Interlab Study: The Results

BioBricks used: BBa_I20270, BBa_R0040, BBa_J36000, BBa_J36001, BBa_J36002, BBa_J36003, BBa_J36004, BBa_J36005

Overview

The iGEM InterLab Study is the currently the largest interlaboratory study in synthetic biology (Beal et al., 2016). The interlab study investigates the replicability of data by providing all participating iGEM teams with protocols for measuring the fluorescence output of a set of genetic devices that have been designed to provide varying levels of Green Fluorescent Protein (GFP) expression. Team Newcastle 2017 followed the Interlab plate reader protocol, which can be found here (link protocol document).

InterLab Measurement Kit

This year, teams were provided with the InterLab Measurement Kit containing fluorescein and LUDOX stocks, along with the devices stored in the Distribution kits. The devices are:

• A positive control: BBa_I20270
• A negative control: BBa_R0040
• Test Device 1: BBa_J36000 (J23101.BCD2.E0040.B0015)
• Test Device 2: BBa_J36001 (J23106.BCD2.E0040.B0015)
• Test Device 3: BBa_J36002 (J23117.BCD2.E0040.B0015)
• Test Device 4: BBa_J36003 (J23101+I13504)
• Test Device 5: BBa_J36004 (J23106+I13504)
• Test Device 6: BBa_J36005 (J23117+I13504)

Protocols

OD600 Reference Point

LUDOX-HS40 was used as a single point reference to obtain a radiometric conversion factor to convert absorbance data into a standard OD600 measurement. The Reference OD600 divided by the Abs600 from four replicates of LUDOX was used to obtain a correction factor for use against the cell based assays.

Fluorescein Standard Curve

A dilution series of fluorescein in four replicates was prepared and measured in the plate reader to obtain a standard curve of fluorescence for fluorescein concentration. This was used to correct cell-based readings to an equivalent fluorescein concentration, and to then convert this to a GFP concentration.

Plate Reader

Competent E. coli DH5α cells were transformed with each of the devices and plated onto LB+Chl agar, and two colonies from each transformation plate were grown overnight in 10 ml LB + Chl in 50 ml Falcon tubes. Protocols for making competent cells and cell transformation can be found here (link to protocol). Fluorescence was measured as specified in the InterLab protocol on a Synergy H1 plate reader.

Results

Raw results can be found here

Figure 1 InterLab results – (A) Growth, (B) fluorescence and (C) calculated µM fluorescein/OD600 of InterLab devices grown at 37°C in LB for 6 h. Measurements from 4 individual plate readings at 0, 2, 4 and 6 h.

Bacterial growth - We found that the negative control, Tests 3, 5 and 6 grew the most in the 6 h experiment, with the positive control and Test 2 growing less so. Test 1 had the least growth.

Fluorescence - Test 2 showed the most fluorescence after 6 h, with Test 4 showing slightly less, followed closely by the positive control, then Tests 1 and 5. Tests 3 and 6 showed very little fluorescence.

FL/OD - Test 1 gave a significantly higher overall uM fluorescein/OD600 ratio compared to all other devices with values of 0.91 and 0.94 from the separate isolates, compared to a range of <0.01 and 0.18 from all other devices.

Variability - As part of the Interlab study, we analysed two separate bacterial transformants in quadruplicate. This allows an examination of the variability between replicates and between colonies. Between replicates, variation was minimal. However, between colonies containing the same device we saw a degree of variability in growth and fluorescence, thus affecting FL:OD. Figure 2 shows high levels of variability between max OD and Abs measurements of Tests 2 and 4. Fluorescence and subsequently FL:OD are directly affected by growth, but factors including inaccuracies in growth set up and the user taking the samples could have also affected these. As plasmid copy number will not be equal, this will also manifest in readings over time.

Figure 2 - Box and whisker plots of (A) max OD and (B) Absorbance values for fluorescence. Maximum values were obtained from all colonies and replicates for each device.

Conclusions and Future Work

After carrying out the InterLab Study we decided to take a high, medium and low expressing device and analyse their sensitivity to changes in environmental conditions. We also looked into how automation could reduce variation between results.

References

Beal, J. et al. (2016) ‘Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli’, PLOS ONE. Edited by D. D. Jones. ACM, 11(3), p. e0150182. doi: 10.1371/journal.pone.0150182.

Interlab Study Improvements: The Results

BioBricks used: BBa_J36001, BBa_J36004, BBa_J36005

Rationale and Aim

Background Information

Interlab Devices in Different Contexts

Standard Assembly Methods

Standard Measurement Methods

Internal Controls

Robust Promoter Characterisation

Conclusions and Future Work

References