The Interlab Study

Introduction

I read the interlab study as 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 value for other scientists is near zero. Standards must be set, in order to embue our results with any useful meaning. Although its motivations are shrouded in mystery, I percieve the interlab study as the first step towards that ultimate goal of blanket standardisation in a 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 two members of the team to pursuing it in parallel to our other endeavours, as a sort of side quest.

The Brief

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. I will quickly describe the nature of these devices.

Interlab Study

Section I: Provenance & Release

Work was done by Dan Goss, Chelsey Tye and Waqar Yousaf with help from Sian Davies and William Rostain as advisers.

Timeline of when protocols were run and measurements were taken is listed below

05/08/2014 Transformed all parts from kit plates, including an RFP-producing control

06/08/2014 Isolated/inoculated one colony from each and and grew them up overnight

07/08/2014 Miniprepped the overnights to secure sufficient plasmid DNA for digest

08/08/2014 Restriction digested parts and linearised plasmid backbones for assembly

08/08/2014 Ligated digested parts to produce devices 2 and 3

08/08/2014 Transformed ligation products over weekend

11/08/2014 Inoculated colonies of ligation products

12/08/2014 Miniprepped ligation products to ascertain plasmid DNA of devices 2/3

12/08/2014 Gel electrophoresis assay of these devices, with positive results

Interlab hiatus period

20/08/2014 Transformation of all devices from plasmid DNA for measurement

21/08/2014 Inoculated colonies of each device (three biological replicates for each)

22/08/2014 Refreshed cultures in M9 minimal media in the morning

22/08/2014 Measured optical density and fluorescence with plate reader overnight

25/08/2014 Collected data from plate reader, but gain was set to 100 so had to repeat

26/08/2014 Inoculated colonies of each device (three biological replicates for each)

27/08/2014 Refreshed cultures in M9 minimal media in the morning

27/09/2014 Measured optical density and fluorescence with plate reader overnight

28/08/2014 Collected data from plate reader and imported into Excel for analysis

Protocols

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