Team:Newcastle/Measurement/Methods
Alternative Roots
Materials and Methods
Materials and Methods used regarding Measurement, Automation and Reproducibility
Bacterial Strains.
Transformations with, and expression of, iGEM test devices and controls were carried out using chemically competent Escherichia coli DH5α. Competency was conferred using the MgCl-CaCl2 method (Sambrook and Russell 2001). Briefly, a single colony of DH5α was incubated in Leuria Bertoni (LB) broth overnight at 37°C with shaking at 220rpm. Overnight culture was diluted 1:100, further incubated until an optical density (OD600nm) of 0.3 – 0.6 was reached and then placed on ice for 30 minutes. Cells were centrifuged at 4000g for 5 minutes at 4°C, resuspended in 0.1M MgCl2 and incubated on ice for 30 minutes. The suspension was centrifuged again as before, resuspended in 0.1M CaCl2 and placed on ice for 30 minutes. Cells were spun down again, resuspended in 0.1M CaCl2 with 15% glycerol and frozen at -80°C.
Plate Reader Set-up
Culture absorbance and fluorescence were measured in 96 well plates using a Thermofisher Varioskan Lux plate reader (Thermofisher scientific) unless stated otherwise. Absorbance was measured at 600nm. GFP Fluorescence was measured at 525nm with excitation at 485nm. RFP fluorescence was measured at 635nm with excitation at 588nm. All readings took place at 25°C after a 5 second 300rpm shake step to homogenise the culture. Readings used a 12nm bandpass width and pathlength correction was disabled, as per the iGEM Interlab study guidelines.
Internal Standard Design
An RFP construct was designed for use as an internal standard for each test device. The RFP construct was designed using Benchling. The parts used for building the RFP construct were Anderson promoter BBa_J23108, RBS BBa_0032, the RFP gene - gained from SnapGene - and double terminator BBa_B0015. Gibson ends were also designed for cloning into pSB1C3 using the NEBuilder DNA assembly tool and the gBlock was synthesised by IDT. The promoter has a measured strength of 0.51 relative to BBa_J23100.
mNeonGreen Design
The mNeonGreen construct was designed for use as an alternate fluorescent reporter for each test device - replacing GFP. The mNeonGreen sequence was codon optimised using Benchling and the Gibson ends were designed using NEBuilder for cloning into pSB1C3. The subsequent sequence was synthesised by IDT.