Inter-Lab Study Worksheet:

For each assay that you perform, fill out the following worksheet. Answer each question with enough detail to allow another person to replicate your measurements without needing to ask you any questions. This does not necessarily mean you need to describe everything in detail---for example, if you use a standard assay, you just need to give enough information to allow another person to use that assay in the same way that you did.

 

Section I: Provenance & Release

Who did the actual work to acquire these measurements?

Our laboratory group carried out the experiments this group consists of Anne Sofie Lærke Hansen, Anne Pihl Bali, Caroline Mosbech, Kristian Barreth Karlsen, Kristian Jensen & Rasmus Bech

 

What other people should be credited for these measurements?

We did all the experimental work for the interlab study ourselves.

Ali Altintas assisted us in setting up the BioLector. Anne Egholm helped us to use the Flow Cytometer.

 

On what dates were the protocols run and the measurements taken?

The BioLector experiment ran: 10th-11th of September 2014

The Flow cytometry experiment was done on the 22nd of September 2014.

 

Do all persons involved consent to the inclusion of this data in publications derived from the iGEM interlab study?

Yes.

 

Section II: Protocols

What protocol did you use to prepare samples for measurement?

Construct strains:

Create multiple strains expressing GFP from different promoters from the Andersen Library. Constructs were prepared with Standard Assembly. (see protocol).

Plasmid were purified with Zyppy™ Plasmid Miniprep Kit from Zymo research following manufacturer's protocol.

Competent DH5α cells were prepared (see protocol).

Purified plasmids were transformed into competent DH5α cells (see protocol).

 

Culture measurements:

We used cell culture tubes (10 mL) containing 2 mL LB medium (with 0.5% NaCl) and 25 μg/mL chloramphenicol for each strain. To each of the tubes, we inoculated cells from a single colony with a sterile inoculation loop and left it overnight at 37°C, shaking at 350 rpm.

The wells of the sterile BioLector plate (48 well FlowerPlate) was prepared with 1450 μL (1500μL for controls) of LB (0.5% NaCl) containing 25 μg/mL chloramphenicol. The following day we transferred 10 μL of each culture into an Eppendorf tube with 990 μL of sterile physiological salt water (creating a 100-fold dilution). We inoculated the individual BioLector wells with 10 μL of the dilution (creating an additional 150-fold dilution). Each strain was grown in triplicates to make sure we had 3 independent measurements and a more precise result. We incubated the plate in the BioLector at 37°C and 1000 rpm for 12.5 hours (stationary phase was reached). During the growth experiment we measured biomass and GFP fluorescence every 5 minutes. Biomass was measured by light scatter at 620 nm, while fluorescence was measured at 520 nm, with an excitation wavelength of 488 nm.

 

Single cell measurements:

2 mL LB (0.5% NaCl) was inoculated with cells from a plate and grown overnight.

The cultures were washed and resuspended in PBS.

The cell suspensions were diluted in PBS to a concentration where the flow cytometer could detect 1000-1500 events per second.

 

 

What sort of instrument did you use to acquire measurements?

What is the model and manufacturer?

Instrument: BioLector (Basic) - Microbioreactor System; Manufacturer: m2p-laps.

Instrument: FACSCalibur Cell Analyzer; Manufacturer: BD Biosciences

 

How is it configured for your measurements?

The BioLector is fitted with filters for measuring biomass (scatter at 620 nm) and GFP (emission at 520 nm, excitation with 488 nm). Settings: Gain: GFP: 80; Biomass: 30; Humidity: controlled at 95%; Shake: 1000 rpm.

The FACSCalibur was set up with a side scatter threshold of 4.5, to filter out events that were likely to be noise. Fluorescence was measured with a log amplification value of 750.

 

What protocol did you use to take measurements?

The BioLector was instructed to take measurements every 4.86 minutes. Settings: GFP: 80; Biomass: 30; Humidity: controlled at 95%. 1.5 mL LB media with relevant antibiotics in each well.

 

The FACS was set up with a threshold on side scatter, as events with low side-scatter are very likely noise, i.e. not actual cells. Each sample was then analysed at 1000-1500 events per second, until a total of 100,000 events was reached.

 

What method is used to determine whether to include or exclude each sample from the data set?

Strains that showed correct digest patterns were included. Strains that were sequenced and showed wrong sequences were excluded. As were cultures that did not grow, and single replicates that showed negligible fluorescence.

 

What exactly were the controls that you used?

DH5α with BBa_E0240 (no promoter) was used to determine cell background fluorescence (without GFP). For the BioLector, clean media (without cells) was used to determine media fluorescence.

 

What quantities were measured?

On the BioLector: GFP fluorescence (488 nm /520 nm) and biomass (scatter at 620nm). The flow cytometer measured forward scatter (cell size), side scatter (cell granularity) and fluorescence.

 

How much time did it take to acquire each set of measurements?

The running time of the BioLector experiment was 12 hours and 30 minutes. One cycle took 4.86 min.

Flow cytometry: The measurements themselves took less than 1 hour.

 

How much does it cost to acquire a set of measurements?

Assuming the lab already have a BioLector. For a full plate with samples: Flowerplate for the BioLector, 170mL LB medium, chloramphenicol, 16 pts. inoculation tubes, tips, Eppendorf tubes approximately $150.

 

What are the practical limits on the number or rate of measurements taken with this instrument and protocol?

BioLector: With the used protocol 48 samples can be measured within short intervals of time, however, 3 of these should be used as control. So when measuring triplicates it is possible to measure 15 different strains in the protocol used for the interlab study. Analysing such a plate takes approximately 12 hours.

 

Section III: Measured Quantities

For each type of quantity measured, report on the following:

What are the units of the measurement?

All measurements are in arbitrary units in both the BioLector and the FACSCalibur.

 

What is the equivalent unit expressed as a combination of the seven SI base units?

Not applicable due to arbitrary units.

 

 

Precision:

What is the range of possible measured values for this quantity, using your instrument as configured for these measurements?

According to the manufacturer, the BioLector has a linear range for OD600 from 0.2 to 600.

 

What are the significant figures for these measurements?

We have fitted a mixed model with promoters as a fixed effect and replicates as a random effect. This model gave a residual standard deviation on fluorescence/OD of 0.040. With this result we can say that a single fluorescence/OD measurement is no more precise than the measured value ± 0.040.

 

Is the precision the same across the entire range? If not, how does it differ?

We have no data to determine if the precision differ through the entire range.

 

 

Accuracy:

When was the instrument last calibrated?

The machine “self-calibrate” before every cycle, meaning the BioLector sensor/probe returns to the original position and measure a fixed known light intensity. It is approximately 5 years old but the filters have been changed 3 years ago.

 

How was the instrument calibrated?

The machine “self-calibrate” before every cycle, meaning the BioLector sensor/probe returns to the original position and measure a fixed known light intensity.

 

Section IV: Measurements

BioLector experiment

For each sample, report:

The following table shows the BioMass and Fluorescence measurements acquired from the BioLector. Each entry in the table is the mean value of all the measurements taken during a 3-hour period, with 4.88 min intervals. The full dataset can be found here.

Name	Biomass	GFP fluorescence	Fluorescence/OD	Note
J23100-1	12.59	2.13	0.1695	Excluded
J23101-1	54.80	30.17	0.5506
J23102-1	33.43	1.27	0.0381	Excluded
J23103-1	62.60	0.38	0.0061
J23106-1	59.10	30.66	0.5188
J23107-1	60.78	13.95	0.2296
J23100-2	57.97	47.45	0.8185
J23101-2	59.11	31.42	0.5315
J23102-2	61.60	18.93	0.3073
J23103-2	65.37	0.42	0.0064
J23106-2	59.04	30.29	0.5130
J23107-2	60.63	13.82	0.2279
J23100-3	61.51	49.65	0.8072
J23101-3	60.83	32.54	0.5349
J23102-3	61.59	21.08	0.3422
J23103-3	65.75	0.39	0.0060
J23106-3	59.63	29.42	0.4934
J23107-3	60.89	13.89	0.2280
J23113-1	69.70	0.43	0.0062
J23114-1	69.60	1.56	0.0225
J23115-1	66.27	1.85	0.0279
J23118-1	50.75	95.47	1.8811
J23119-1	46.99	17.50	0.3724
No promoter-1	64.81	0.39	0.0060
I20260-1	61.36	19.75	0.3219
J23113-2	72.55	0.45	0.0062
J23114-2	71.19	1.58	0.0223
J23115-2	68.07	1.88	0.0276
J23118-2	50.15	92.32	1.8410
J23119-2	47.35	17.18	0.3629
No promoter-2	67.06	0.38	0.0057
I20260-2	62.79	18.62	0.2966
J23113-3	77.21	0.43	0.0056
J23114-3	76.52	1.63	0.0213
J23115-3	72.29	1.92	0.0265
J23118-3	52.00	96.50	1.8556
J23119-3	47.13	16.81	0.3567
No promoter-3	70.07	0.39	0.0056
I20260-3	64.47	17.33	0.2688

 

For each group of replicates, report:

The data was analyzed by fitting a statistical mixed model to the data, using the lmerTest package in R. The different promoters were modeled as a fixed effect, and replicates were modeled as a random effect. The estimated means for each promoter and the standard error of the mean can be seen in the table and chart below.

Strain	Mean	Standard Error	Background-subtracted mean
J23100	0.91	0.01300	0.9050
J23101	0.61	0.01160	0.5980
J23102	0.37	0.01300	0.3610
J23103	0.01	0.01160	0.0005
J23106	0.58	0.01160	0.5690
J23107	0.26	0.01160	0.2500
J23113	0.01	0.01160	0.0003
J23114	0.03	0.01160	0.0183
J23115	0.03	0.01160	0.0243
J23118	2.14	0.01160	2.1300
J23119	0.42	0.01160	0.4120
I20260	0.32	0.01160	0.3080
E0240	0.0079	0.00821

The background-subtracted values were calculated by subtracting the mean of E0240 from the other means.

The replicate standard deviation was estimated to be 0.014, meaning that the effect of using multiple replicates contributes a random effect distributed with this standard deviation.
The residual standard deviation was estimated to be 0.040, meaning that the random effect of taking multiple measurements on the same sample has this standard deviation.

Which, if any, of the samples are excluded and why

In two of the triplicate sets, one outlier was excluded: For the promoter J23100, one replicate did not grow very much. For promoter J23102 one replicate had negligible fluorescence.

Flow cytometry

Histograms of fluorescence values for each construct are shown below. At least 100,000 cells were measured for each construct. Values on the X-axes are log10-values of the fluorescence measurements.
For each construct, a threshold was determined to split the measurements into high-fluorescence and low-fluorescence cells. The high-fluorescence sub-datasets were log10-transformed and a normal distribution was fitted. Mean and standard deviation for the log-normal distributions are reported below each histogram. The vertical red line indicates the threshold.

Mean: 2.41
SD: 0.25

Mean: 1.02
SD: 0.27

Mean: 3.05 SD: 0.24

Mean: 2.32 SD: 0.48

Mean: 2.33 SD: 0.20