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

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.

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

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.

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

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.

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.

• 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)

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.

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.

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.

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.

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

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