbance and fluorescence (Neoteck-Tecan-INFINITE M100) without a pathlength correction. The temperature was set to 25̊C, under shaking with a duration of 3 sec and an amplitude of 2 mm. We went through a tutorial and learned how to operate the machine.
In addition, we used the following reagents (Partially supplied by iGEM):

1.0 ml LUDOX CL-X.
150 μL Silica Bead (microsphere suspension).
Fluorescein (powder, in amber tube)
iGEM Parts Distribution Kit Plates.
1 x PBS (phosphate buffered saline, pH 7.4 - 7.6.
ddH2O
Competent cells (Escherichia coli DH5α).
LB (Luria Bertani) media.
Chloramphenicol
96 well plates, black with clear flat bottom.

Safety procedures

As part of an ongoing work in a biological laboratory, safety gear was used to work with bacteria. The work was done with closed shoes, long pants, gloves and lab coats.
In order to maintain a clean environment, and sterility between the samples the work was performed next to a flame, and all the disposable parts were thrown after a single use into a biological waste bin.
Before and after work, work surfaces were sterilized with 70% alcohol.

Protocols

Calibration 1: OD600 Reference point - LUDOX Protocol:
100 μL LUDOX were added into wells A1, B1, C1, D1.
100 μLof ddH2O were added into wells A2, B2, C2, D2

	LUDOX CL-X	H₂O
Replicate 1	0.0561	0.029
Replicate 2	0.0522	0.0266
Replicate 3	0.0554	0.033
Replicate 4	0.0534	0.0292
Arith. Mean	0.054	0.029
Corrected Abs₆₀₀	0.025
Reference OD₆₀₀	0.063
OD₆₀₀/Abs₆₀₀	2.538

Table 1: OD₆₀₀/Abs₆₀₀ measurements.
The correction factor is 2.538.

Calibration 2: Particle Standard Curve - Microsphere Protocol

The Silica Beads from the InterLab test kit were vigorously vortexed for 30 seconds.
96 μL microspheres were pipetted into a 1.5 mL Eppendorf tubes.
904 μL of ddH2O were added to the microspheres.
The Microsphere Stock Solution was vortexed well.
200 μL of Microsphere Stock Solution were transferred into each well in column 1.
100 μL of ddH2O were added into each well (2-12) in the corresponding row.
Serial dilution by transfer of 100 μL from column to column with good mixing.
Absorbance measurements of all the samples in the plate reader in 600 nm.

Figure 1: Particle Standard Curve

Figure 2: Particle Standard Curve log scale.

We got a linear correlation between the Abs600 and the amount of particle.

Calibration 3: Fluorescence standard curve - Fluorescein Protocol

Fluorescein kit tube was spun-down to make sure pellet is at the bottom of the tube.
10x fluorescein stock solution (100 μM) was prepared by resuspending fluorescein in 1.0 mL of 1xPBS.
10x fluorescein stock solution was diluted with 1x PBS to make a 1x fluorescein solution with a concentration 10 μM.
200 μL of 1x fluorescein was transferred into each well in column 1.
Serial dilution by transferring 100 μL from column to column with good mixing.
Fluorescence measurements of all the samples in the plate reader.

When we got the feedback about out results, we were told that there was a problem with the measurements in which the positive and the negative results were too close, so we repeated the measurements.
After the second repetition of the measurements, we produced the results for the Excel sheet and the results were presented below in figures 3 and 4:

Figure 3: Fluorescein Standard Curve

Figure 4: Fluorescein Standard Curve log scale

The Fluorescence standard curve of Fluorescein (figure 3) allows us to convert cell-based fluorescence reading originating from GFP fluorescence (at the same wavelengths) readings to equivalent fluorescein concentrations found in the calibration curve.

Cell measurement protocol:
Day 1:
Escherichia coli DH5α was transformed with the following parts all harboured in pSB1C3 plasmids as indicated in table 2:

Device	Part Number	Plate	Plate Location
Negative control	BBa_R0040	Kit Plate 7	Well 2B
Test Device 1	BBa_J364000	Kit Plate 7	Well 2F
Test Device 2	BBa_J364001	Kit Plate 7	Well 2H
Test Device 3	BBa_J364002	Kit Plate 7	Well 2J
Test Device 4	BBa_J364007	Kit Plate 7	Well 2L
Test Device 5	BBa_J364008	Kit Plate 7	Well 2N
Test Device 6	BBa_J364009	Kit Plate 7	Well 2P

Table 2: plasmids list and locations

Day 2:
2 colonies from each of the transformation plates were picked and inoculated in 10.0 mL LB medium + Chloramphenicol.
The cells were incubated overnight at 37°C and 220 rpm shaking.

Day3: Cell growth, sampling, and assay

1:10 dilution of each overnight culture in LB+Chloramphenicol.
Abs600 measurement of these 1:10 diluted cultures.
Dilution of the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol.
500 µL samples of the diluted cultures at 0 hours were taken into 1.5 ml Eppendorf tubes and placed on ice.
The remainder of the cultures incubated at 37°C and 220 rpm for 6 hours.
500 µL samples of the cultures at 6 hours of incubation were taken into 1.5 ml Eppendorf tubes and placed on ice.
Abs600 and fluorescence measurement of all the samples- the results were added to the Excel sheet.

Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures:
Step 1: “Starting Sample” Preparation

The overnight culture of the Positive Control (BBa_I20270) and the Negative Control (BBa_R0040) were diluted 1:8: 25 μL culture to 175 μL LB + Cam in a well of a 96-well plate.
OD₆₀₀ measurement of the samples (include blank media measurement).
Dilution of the overnight culture to OD₆₀₀ = 0.1 in 1.0 mL of LB + Cam media according the calculation of (C1)(V1) = (C2)(V2).
The dilution cultures were added in triplicate samples into the 96 well-plates. Each well had 200 μL.
OD₆₀₀ measurement of the samples.

Step 2: Dilution Series

Serial Dilution was made to the triplicate Starting Samples of the previous step according to the instruction of the protocol.
From each sample, 3 dilutions (8*10-3 , 8*10-4, 8*10-5) were plated on LB + Cam plate.
Incubation of the plates at 37°C overnight.
Colonies were counted after 18-20 hours of growth.

Step 3: CFU/mL/OD Calculation

The colonies on each plate were counted.
The colony count was multiplied by the Final Dilution Factor on each plate as shown in Table 3:

(note: CFU values presented below are in 10^8 scale)

Plate number	Number of colonies	CFU*10^8
1.1 dilution 3	>300
1.1 dilution 4	82	0.656
1.1 dilution 5	25	2
1.2 dilution 3	>300
1.2 dilution 4	102	0.816
1.2 dilution 5	8	0.64
1.3 dilution 3	>300
1.3 dilution 4	126	1.008
1.3 dilution 5	22	1.76
2.1 dilution 3	>300
2.1 dilution 4	61	0.488
2.1 dilution 5	5	0.4
2.2 dilution 3	237	0.1896
2.2 dilution 4	42	0.336
2.2 dilution 5	7	0.56
2.3 dilution 3	>300
2.3 dilution 4	85	0.68
2.3 dilution 5	10	0.8
3.1 dilution 3	>300
3.1 dilution 4	73	0.584
3.1 dilution 5	2	0.16
3.2 dilution 3	>300
3.2 dilution 4	52	0.416
3.2 dilution 5	4	0.32
3.3 dilution 3	>300
3.3 dilution 4	64	0.512
3.3 dilution 5	6	0.48
4.1 dilution 3	>300
4.1 dilution 4	38	0.304
4.1 dilution 5	14	1.12
4.2 dilution 3	>300
4.2 dilution 4	50	0.4
4.2 dilution 5	7	0.56
4.3 dilution 3	292	0.2336
4.3 dilution 4	36	0.288
4.3 dilution 5	0	0

Table 3: CFU counts under different dilutions

These results provide us a direct correlation between actual concentrations of bacteria (actual number) to OD₆₀₀.

Final remarks

As a collaboration with the HebrewU iGEM team, we provided their team the Escherichia coli DH5α strain for their InterLab study. Representatives of this team came to our lab and we were glad to give them a tour.

In conclusion, we hope that our measurements and results will provide the required data, which will help to achieve the final goal of this InterLab experiment.

It was a wonderful experience to work on an international, collaborative study and help to the synthetic biology field develop.

Figure 5: Us, hard at work on the Interlab measurements

OriginALS

Team:BGU Israel/InterLab

Equipment

Safety procedures

Protocols

Final remarks