Team:Oxford/Test/Interlab Study1
Interlab Study
Introduction
Overview
One of the fundamental principles of synthetic biology is the characterization of standard biological parts. iGEM HQ's International InterLab Measurement Study is an endeavour at achieving the large-scale characterization of a set of biological devices which has been previously designed and catalogued in the BioBricks Registry. In this study, iGEM teams from across the world set out to measure the fluorescence of model organisms which have been transformed with devices comprising the same green fluorescent protein (GFP) gene expressed at different levels by being fused to promoters of different strengths. This would provide a large dataset from which a more accurate analysis that accounts for lab-to-lab variations can be drawn.
The promoters characterized in the 2015 InterLab Study are three constitutive promoters of different strengths found within the Anderson Promoter Collection, which were included in the 2015 iGEM Distribution Kit. We assembled each of these three promoters upstream of a given GFP-encoding BioBrick and transformed each of the composite parts into three different strains of E. coli to measure their respective fluorescence levels. We measured fluorescence levels using a 96-well microplate reader, a flow cytometer, as well as a confocal microscope.
Key Results and Findings
Through the data obtained from our microplate reader, we found that the strengths of the three promoters investigated are in the same order as Anderson et al's initial characterization across the three E. coli strains which we studied. The relative fluorescence magnitudes we measured however do not agree with the ratios presented in the Anderson study.
To eliminate the dependence on equipment parameters associated with arbitrary fluorescence units, we calibrated our fluorescence readings against different concentrations of a fluorescent chemical standard, sodium fluorescein, and expressed fluorescence intensity in units of its concentration.
Methods and Materials
Bacterial Strains and Growth Conditions
E. coli DH5α was used for all cloning purposes. E. coli DH5α, E. coli MG1655, and E. coli RP437 ∆FliC were used as expression hosts. Cultures for cloning were grown in Lysogeny Broth (LB) at 37°C, whereas for GFP expression cultures were grown in Knight's M9 Supplemented Media at 37°C. Stationary cultures for expression measurement were always grown overnight for 18 hours at 37°C in 5mL chloramphenicol-supplemented media contained in glass test tubes oriented to be standing at an angle with 225 rpm orbital shaking.
Plasmid Constructions and Gene Cloning
The selected Anderson promoters were fused to the designated GFP coding sequence using the BioBrick assembly method (step-by-step experimental detail as well as gel electrophoresis images can be found at the InterLab section of our lab notebook).
Briefly, the promoter-containing plasmids BBa_K823005, BBa_K823008, BBa_K823013, and RBS-GFP-containing plasmid BBa_I13504 were obtained from the 2015 iGEM Distribution Kit and transformed into E. coli DH5α. Upon plasmid extraction (using Omega Biotek E.Z.N.A. Plasmid DNA Mini Kit I, Spin Protocol according to manufacturer specifications), restriction digest was performed on the SpeI and PstI sites of the promoter plasmids to linearize them, and the XbaI and PstI sites of the RBS-GFP plasmid to isolate the RBS-GFP sequence. Subsequent ligations were performed to insert the isolated RBS-GFP sequence into the linearized promoter plasmids, yielding the composite parts BBa_K823005+I13504, BBa_K823008+I13504, and BBa_K823013+I13504, each containing an 8bp assembly scar sequence TACTAGAG between the promoter sequence and the ribosome binding site. The identities of the plasmids were confirmed through sequencing (SourceBioScience, UK). The sequencing data is available for perusal in the Supplementary Information section below.
The three composite parts were then each transformed into the three E. coli expression host strains.
Experimental Controls
Calibration against Chemical Standard