Introduction

The interlab study is an attempt by iGEM HQ to conduct a comparative analysis of the different methods employed by the varied and diverse teams internationally to arrive at useful data. A key problem in science is ascertaining absolute measurements; there is no point in one measuring the fluorescence of some given part, only to arrive at arbitrary units whose meaning to other scientists is near zero. Standards must be set, in order to embue our results with any useful meaning. The interlab study is a step towards that ultimate goal of blanket standardisation in the synthetic biological context.

It is suggested that a team conduct the interlab study as a preamble to the main event. However, we are the first example of an iGEM team at Warwick, and coalesced rather late in the day. Hence we decided just to dedicate three members of the team to pursuing it in parallel to our other endeavours, as a side quest. It was primarily undertaken by Dan Goss , Waqar Yousaf and Chelsey Tye , with support from advisers Will Rostain and Sian Davies . All consent is given in accordance with the WTFPL license!

The experimental timeline

The remit of the interlab study boils down to constructing and characterising, albeit minimally, three devices. They share a lot of similarities, and the objective is obviously not to create some wacky new form of life, but to measure well characterised and well understood parts in order to measure the measuring equipment, as it were.

Therefore, over a period of about 1 month, we engineered the three devices from the brief via transformation, miniprep, digestion, ligation, and all the protocols you would expect (more on that below). What follows is a timetable of our experimental work in the wet lab:

Protocols and methodology

Many of the protocols mentioned above which we used were harvested straight from the iGEM website, but we also used content from previous iGEM teams and instructions packaged with kits. The specific materials and procedures can be accessed through clicking the relevant hyperlink:

• Transformation
• Growing (which I refer to mostly as inoculation)
• Miniprep
• Restriction digest
• Ligation
• Gel electrophoresis
• 3A Assembly basically comprises digestion and ligation

To acquire the measurements necessary, we used a microplate reader, rather than a flow cytometer or something similar. To do this, we used the Tecan Infinite F500 , seen below.

This machine is very flexible, supporting various different plate sizes (from 6- to 1536-well plates!) and various modes of measurement (supporting absorbance wavelength from 230-1000nm and excitation wavelengths from 230-900nm). It is configured in to take the readings we were after using the following setup:

• 96 well plate (see diagram below)
• Temperature: 37 degrees Celsius
• Absorbance measurement wavelength: 600nm (OD)
• Excitation wavelength: 465nm (GFP)
• Emission wavelength: 530nm (GFP)
• GFP gain: 35

We then ran the programme ‘GFPandOD_overnight’, for which one cycle runs like so:

1. Orbital shaking (amplitude 2.5mm, frequency 246.7rpm) for 200s
2. Wait for 20s
3. Repeat shaking for 200s
4. Wait for 2s
5. Repeat shaking for 200s
6. Take measurements (takes about four minutes)

This whole process takes 15 minutes per cycle, and would run 80 cycles, coming to 20 hours, unless interrupted. Generally the optical density (that is, the growth of cells) has maxed out earlier than that. I stopped my measurements after 14 hours because growth had reached a plateau.

In terms of the protocol for preparing the samples and the plate for the reader, the first step was to concoct some M9 minimal media in which to refresh inoculated overnights before putting them into the plate reader. For 200ml of M9, combine the ingredients given, paying attention to amounts, concentrations and preparation advice given, and make up with H2O:

Measurements and results