Aim & Concept

In a field as young as synthetic biology, it remains important to improve measurement techniques to ensure quality standards for methods commonly used. iGEM headquarters reaches out to its vast pool of researchers to conduct Interlab studies to collect data in its Fourth International InterLaboratory Measurement Study. By comparing data from teams all around the world, iGEM contributes to reproducible and comparable results. Our team participated in this year’s fluorescence of GFP measurements. Reliable data for GFP is of high value for the scientific community, as it is one of the most commonly used fluorescence marker proteins in the field.
The experiment was divided in 3 major parts: The transformation of the devices that were to be measured (8 in total) in DHα E- coli, the establishment of standard curves with LUDOX and fluorescein and the actual measurement of the devices over 6 hours growth time.

Material & Methods

Six test devices where used for measuring (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005), as well as a positive (BBa_I20270) and negative (BBa_R0040) control. All devices carry a chloramphenicol resistance that allows positive selection. The devices were transformed into in DHα E- coli and two colonies where picked for overnight growth in 5 ml LB + Chloramphenicol to grow the cells overnight (16-18 hours) at 37°C and 220 rpm. The cultures were then diluted to an OD of 0.02 in 12 ml LB medium + antibiotic in a 50 ml falcon tube, which was covered in tin foil to avoid exhaustion of the fluorescence proteins.
During the incubation at 37 °C at 220 rpm, the OD and the fluorescence were measured at 0, 2, 4 and 6 hours after standard measurements. For OD600, the reference point was obtained by measuring H2O and LUDOX-S40 (from the kit); while the fluorescence standard was obtained by measuring a dilution series of fluorescein in a 96-well plate in Fluoroskan Ascent 1.6.

Results

Six test devices where used for measuring (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005), as well as a positive (BBa_I20270) and negative (BBa_R0040) control. All devices carry a chloramphenicol resistance that allows positive selection. The devices were transformed into in DHα E- coli and two colonies where picked for overnight growth in 5 ml LB + Chloramphenicol to grow the cells overnight (16-18 hours) at 37°C and 220 rpm. The cultures were then diluted to an OD of 0.02 in 12 ml LB medium + antibiotic in a 50 ml falcon tube, which was covered in tin foil to avoid exhaustion of the fluorescence proteins.
During the incubation at 37 °C at 220 rpm, the OD and the fluorescence were measured at 0, 2, 4 and 6 hours after standard measurements. For OD600, the reference point was obtained by measuring H2O and LUDOX-S40 (from the kit); while the fluorescence standard was obtained by measuring a dilution series of fluorescein in a 96-well plate in Fluoroskan Ascent 1.6.

Figures

Fluorescein standard curve:

Fluorescein standard curve on Logarithemic scale:


Optical density of cell suspension over time