InterLab

Overview

Synthetic biology requires reliable and repeatable measurement. However, the ability to repeat measurements in different labs has been difficult for different units or different ways may be used in various groups. The InterLab Study is part of the Measurement Committee’s continuing effort to develop a standard measurement procedure for green fluorescent protein (GFP). In order to improve reproducibility, we used plate readers to take measurements of fluorescence and absorbance this year.

Methods and Materials

OD600 Reference Point

Materials

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

Methods

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

Particle Standard Curve

Materials

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

Methods

Prepare the Microsphere Stock Solution

Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
Add ddH₂O to the microspheres
Vortex well.

Prepare the Microsphere Stock Solution
Measure Abs600 of all samples in instrument

Fluorescence standard curve

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

Cell Measurements

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

Day 1: transform Escherichia coli DH5α with given plasmids (all in pSB1C3)
Day 2: Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells
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).

Colony Forming Units per 0.1 OD600 E. coli cultures

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: