Revision as of 01:27, 16 October 2014 by Waqar (Talk | contribs)

The Interlab Study


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.

Device 1: BBa_I20260

This device is fully composed and readily available in Kit Plate 4 of the 2014 distribution. The parts in this kit plate are contained within non-standard plasmids, as opposed to the classic pSB1C3 in which parts must be delivered to iGEM HQ. In this case, pSB3K3. There is a logic to the plasmid nomenclature ; here the capital K denotes the plasmid's antibiotic resistance to kanamycin. This is the low hanging fruit! As you can see below, it is composed of a promoter, a ribosome binding site (or RBS), a GFP gene and two terminators, one each for ending transcription and translation (INSERT APPROPRIATE PICTURE).

Device 2: BBa_J23101 + BBa_E0240

Warwick iGEM • Interlab Study Section I: Provenance & Release Q. Who did the actual work to acquire these Measurements? Dan Goss Chelsey Tye Waqar Yousaf Q. What other people should be credited for these measurements? Sian Davies William Rostain Q. Dates protocols were run and measurements taken? Date Protocols and measurements 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 Q. Do all persons involved consent to the inclusion of this data in publications derived from the iGEM interlab study? Yes, we all give consent for inclusion of this data in publications. Section II: Protocols Q. What protocol did you use to prepare samples for measurement? 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 Q. What sort of instrument did you use to acquire measurements? We used a microplate reader, rather than a flow cytometer or something similar. Q. What is the model and manufacturer? The Tecan Infinite F500.