Materials

• 1 mL LUDOX (provided in kit)
• ddH₂O
• 96 well plate

Methods

1. Add 100 μl LUDOX into wells A1, B1, C1, D1
2. Add 100 μl of dd₂O into wells A2, B2, C2, D2
3. Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements

Materials

• Fluorescein (provided in kit)
• 10 mL 1xPBS (phosphate buffered saline)
• 96 well plate, black with clear flat bottom

Methods

1. Prepare the Microsphere Stock Solution
1. Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
2. Add ddH₂O to the microspheres
3. Vortex well.
2. Prepare the Microsphere Stock Solution
3. Measure Abs600 of all samples in instrument

Materials:

• Fluorescein (provided in kit)
• 10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline)
• 96 well plate, black with clear flat bottom

Methods:

• Prepare the Fluorescein Stock Solution
• Measure fluorescence of all samples in instrument

Materials

• Competent Cells (Escherichia coli strain DH5α
• LB (Luria Bertani) media
• Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
• 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
• Incubator at 37°C
• Ice bucket with ice
• Micropipettes and tips
• 96 well plate, black with clear flat bottom preferred
• Devices (from Distribution Kit, all in pSB1C3 backbone):

Device	Part Number	Plate	Location
Negative control	BBa_R0040	Kit Plate 7	Well 2D
Positive control	BBa_l20270	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

Methods

1. Day 1: transform Escherichia coli DH5α with given plasmids (all in pSB1C3)
2. Day 2: Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells
3. Day 3: Cell growth, sampling, and assay
   1. Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
   2. Measure Abs 600 of these 1:10 diluted cultures
   3. Dilute the cultures further to a target Abs600 of 0.02 in 50 mL falcon tube
   4. Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation.Place the samples on ice.
   5. Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
   6. Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice.
   7. Measure samples (Abs fluorescence measurement).

Step 1: Starting Sample Preparation

• Measure the OD600 of the cell cultures and dilute to the linear detection range of the plate reader, e.g. to 0.05 – 0.5 OD600 range.
• Dilute the overnight culture to OD600 = 0.1 in 1mL of LB + Cam media. Do this in triplicate for each culture.
• Check the OD600 and make sure it is 0.1.

Step 2: Dilution Series Instructions

For each Starting Sample (total for all 12 showed in italics in paraenthesis):

• We need 3 LB Agar + Cam plates (36 total).
• Prepare three 2.0 mL tubes (36 total) with 1900 ºL of LB + Cam media for Dilutions 1, 2, and 3 (see figure below).
• Prepare two 1.5 mL tubes (24 total) with 900 ºL of LB + Cam media for Dilutions 4 and 5 (see figure below)..
• Label each tube according to the figure below (Dilution 1, etc.) for each Starting Sample.
• Pipet 100 ºL of Starting Culture into Dilution 1. Discard tip. Do NOT pipette up and down. Vortex tube for 5-10 secs.
• Repeat Step 5 for each dilution through to Dilution 5 as shown below.
• Aseptically spead plate 100 ºL on LB + Cam plates for Dilutions 3, 4, and 5.
• Incubate at 37°C overnight and count colonies after 18-20 hours of growth.

Step 3: CFU/mL/OD Calculation Instructions

Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture can be calculated as follows:

• Count the colonies on each plate with fewer than 300 colonies.
• Multiple the colony count by the Final Dilution Factor on each plate.

Averaged OD measurements of the 4 replicates for each sample, taken at two hour intervals.