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Team:Warwick/Interlab
From 2014.igem.org
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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:
| 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 |
| ... | Hiatus period (focusing on other work!) |
| 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 |
| 27/08/2014 | Measured optical density and fluorescence with plate reader overnight |
| 28/08/2014 | Collected data from plate reader and imported into Excel for analysis |
| 29/08/2014 | The end! |
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
Measurements and results
