Team:UNSW Australia/InterLab

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Interlab

Introduction

The interlab study was an experiment that was run by the iGEM measurement committee across many of the labs competing in the iGEM competition. The aim was to run one standardised experiment across all of the labs, alongside a set of calibrations in the hope of achieving equivalent results from all participating laboratories. After performing standardisations for optical density and fluorescence, Escherichia coli (E. coli) DH5α cells were transformed with GFP expression plasmids and their optical density and fluorescence recorded. A count of colony forming units of the transformed E. coli was also used as an alternative indication of cell density.

Calibrations

First, a 45% colloidal silica suspension was used to measure the optical density (OD) of a known substance, seeing as this suspension was sent out by iGEM, making it consistent across all participating laboratories. This first measurement allows future measurements to be corrected for the path length, a factor that varies in spectrophotometry if the volume per well changes.

The second calibration was completed using microspheres of silica. These beads are of a comparable size to the bacterial cells used later in the study, and refract light in a similar way. In creating a serial dilution of these beads, a standard curve was generated so that future absorbance values could be converted to an equivalent number of cells in solution.

The third and final calibration involved the measurement of a serial dilution of the chemical fluorescein. A second standard curve was generated, allowing for the conversion of fluorescence readings into a standardised value, given that the concentration of fluorescein is known.

Table 1: Fluorescence values output from serial dilution of fluorescein. 1 in 10 dilutions were made from 10 µM fluorescein on the left, through to phosphate-buffered saline (PBS) on the right.

Transformations, Fluorescence and Colony Counts

E. coli DH5α cells were transformed with a positive control, GFP producing plasmid, a negative control, as well as a series of GFP expression plasmids that vary in their levels of expression. Transformation was first attempted with our team's protocols on cells we had made competent however we were unsuccessful. Due to the low amount of DNA per well, the second attempt was made following the iGEM protocols and with commercial competent cells. This method yielded successfully transformed cells for all of the parts of the study.

The bacteria were each grown overnight, diluted to a standardised starting OD, and allowed to grow, their fluorescence and OD measured at the start and end of the period of growth. The calibration protocols conducted previously allowed us to convert these results to a set of values, hopefully equivalent across all participating laboratories.

The final experiment in this series involved growing each of the transformed bacteria, diluting them to a standardised OD, and plating to determine the number of colony forming units present in the original culture. This allows for the conversion between OD measurements and a known concentration of cells.

Figure 1: Fluorescence of GFP expressed cultures observed under UV light.

Discussion

A key issue facing the scientific community is the reproducibility of experiments across different laboratories. The interlab study proposes an outstanding initiative in combating such discrepancies. Our team firmly believes that these standardisation techniques should be applied to all experiments where possible to increase the repeatability of results across laboratories.

We faced difficulties in transforming the bacterial cells with such a small amount of DNA as our methods normally use a substantial amount more. As previously mentioned, we were able to overcome this using the iGEM protocol for transformation and comercial competent cells. Once transformed, we also experienced issues in getting the bacteria to grow sufficiently in the 6 hour period. This was later found to be an issue with the luria broth used for the experiment, although this issue was not resolved before the conclusion of the interlab study. The calibration protocols employed should have accounted for this low level of growth as results were standardised based on a known OD.

Our team followed the protocols of the interlab study so that all results achieved would be valid across all participating laboratories, however the amount of plastic waste generated was noted by several team members. Several suggestions for future studies were made, such as diluting directly into reuseable cuvettes, rather than disposable 2.0 mL tubes.