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Revision as of 15:35, 17 October 2018
Alternative Roots
Materials and Methods
Implementing the New Devices
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.
Internal Standard & mNeonGreen Analysis
Analysis of the internal standards involved comparing the original InterLab test device plasmids against the new internal standard plasmids. Wells A-D represented the RFP containing E. coli and wells E-H represented the original test device containing E. coli. Column 9 wells A-H contained an LB+cam blank. The microtiter plate was incubated for 24 hours in the plate reader with Abs600, fluorescence (GFP): Excitation 485, Emission 420 and fluorescence (RFP): Excitation 588, emission 635 measured every 15 minutes following a short shake at 420 rpm at a low shake diameter.
Three further Interlab studies were carried out for mNeonGreen expressing E. coli DH5-alpha and those containing the original test devices, using the same conditions as the original study. The results fluorescence/OD, MEFL/particle and mean standard error of the mNeonGreen study was compared to the original Interlab.
Bio-Design Automation
Transformation Buffer Preparation
Primary transformation buffers (TB) before optimisation were CCMB80 protocol and a CaCl2-MgCl2 protocol. CCMB80 buffer was made from the following: 10 mM KOAc, 80 mM CaCl2, 20 mM MnCl2, 10 mM MgCl2, 10% glycerol and pH was adjusted to 6.4 with 0.1 N HCl. For the CaCl2-MgCl2 protocol, a 100 mM CaCl2 and a 100 mM MgCl2 solution were made. For preliminary DoE scoping experiments, concentrated stock solutions were made up of each individual reagent, buffer or compound. These stocks were used for the low, medium and high scoping (Table 1), buffer (Table 2), wash step (no wash, 1 wash or 2 wash) and cryoprotectant experiments (Medium TB with either DMSO 7.5% or glycerol 18%). All buffers were filter sterilised using Soft-Ject® syringes with Minisart 0.2 um filter (bar DMSO which was filtered with a DMSO-Safe Acrodisc® filter) and stored in 30 mL sterile universal tubes at 4 ℃. Fresh stocks were made when solutions ran out, however concentrations and storage remained the same.
Plasmid Preparation
RFP test and GFP (TD4) plasmids were purified using a QIAprep Spin Miniprep Kit (Qiagen). One colony of transformed E. coli was grown at 37 ℃ @ 220 rpm O/N (16 hours) in 5 mL SOB media in a 50 mL falcon tube. 1 mL was then aliquot into four different 2 mL microcentrifuge tubes. The official protocol was followed, including the addition of RNase A, LyseBlue reagent and 100% EtOH to the appropriate buffers. Instead of using Buffer EB to elute the DNA in step 10, sterile ddH2O was used to remove any potential downstream interactions that the buffer could have during analysis and transformation. The elution was run through the QIAgen 2.0 spin column a second time to maximise the yield. To determine plasmid concentration, a Qubit dsDNA BR Assay Kit (Invitrogen) was used following its standard protocol. 100 pg/µL stocks of both RFP and GFP were made with ddH2O and frozen at -20 ℃.
Manual Competent Cell and Transformation Workflow
Competent cells for the iGEM Interlab were made using the CCMB80 buffer (iGEM 2018) and CaCl2-MgCl2 buffer protocol. For downscale experimentation from 250 mL tube to 96 well plate, as well as wash step experiments, CaCl2-MgCl2 buffer was used. For all other experiments, the competent cells were made using a modified 0 wash protocol with varying TBs. The 0 wash protocol was an adapted version of the CaCl2-MgCl2 protocol. 1 colony was used to inoculate 10 mL of SOB in a 50 mL falcon tube for an O/N culture. 1 mL of the O/N culture was then used to inoculate 50 mL SOB in a 250 mL baffled conical flask the following morning. Once OD600 of 0.4-0.6 reached, 1.5 mL of culture was aliquoted into 2 mL microcentrifuge tubes and chilled on ice for 15 minutes, before being pelleted by a 5417R microcentrifuge (Eppendorf) at 3500 RCF for 5 minutes at 4 ℃. Pellets were then resuspended as 100 µL aliquots, made with TB, and with cryoprotectants glycerol or DMSO added if TB did not already contain one. These aliquots could then be frozen or used after a 40 minutes ice incubation. For the transformations, there was no variance in protocol, with the following standard transformation workflow being used for all transformations. Competent E. coli DH5α aliquots were thawed on ice for 15 minutes before 1 µL plasmid DNA added (plasmid concentration range from 10 pg/µl – 50 ng/µl). E. coli was then incubated on ice for 40 minutes before a heat shock at 42 ℃ for 45 seconds was carried out. After heat shock, cells were returned to ice for at least 5 minutes. For 2 mL Eppendorf’s, 900 µL preheated SOB (37℃) was then added to aliquots. For 96 well microtiter plates, 150 µL preheated SOB was added. For 2 mL microcentrifuge transformations, if ampicillin was the plasmids selection agent, incubation recovery times were reduced to 40 minutes at 37 ℃ @ 220 rpm. For chloramphenicol, incubation recovery times were extended to 2 hours. For 96 well plate transformations not following automated protocol, incubation recovery times were kept the same for the respective antibiotics, however plates were incubated in plate reader at 37 ℃ with 600 rpm shake and low force. Using automation, 96 wells plates were incubated at 37 ℃ static with TempDeck (Opentrons, United States).
To assess transformation efficiency, 50 µL of transformed culture was spread on SOB+CAM agar plates and incubated O/N at 37℃. Colonies were counted and TrE was determined by CFU. The calculation is as follows:
