All of 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

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

• 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:


Raw Results

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)

Raw Results

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





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