Team:Oxford/InterLab

Interlab

Overview

This year is the 5th installment of iGEM’s interlab; a powerful joint venture undertaken by hundreds of iGEM teams to gather data with the hope to create tools to help future iGEM teams as well as the the Synthetic Biology community as a whole.

Across Synthetic Biology a commonly used method to test biological constructs is to tag them with fluorescent proteins such as GFP (Green Fluorescent Protein) and then measure their fluorescence on plate readers. This fluorescence can then be correlated to the rest of the construct but currently it is difficult to compare this data with other labs due to different units. It is the focus of Interlab to attempt to combat this by developing a detailed protocol that allows the measurement to be made in absolute units, therefore allowing comparison between all labs. This year, we attempt to assist in this by answering the following question:

Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?

Currently, Fluorescence, being an aggregate measurement over the whole population of cells, is normalized by number of cells to find a mean.. This is done by also measuring the the absorbance or optical density of the cells at 600nm (OD600) which is then used as an approximation for the cell population size. However it is the accuracy of this measurement which causes too much variability in measurements between labs so we are looking to develop a more direct method to measure cell count, formally Colony Forming Units (CFUs).

Experiments

2 Orthogonal approaches:

1. Converting between absorbance of cells to absorbance of a known concentration of beads. Silica beads the same size and shape as E.coli cells should act in the same way when used in absorbance measurements. Hence by using known concentrations of silica beads, we can accurately convert cell OD600 measurements to cell concentrations and hence cell counts.


2. Counting colony forming units (CFUs) from the sample. Since a colony on a plate can be grown from one cell, an easy method to count how many living cells are in a particular medium would be to plate an aliquot of it and count how many colonies form. We can then use the absorbance measurements made from the same samples to convert between absorbance and CFUs.

If the data from these 2 experiments appear reproducible across the range of iGEM teams undertaking the study, we will have helped create a new powerful technique for the Synthetic Biology community as a whole.

Protocols

An important criteria for the interlab to be valid is that all teams used the same protocol in carrying out their measurements. These protocols can be found on the following link: https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf

Results

Initally the transformation of all plasmids gave colonies on LB+Chlor plates but we had issues with some devices (1,4 &5) failing to grow during the 6 hour incubation periods during day 2 of the plate reader protocol. After a few attempts and the same failed results we resorted back to transforming directly from the kit plates again. By now, we were running low on many of the supplied materials - namely the kit plate plasmids and the silica beads to calibrate the plate reader. This led to us collaborating with the University of Warwick iGEM team where they gave us their spare materials and we gave them other iGEM supplied plasmids that they required which were unrelated to Interlab.

After the second round of transformations we were able to conduct the whole protocol and get results that passed common sense checks and these were what we eventually submitted.

Evaluation

After completing the Interlab project here are our thoughts:

• To try to develop a protocol which ensures results can be compared between scientists, the Interlab protocol requires all the plate readers must be calibrated against known concentrations of Fluorescein. These preset concentrations resulted in high fluorescence requiring a low gain to cover the range. This resulted in the measurements made of the cells being incredibly low, making noise an appreciable factor. Hence we would suggest either altering the preset concentrations to be lower or finding an alternative method of carrying out this calibration.


• We could not understand the reasoning for growing the cells in foil-wrapped falcon tubes and felt this was an unnecessary step and time/material wasting.


• We believe that since the Interlab protocol is meant be designed to be as useful as possible for scientists around the world, it should be altered to reflect how most growth assays are currently performed which is to prepare a plate and grow the samples in the plate reader whilst taking regular measurements instead of growing large stocks of the samples and taking aliquots to measure. This would not only be more relevant but also a lot less labour and material intensive.

We were glad to participate in the Interlab study as not only did it count as a Bronze award criterion but many of the skills used in the study were directly applied to our own project, namely the use of plate readers to measure absorbance and fluorescence to characterise our parts. Not only did it help us get acquainted with the machines but there was also valuable information about how much samples should be diluted to get to particular starting OD’s.

We hope that the data gathered by us proves useful for the study.