Team:UC Davis/InterLab

iGEM

interlab

InterLab

I. Introduction

Interlab studies are a key component of advancing the field of synthetic biology. In past years, participants in the iGEM competition have conducted large-scale studies at research labs across multiple continents, with the purpose of seeking a better understanding of reproducibility and variation in synthetic biology [1].

One of the most ubiquitous reporter genes used in synthetic biology is green fluorescent protein (GFP). Currently, it can be difficult to directly compare measurements of GFP expression between different labs, or even the same lab over time. This is, in part, due to variation in protocols and equipment used to make these measurements [2]. The purpose of the 2018 iGEM interlab study was to gain better understanding of variation and calibration in order to increase the reproducibility of GFP reporter gene expression level characterization [3].

II. Materials

1. Tecan infinite M200 microplate reader machine (Top optics was used for this experiment)

2. Costar® 96 well black with clear bottom assay plates

Table 1: Settings used for Absorbance

Mode	Absorbance
Wavelength	600nm
Bandwidth	9nm
Number of Flashes	25
Settle time	0ms
Temperature	25°C

Table 2: Settings used for Fluorescence

Mode	Fluorescence
Excitation Wavelength	485nm
Emission Wavelength	525nm
Excitation Bandwidth	9nm
Emission Bandwidth	20nm
Gain	50
Number of Flashes	25
Settle time	0ms
Integration time	20µs
Temperature	25°C

III. Methods

Calibration 1: OD600 Reference Point– LUDOX

We used LUDOX CL-X, a 45% colloidal solution of silica, to obtain a OD600 reference point. This reference point was used to transform Abs600 values obtained from our plate reader to corresponding OD600 values. 100 µL of LUDOX solution was aliquoted into a well plate, in 4 replicates. 4 replicate wells of molecular-grade water were used as a blank. Absorbance data was taken at 600 nm. All subsequent measurements were taken using Costar® 96 well black with clear bottom assay plates (Costar® Cat. 3631, Corning, NY) and settings on the Tecan infinite M200 microplate reader machine.

Calibration 2: Particle Standard Curve – Microsphere Protocol

We prepared an 11 step dilution series of monodisperse silica microspheres and measured the Abs600 in our plate reader using the specified settings. A serial dilution was prepared by consecutively transferring 100 μl from column to column with good mixing and using 100 μl of ddH2O as the blank. Measurements were made using the microplate reader.

Calibration 3: Fluorescence Standard Curve – Fluorescein

We prepared a dilution series of fluorescein in four replicates and measured fluorescence in our plate reader using the specified settings. 10x fluorescein stock solution (100 uM) was prepared by resuspending in 1 mL of 1x PBS. After the fluorescein was fully dissolved, the stock solution was diluted to 1x and an 11-step, 2-fold serial dilution of fluorescein was carried out and measurements were made using the microplate reader.

Transformations

After calibrating our plate reader, we performed a heat shock transformation of DH5-Alpha E. coli with the plasmids supplied in the Distribution Kit (Table 3). Plasmids from the Distribution Kit were resuspended following the iGEM resuspension protocol [4]. After transformation, cells were grown for 15 hours in presence of selection (chloramphenicol).

Device	Part Number	Plate	Location
Negative Control	BBa_R0040	Kit Plate 7	Well 2D
Positive Control	BBa_I20270	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

Device Measurement – Abs600 and Fluorescence

Two colonies of each construct were selected and grown overnight in 5ml LB liquid media containing 25 µg/mL of chloramphenicol. A 1:10 dilution of each colony was made with 0.5 mL of culture into 4.5 mL of LB + chloramphenicol. The dilutions were plated and Abs600 was measured using the plate reader. The cultures were further diluted to a target Abs600 value of 0.02, with a final volume of 12 mL LB + chloramphenicol.

Equations used for dilution:

Volume_i = (0.02*12)/ Abs_600x

Abs_600x = Abs_cell - Abs_blank

At 0 hours, 500 µL samples were removed from each culture, for a total of 16 samples. These samples were placed on ice. The remaining portion of the cultures were incubated for an additional 6 hours at 37℃ and 220 rpm. At 6 hours, 500 µL samples were removed from each culture, for 16 additional samples, and a total of 32 samples taken over two time points. 100 µL of each sample was loaded into a well plate in 4 replicates. Abs600 and fluorescence measurements were taken. There were 144 resulting data points, representing the 8 constructs and 1 well of LB and chloramphenicol as a control, with 2 colonies each, in 4 replicates per colony, at 2 time points.

Device Measurement – Colony Forming Units per 0.1 OD600 E. coli cultures

OD600 values were calibrated to Colony Forming Unit (CFU) counts using the following protocol. The OD600 value was measured for the 2 colonies of the positive control and for the 2 colonies of the negative control. 25 µL of culture was added to 175 µL of LB with chloramphenicol in a black 96 well plate with a clear, flat bottom. 200 µL of blank LB with chloramphenicol was used as the control. The OD600 was then diluted with LB with chloramphenicol to .1 using the formula given in the protocol. Triplets of the diluted cultures were plated on the same plate used before and the OD600 was measured to ensure an OD of .1. Equation used for OD600 of .1 dilution:

(.1)(1000µL)/((OD600-blank OD600)x8)=µL of culture needed

1000µL-(µL of culture needed)=µL of media needed

For each culture, 3 solutions with dilution factors of 8 x 104, 8 x 105, and 8 x 106, respectively, were plated on LB agar plates with chloramphenicol and grown for 18 hours at 37º Celsius. After 18 hours, visible colonies were counted on each plate.

Equation used to calculate CFU count:

# Colonies * Final Dilution Factor = CFU/mL

IV. Results

Click here to view Excel sheet

Calibration 1: OD600 Reference Point– LUDOX

Table 4: Ludox Protocol

	LUDOX CL-X	H2O
Replicate 1	0.052499998	0.033300001
Replicate 2	0.054099999	0.0341
Replicate 3	0.0548	0.0341
Replicate 4	0.054099999	0.044199999
Arith. Mean	0.054	0.036
Corrected Abs600	0.017
Reference OD600	0.063
OD600/Abs600	3.610

After measuring the absorbance of the prepared LUDOX solution, we calculated the arithmetic mean of the absorbance across 4 replicates (see figure 5). We arrived at a corrected value for the Abs600 of our LUDOX solution by subtracting the arithmetic mean of the ddH2O blanks from the arithmetic mean of our LUDOX solution replicates. This gave us a corrected Abs600 value of 0.017. iGEM supplied us with a reference OD600 of 0.063. By dividing the reference OD600 by our measured Abs600 value, we arrived at a conversion factor of 3.610.

Calibration 2: Particle Standard Curve – Microsphere Protocol

The microsphere calibration is used to estimate the number of cells based off of recorded Abs 600. As particle count increases, the Abs 600 increases non-linearly.

Calibration 3: Fluorescence Standard Curve – Fluorescein Protocol

As the dilution factor of the Fluorescein decreases there is a linear increase in the concentration of Fluorescein.

Device Measurement – Abs600 and Fluorescence

Abs600 Raw Measurements

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0.0721	0.0801	0.0674	0.0671	0.0636	0.0659	0.0711	0.0662	0.0496
Colony 1, Replicate 2	0.0752	0.084	0.0693	0.0723	0.0676	0.0712	0.0771	0.0691	0.0489
Colony 1, Replicate 3	0.0717	0.0835	0.0701	0.069	0.0652	0.0699	0.0748	0.0704	0.0491
Colony 1, Replicate 4	0.0733	0.084	0.0716	0.0708	0.0689	0.0679	0.0719	0.0735	0.0494
Colony 2, Replicate 1	0.0771	0.0792	0.0844	0.0723	0.0683	0.0599	0.0628	0.0726	0.0493
Colony 2, Replicate 2	0.0765	0.0776	0.0814	0.0707	0.0676	0.0576	0.0646	0.0724	0.0495
Colony 2, Replicate 3	0.0773	0.079	0.0796	0.0712	0.0687	0.0575	0.065	0.0725	0.0494
Colony 2, Replicate 4	0.0776	0.0786	0.0801	0.0699	0.0652	0.0562	0.0643	0.0695	0.049

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0.3669	0.3528	0.0889	0.3391	0.3278	0.1548	0.0777	0.3385	0.0484
Colony 1, Replicate 2	0.3634	0.3661	0.0895	0.3397	0.3274	0.1597	0.0808	0.3558	0.0488
Colony 1, Replicate 3	0.3691	0.3646	0.0916	0.3617	0.3416	0.1617	0.0812	0.3566	0.0506
Colony 1, Replicate 4	0.3724	0.3696	0.0919	0.3463	0.3547	0.1594	0.0851	0.3706	0.0489
Colony 2, Replicate 1	0.3657	0.3674	0.1092	0.333	0.348	0.1223	0.068	0.3655	0.0491
Colony 2, Replicate 2	0.3872	0.3501	0.1088	0.3549	0.3468	0.1256	0.0734	0.3796	0.0492
Colony 2, Replicate 3	0.3745	0.3864	0.1122	0.3617	0.3372	0.1217	0.0709	0.3749	0.0493
Colony 2, Replicate 4	0.3774	0.3553	0.1099	0.363	0.3352	0.1217	0.0691	0.378	0.0483

After six hours, the cultures showed increased fluorescence, due to increased production of GFP.

Fluorescence Raw Measurements

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	67	108	173	98	67	147	224	80	70
Colony 1, Replicate 2	66	108	173	99	68	149	222	80	66
Colony 1, Replicate 3	66	111	166	97	64	144	218	81	64
Colony 1, Replicate 4	65	109	170	99	67	142	209	80	64
Colony 2, Replicate 1	65	100	237	94	65	101	158	83	62
Colony 2, Replicate 2	67	99	237	97	64	102	167	84	64
Colony 2, Replicate 3	66	101	232	92	64	98	167	83	66
Colony 2, Replicate 4	65	104	233	101	62	99	175	77	62

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	72	531	400	813	80	701	342	342	67
Colony 1, Replicate 2	75	561	421	794	77	692	351	354	69
Colony 1, Replicate 3	75	553	423	856	80	706	370	358	73
Colony 1, Replicate 4	76	565	403	809	78	729	350	357	65
Colony 2, Replicate 1	71	575	573	699	78	404	241	360	64
Colony 2, Replicate 2	74	553	536	729	78	420	233	374	67
Colony 2, Replicate 3	74	617	584	762	77	444	245	370	65
Colony 2, Replicate 4	70	596	586	810	76	407	249	376	64

Colony Forming Protocol

CFU per 1mL

Dilution factor	PA1	PA2	PA3	PB1	PB2	PB3	NA1	NA2	NA3	NB1	NB2	NB3
8 x 10^4	5280000	5040000	9760000	4160000	7440000	4720000	12400000	9040000	7520000	11520000	2400000	22400000
8 x 10^5	2400000	4000000	9600000	4800000	800000	3200000	28000000	27200000	9600000	38400000	32800000	58400000
8 x 10^6	0	0	0	0	0	0	64000000	0	0	40000000	8000000	40000000

The number of colonies on each plate was multiplied by the dilution factor of the respective plate to obtain the CFU value.

V. References

[1] Dy, Aaron. “Student Teams Take on Synbio Reproducibility Problem | PLOS Blogs Network.” PLOS Synbio, PLOS, 21 Nov. 2016, blogs.plos.org/blog/2016/11/21/student-teams-take-on-synbio-reproducibility-problem/.

[2] Beal, J., Haddock-Angelli, T., Gershater, M., Mora, K. D., Lizarazo, M., Hollenhorst, J., & Rettberg, R. (2016). Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. Plos One, 11(3). doi:10.1371/journal.pone.0150182

[3]“Fifth International Interlab Measurement Study.” IGEM, IGEM Organization, 2018.igem.org/Measurement/InterLab.

[4] “Help:2018 DNA Distribution.” IGEM, IGEM Organization, parts.igem.org/Help:2018_DNA_Distribution.

iGEM-Footer