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OVERVIEW

A challenge of synthetic biology is repeating measurements in different laboratories. For example, fluorescence data is difficult to compare either because it is reported in different units, or because different groups handle raw data differently. iGEM’s Measurement Committee thus aims to use the InterLab Study to eventually develop absolute units for measurements of green fluorescent protein (GFP) in a plate reader. This will improve the measurement tools of synthetic biologists.

This year, the Committee aims to discover if it is possible to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of optical density (OD). For this, we were required to measure the cell density of Escherichia coli (E. coli) DH5⍺ cells using the methods below.

Method 1: Converting between absorbance of cells to absorbance of a known concentration of beads

In the first method, silica beads are used to estimate the actual amount of cells during fluorescence measurement. These beads are modeled after a typical E. coli cell and are thus expected to scatter light in a similar way to E. coli cells. As a sample of these silica beads gives a consistent and known absorbance measurement at 600 nm, absorbance measurements from a sample’s cell density can be converted into an “equivalent concentration of beads” measurement that should be more universal and comparable between different labs.

Method 2: Counting colony-forming units (CFUs) from the sample

In the second method, cell concentration is approximated is by plating a known volume of the sample and letting bacterial colonies grow. As each bacterial colony is assumed to represent a single cell (for cells that do not stick together), the cell concentration in the sample is then directly proportional to the number of CFUs. Using a scaling factor computed from negative and positive control CFUs, a conversion factor from absorbance to CFU can be computed.

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PARTS RECEIVED

Device	Part Number	Usage
Negative control	BBa_R0040	TetR repressible promoter, medium strength promoter
Positive Control	BBa_I20270	Promoter MeasKit (J23151)
Test Device 1	BBa_J364000	GFP expressing constitutive device
Test Device 2	BBa_J364001	GFP expressing constitutive device
Test Device 3	BBa_J364002	GFP expressing constitutive device
Test Device 4	BBa_J364007	Expresses GFP under the control of a constitutive promoter
Test Device 5	BBa_J364008	Expresses GFP under the control of a constitutive promoter
Test Device 6	BBa_J364009	Expresses GFP under the control of a constitutive promoter

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EXPERIMENTS

PLATE READER SETUP

Abs600

Wavelength: 600nm Read Speed: Normal Delay: 100 msec

Fluorescence

Excitation: 485 Emission: 525 Optics: Top Gain: 50 Light Source: Xenon Flash Lamp Energy: High Read Speed: Normal Delay: 100 msec Read Height: 7 mm

OD600 REFERENCE POINT (CALIBRATION 1)

Materials

• 1 ml LUDOX CL-X
• ddH₂O
• 96-well plate (black)

Method

1. 100 µl of LUDOX was added into wells A1, B1, C1 and D1.
2. 100 µl of ddH20 was added into wells A2, B2, C2 and D2.
3. Abs_600nm was measured for all samples.

Table 1: Abs₆₀₀ of LUDOX CL-X and blank replicates. Their corresponding arithmetic means were calculated and included, along with the corrected Abs₆₀₀, reference OD₆₀₀ and conversion factor.

Results	LUDOX CL-X	H2O
Replicate 1	0.056	0.037
Replicate 2	0.056	0.038
Replicate 3	0.056	0.038
Replicate 4	0.055	0.038
Arth. Mean	0.056	0.038
Corrected Abs600	0.018
Reference OD600	0.063
OD600/Abs600	0.063

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PARTICLE STANDARD CURVE (CALIBRATION 2)

Materials

• 300 µl Silica beads (4.7 x 108 microspheres)
• ddH₂O
• 96-well plate (black)

Methods

(A) To prepare the Microsphere Stock Solution

1. Tube labelled “Silica Beads” was vortexed vigorously for 30 s.
2. 96 µl of microspheres was immediately pipetted into a 1.5 ml eppendorf tube.
3. 904 µl of ddH₂0 was added to the microspheres. The eppendorf was vortexed well.

Figure 1: Graph of Abs₆₀₀ against particle count.

(B) To prepare the serial dilution of microsphere

1. 100 µl of ddH20 was added into wells A2, B2, C2, D2...A12, B12, C12, D12.
2. The microsphere stock solution was vortexed vigorously for 10 s before immediately adding 200 µl of microspheres into A1.
3. 100 µl of microsphere stock solution was transferred from A1 to A2.
4. Mix A2 by pipetting up and down 3 times and transfer 100 µl into A3.
5. The subsequent dilutions were prepared as illustrated on Image A (below).
6. Samples were re-mixed immediately before putting it in the plate reader. Fluorescence_(Abs600) all samples were measured.
7. Fluorescence (Abs₆₀₀ ) of all samples were measured. Our results are reflected by Fig. 2 and Fig. 3.

Figure 2: Graph of Abs₆₀₀ against particle count, logarithmic scale.

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FLUORESCENCE STANDARD CURVE (CALIBRATION 3)

Materials

• Fluorescein
• 10 ml 1X PBS pH 7.4 - 7.6
• 96-well plate (black)

Methods

(A) To prepare the fluorescein stock solution

1. The fluorescein kit tube was spun down to make sure that the pellet was collected at the bottom of the tube.
2. 10X fluorescein stock solution (100 µM) was prepared by resuspending fluorescein in 1 ml of 1X PBS. Fluorescein was checked to be properly dissolved in PBS by checking for no more visible particulates in the pipette tip when resuspending.
3. 10X fluorescein stock solution was diluted with 1X PBS to make a 1X fluorescein solution (10 µM): 100 µl of 10X fluorescein stock solution was mixed with 900 µl of 1X PBS.

Figure 3: Graph of fluorescence against fluorescein concentration.

(B) To prepare the serial dilution of fluorescein

1. 100 µl of PBS was added into wells A2, B2, C2, D2...A12, B12, C12, D12.
2. 200 µl of 1X fluorescein stock solution was added into A1, B1, C1 and D1.
3. 100 µl of 1X fluorescein stock solution was transferred from A1 to A2.
4. Mix A2 by pipetting up and down 3 times and transfer 100 µl into A3.
5. The subsequent dilutions were prepared as illustrated by Fig. 4.
6. Fluorescence of all samples were measured, and our results are reflected by Fig. 5.

Figure 4: Graph of fluorescence against fluorescein concentration, logarithmic scale.

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CELL MEASUREMENTS

Materials

• Competent cells (Escherichia coli strain DH5α)
• Luria Bertani (LB) media
• Chloramphenicol (stock concentration 25 mg/ml dissolved in ethanol)
• 50 ml Falcon tube (wrapped with a thick layer of paper towel to block light)
• 1.5 ml eppendorf tubes
• Incubator at 37 °C
• Ice bucket with ice
• 96-well plate (black)

Methods

Day 1: Transforming Escherichia coli strain DH5α with devices provided in the Distribution Kit

1. Each device (powder form) was resuspended in 10 µl of ddH₂O.
2. 1 µl of each respective plasmid was transformed into 50 µl E. coli DH5α via electroporation.
3. 500 µl of LB was immediately added into each tube for recovery of the transformed cells.
4. Each sample tube was incubated with shaking at 37 °C for 30 mins before 100 µl of each sample was plated onto LB + Chlor agar plates and grown overnight at 37 °C.

Day 2: Selecting Colonies and Growing Cells Overnight

1. 2 colonies were selected for being both relatively bigger and more separated from the other colonies.
2. The selected colonies were then inoculated into 5 ml of LB + Chlor and grown overnight at 37 °C at 220 rpm.

Day 3: Cell Growth, Sampling and Assay

Part 1: Abs₆₀₀nm and Fluorescence Measurement

1. A cell stock of each overnight culture was made in glycerol for storage, in case there is a need to use them again. To make this stock, 850 µl of culture was added to 350 µl of glycerol.
2. A 1:10 dilution of each overnight culture was made in LB + Chlor (0.5 mL culture + 4.5 mL media).
3. Abs₆₀₀nm of the 1:10 diluted cultures were measured.
4. Cultures were diluted further to a target Abs600nm of 0.02 in a final volume of 12 ml LB + Chlor in a 50 ml falcon tube that was covered with tissue paper.
5. 500 µl of samples of the diluted cultures at 0 h were transferred into 1.5 ml eppendorf tubes labelled A and B. The tubes were placed on ice until they were ready to be laid out according to the plate diagram to measure fluorescence and Abs600. Fluorescence readings at T = 0 h are shown in Fig. 6.
6. The rest of the cultures were incubated at 37 °C and 220 rpm for 6 hours.
7. After the 6-hour-incubation, 500 µl of these cultures were transferred into 1.5 ml eppendorf tubes before being laid out according to the plate diagram below. The samples’ fluorescence and Abs600nm were measured again. Fluorescence readings at T = 6 h are shown in Fig. 7.

Part 2: Colony Forming Units per 0.1 OD600 E. coli Cultures

Only Positive Control (BBa_I20270) cultures and Negative Control (BBa_R0040) were involved in this part.

1. Overnight cultures were diluted 10-fold in LB + Chlor media to ensure they lay in the linear detection range of our plate reader.
2. The OD₆₀₀nm of cell cultures were measured. Our results are reflected by Fig. 8.
3. Overnight cultures were diluted to OD₆₀₀ = 0.1 in 1 ml of LB + Chlor media. Each culture was done in triplicate.
4. Diluted overnight cultures were checked to ensure that OD600 = 0.1, excluding the blank measurement
5. For each starting sample, serial dilutions were prepared as shown in Fig. 9.
6. 100 µl of Dilutions 3, 4 and 5 were aseptically spread on LB + Chlor agar plates.
7. The plates were incubated overnight at 37 °C.
8. Colonies on each plate were counted. Our results are reflected by Fig. 10.

Table 2: Fluorescence raw readings.

Hour 0	Nge. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	34	76	361	122	53	318	289	88	47
Colony 1, Replicate 2	71	67	418	131	42	338	283	57	34
Colony 1, Replicate 3	52	66	386	127	44	309	304	90	27
Colony 1, Replicate 4	53	101	391	112	45	367	314	94	33
Colony 2, Repplicate 1	65	61	357	100	8	329	277	76	35
Colony 2, Replicate 2	47	83	389	144	24	339	2278	91	38
Colony 2, Replicate 3	79	74	377	138	33	335	280	85	45
Colony 2, Replicate 4	61	65	350	142	45	327	282	72	31
Hour 6	Nge. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	36	461	1252	920	58	2389	338	436	35
Colony 1, Replicate 2	45	500	1351	988	55	2621	354	433	54
Colony 1, Replicate 3	53	501	1390	1014	60	2757	393	446	24
Colony 1, Replicate 4	61	488	1332	1039	36	2793	351	448	31
Colony 2, Repplicate 1	30	429	971	821	44	2142	371	437	8
Colony 2, Replicate 2	55	516	1199	832	96	2390	378	401	24
Colony 2, Replicate 3	38	483	1239	838	74	2401	425	423	40
Colony 2, Replicate 4	49	460	1129	896	57	2528	384	423	51

Table 3: OD₆₀₀ raw readings.

Hour 0:	Nge. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0.061	0.06	0.061	0.063	0.059	0.059	0.063	0.061	0.043
Colony 1, Replicate 2	0.062	0.062	0.063	0.063	0.063	0.062	0.065	0.063	0.043
Colony 1, Replicate 3	0.063	0.062	0.064	0.065	0.064	0.061	0.063	0.061	0.043
Colony 1, Replicate 4	0.062	0.062	0.065	0.063	0.063	0.062	0.063	0.062	0.043
Colony 2, Repplicate 1	0.06	0.063	0.064	0.063	0.062	0.062	0.062	0.064	0.045
Colony 2, Replicate 2	47	83	389	144	24	339	2278	91	38
Colony 2, Replicate 3	0.061	0.063	0.061	0.065	0.061	0.065	0.06	0.062	0.046
Colony 2, Replicate 4	0.061	0.063	0.061	0.065	0.061	0.065	0.06	0.062	0.046
Hour 6	Nge. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0.428	0.415	0.174	0.445	0.455	0.418	0.074	0.432	0.042
Colony 1, Replicate 2	0.452	0.435	0.19	0.481	0.481	0.46	0.08	0.469	0.042
Colony 1, Replicate 3	0.459	0.434	0.198	0.495	0.487	0.462	0.081	0.46	0.042
Colony 1, Replicate 4	0.461	0.439	0.185	0.495	0.479	0.477	0.076	0.467	0.043
Colony 2, Repplicate 1	0.446	0.414	0.138	0.434	0.443	0.406	0.079	0.419	0.043
Colony 2, Replicate 2	0.467	0.447	0.149	0.452	0.484	0.437	0.076	0.435	0.043
Colony 2, Replicate 3	0.496	0.448	0.153	0.449	0.485	0.455	0.082	0.435	0.043
Colony 2, Replicate 4	0.483	0.444	0.148	0.475	0.467	0.455	0.079	0.409	0.04

Table 4: Counts for colony-forming units (CFUs).

D4	8 x 105	NCAI	NCA2	NCA3	NCB1	NCB2	NCB3	Avg NCA	Avg NCB
		445	286	649	173	608	945	4.60E + 02	5.75E + 02
		PCAI	PCA2	PCA3	PCB1	PCB2	PCB3	Avg PCA	Avg PCB
		195	219	245	232	250	253	2.20E + 02	2.45E + 02
D5	8 x 105	NCAI	NCA2	NCA3	NCB1	NCB2	NCB3	Avg NCA	Avg NCB
		49	32	89	32	153	418	5.67E + 01	2.01E + 02
		PCAI	PCA2	PCA3	PCB1	PCB2	PCB3	Avg PCA	Avg PCB
		36	54	35	29	4	27	4.17E + 02	2.00E + 02

Table 5: Cell concentration calculated by multiplying CFUs with the corresponding dilution factors.

D4	8 x 105	NCAI	NCA2	NCA3	NCB1	NCB2	NCB3	Avg NCA	Avg NCB
		3.52E + 09	2.24E + 09	5.14E + 09	1.41E + 09	4.72E + 09	7.49E + 09	3.64E + 09	4.54E + 09
		PCAI	PCA2	PCA3	PCB1	PCB2	PCB3	Avg PCA	Avg PCB
		1.61E + 09	1.95E + 09	2.11E + 09	1.70E + 09	1.85E + 09	2.25E + 09	1.89E + 09	1.93E + 09
D5	8 x 105	NCAI	NCA2	NCA3	NCB1	NCB2	NCB3	Avg NCA	Avg NCB
		3.88E + 09	2.51E + 09	7.05E + 09	2.61E + 09	1.19E + 10	3.31E + 10	4.48E + 09	1.59E + 10
		PCAI	PCA2	PCA3	PCB1	PCB2	PCB3	Avg PCA	Avg PCB
		2.97E + 09	4.80E + 09	3.01E + 09	2.13E + 09	2.96E + 08	2.40E + 09	3.59E + 09	1.61E + 09

Table 6: CFU/mL/OD.

	Avg of D4 and D5
NCA	4.06E + 09
NCB	1.02E + 10
PCA	2.74E + 09
PCB	1.77E + 09

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FLOW CYTOMETRY

Materials

1. SpheroTech Rainbow calibration beads, type RCP-30-5A (Lot Number: AAF02)
2. Flow cytometer set to collect 10,000 events per well or a read time of 1 min per well

Methods

1. A sample of SpheroTech beads was prepared according to the manufacturer instructions
2. Plate setup was as shown below.

CLOSE

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DISCUSSION

Abs₆₀₀ nm

We inferred the rate of colony growth from net Abs600 values - a common method for measuring cell concentration. All cells except for cells transformed with Devices 1 and 5 had comparable rates of growth. In cells transformed with Devices 1 and 5, the increase in net Abs600 was significantly slower than cells transformed with the rest of the devices (Fig. 5, 6).

Figure 5: Graph comparing the net Abs₆₀₀ at T = 0 h for each colony using each control/device.

Figure 6: Graph comparing the net Abs₆₀₀ at T = 6 h for each colony using each control/device.

µM Fluorescein per OD

Cells transformed with Devices 1, 4 and 5 had the highest fluorescein readings per OD (Fig. 7, 8). The µM of fluorescein per OD of cells transformed with Device 3 were very low and at levels comparable to cells transformed with the negative control. On the other hand, cells transformed with Devices 2 and 6 had similar µM fluorescein per OD to that of cells transformed with the positive control. Similar trends were observed for Molecules of Equivalent Fluorescence Level (MEFL)/particle (Fig. 9, 10).

Figure 7: Graph comparing the net mM fluorescein per OD at T = 0 h for each colony using each control/device.

Figure 8: Graph comparing the net mM fluorescein per OD at T = 6 h for each colony using each control/device.

Figure 9: Graph comparing the Molecules of Equivalent Fluorescent Level (MEFL) per particle at T = 0 h for each colony using each control/device.

Figure 10: Graph comparing the Molecules of Equivalent Fluorescent Level (MEFL) per particle at T = 6 h for each colony using each control/device.

Derivations/Inferences made about devices

Despite growing at a considerably slower rate, fluorescein readings per OD of cells transformed with Devices 1 and 5 were among the highest. The high GFP production in cells transformed with Devices 1 and 5 might have caused a unhealthy metabolic stress on the cells, which, in turn, could have impeded the growth of the cells.

Cells transformed with Device 4 had a slightly lower fluorescein reading per OD as compared to cells transformed with Devices 1 and 5. Since there was no compromise on the cell growth rate despite the high GFP production, the slightly lower GFP production (as compared to cells transformed with Devices 1 and 5) was inferred to have permitted the cells to grow at normal rates, leading to an overall higher net fluorescein reading.

Cells transformed with Device 3 was observed to produce very little to no GFP. Promoter within Device 3 might have been repressed by a molecule present in the cells, or not activated, to result in minimal GFP expression.

Conclusion about devices

Devices 1 and 5 were inferred to have highest promoter strength upstream of the GFP gene. While they produced the highest fluorescein readings per OD, overexpression of GFP as a result of Devices 1 and 5 do not seem to be healthy for cells: cells were not observed to be able to cope with the overly high expression of GFP. In the same argument, Device 4 seemed to be the most advantageous for the experimenter: there was a high GFP production without a compromise of growth rate. Cells transformed with Device 4 seemed to be able to manage the level of metabolic stress and grow normally.

CFU/mL/OD

Based on the data, NCA, NCB, PCA, PCB averages were computed based on counted CFU units. The percentage errors computed based on the known concentrations of beads against their respective averages showed high error levels.

Based on the data, NCA, NCB, PCA, PCB averages were computed based on counted CFU units. The percentage errors computed based on the known concentrations of beads against their respective averages showed high error levels (Table 7).

Table 7: Percentage errors of calculated based on the known concentrations of beads against their respective averages.

	Avg Colony Count	% error
NCA	4.6E + 09	1,132.24
NCB	1.02E + 10	3,018.92
PCA	2.74E + 09	803.42
NCB	1.77E + 09	432.61

This suggests that the concentration of microsphere beads cannot be used to reliably predict cell concentration levels. It is possible that the silica beads are not a good model of E. coli cells and thus were unable to reproduce results characteristic of the cells.

Our team proceeded to calculate a conversion factor k to relate cell concentration to optical density.

Table 8: Conversion factor from absorbance to CFU was computed for each sample.

	OD600/Abs600	Cell Conc/OD	CFU/Cell density	Conversion Factor
NCA	3.5	4.11E + 08	1.00E - 04	1.44E + 05
NCB	3.5	1.05E + 10	1.00E - 04	3.68E + 06
PCA	3.5	2.60E+09	1.00E-04	9.10E+05
NCB	3.5	1.84E+09	1.00E-04	6.44E+05

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CONCLUSION

The results from our experiment seem to indicate that normalizing fluorescence measurements to absolute cell count using the Study’s two methods will not be able to reduce lab-to-lab variability because counting colony-forming units do not return the expected cell concentration, i.e. the cell concentration modeled by the silica beads in Method 1. While both methods cannot be used independently to establish a robust fluorescence measurement system, it may be possible that lab-to-lab variability can be reduced if a different method of normalizing to absolute cell count is devised, replacing Method 1, Method 2, or both.

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