Revision as of 12:39, 17 October 2018

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 & mNeonGreen 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.

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.

Cloning of New Devices into pSB1C3

Plasmid vectors were purified from E. coli via miniprep (Qiagen)and the concentration for each mini-prepped test device was determined using a Qubit fluorometer and diluted to 0.5 ng/µl. The diluted pSB1C3 vectors were linearised using a 2 step PCR system following a Q5 Polymerase protocol (NEB). This protocol utilised forward and reverse primers with Tm values of 72°C. The internal standard and mNeonGreen primers were designed by using the NEB Tm calculator and Benchling. The Internal Standard bind in a non-coding region of the pSB1C3 vector – a region between the chloramphenicol resistance gene and the ORI. The mNeonGreen primers consisted of 6 reverse primers, one complimentary to each test device promoter, and a single forward primer over the terminator. The amplified DNA was then digested with DpnI, heat treated to inactivate the enzyme and assembled via Gibson Assembly using the NEBuilder HiFi DNA Assembly Kit. Following their protocol, a 2-fragment reaction with 0.5 pmol of DNA in a 2:1 insert to vector ratio was done and transformants were plated onto agar plates with the appropriate antibiotic (LB+cam for each test device and LB+amp for the controls). Following growth of colonies, plasmid DNA was miniprepped from DH5-α transformed with both the internal standard and the mNeonGreen vector and sequenced to verify presence of the genes.

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                  <h3 class="subhead">Cloning of New Devices into pSB1C3</h3>
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-<p>Plasmid vectors were purified from <I>E. coli<I> via miniprep (Qiagen)and the concentration for each mini-prepped test device was determined using a Qubit fluorometer and diluted to 0.5 ng/µl. The diluted pSB1C3 vectors were linearised using a 2 step PCR system following a Q5 Polymerase protocol (NEB). This protocol utilised forward and reverse primers with Tm values of 72°C. The internal standard and mNeonGreen primers were designed by using the NEB Tm calculator and Benchling. The Internal Standard bind in a non-coding region of the pSB1C3 vector – a region between the chloramphenicol resistance gene and the ORI. The mNeonGreen primers consisted of 6 reverse primers, one complimentary to each test device promoter, and a single forward primer over the terminator. The amplified DNA was then digested with DpnI, heat treated to inactivate the enzyme and assembled via Gibson Assembly using the NEBuilder HiFi DNA Assembly Kit. Following their protocol, a 2-fragment reaction with 0.5 pmol of DNA in a 2:1 insert to vector ratio was done and transformants were plated onto agar plates with the appropriate antibiotic (LB+cam for each test device and LB+amp for the controls). Following growth of colonies, plasmid DNA was miniprepped from DH5-α transformed with both the internal standard and the mNeonGreen vector and sequenced to verify presence of the genes. </p>
+<p>Plasmid vectors were purified from <I>E. coli</I> via miniprep (Qiagen)and the concentration for each mini-prepped test device was determined using a Qubit fluorometer and diluted to 0.5 ng/µl. The diluted pSB1C3 vectors were linearised using a 2 step PCR system following a Q5 Polymerase protocol (NEB). This protocol utilised forward and reverse primers with Tm values of 72°C. The internal standard and mNeonGreen primers were designed by using the NEB Tm calculator and Benchling. The Internal Standard bind in a non-coding region of the pSB1C3 vector – a region between the chloramphenicol resistance gene and the ORI. The mNeonGreen primers consisted of 6 reverse primers, one complimentary to each test device promoter, and a single forward primer over the terminator. The amplified DNA was then digested with DpnI, heat treated to inactivate the enzyme and assembled via Gibson Assembly using the NEBuilder HiFi DNA Assembly Kit. Following their protocol, a 2-fragment reaction with 0.5 pmol of DNA in a 2:1 insert to vector ratio was done and transformants were plated onto agar plates with the appropriate antibiotic (LB+cam for each test device and LB+amp for the controls). Following growth of colonies, plasmid DNA was miniprepped from DH5-α transformed with both the internal standard and the mNeonGreen vector and sequenced to verify presence of the genes. </p>
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