Experiments

Stock Solutions

Antibiotic Stocks:

Chemical and Reagent Stocks:

Cell free lysate:

Equipment

Mfr. Protocols

The following kits were used according to the manufacturer’s recommendations. Variations from the recommended protocol are noted as appropriate.

Restrict Digest

Geneblocks (pmntR-riboswitch-deGFP, pmntR-riboswitch-NanoLuc, and T7-riboswitch-sfGFP) were cloned into a linearized pSB3K3 backbone. To obtain the linearized backbone, 1mg of pSB3K3 plasmid DNA was digested with EcoRI and SpeI-HF. Single digests were used to confirm each enzyme cut the plasmid backbone as expected. Digests were set up using 1 microgram of plasmid DNA and digested for 3hr at 37°C in a thermocycler followed by heat-inactivation of restriction enzymes at 80°C for 20 min. The table below shows a typical digest. (The amount of enzyme was adjusted based on the amount of template used.) Subsequently, 50 µL of each reaction were run on a 0.8% agarose gel and imaged on a Fuji LAS 4000 and the desired linearized backbone was purified using the Monarch® DNA Gel Extraction Kit using the manufacturer's protocol.

The above was also used to screen individual colonies from HiFi cloning to confirm insertion of the desired geneblock (pmntR-riboswitch-deGFP, pmntR-riboswitch-NanoLuc, T7-riboswitch-sfGFP and pmntR-riboswitch-eforRed).

HiFi Cloning

The linearized pSB3K3 backbone and individual geneblock (pmntR-riboswitch-deGFP, pmntR-riboswitch-NanoLuc, T7-riboswitch-sfGFP or pmntR-riboswitch-eforRed) were assembled and transformed into NEB5α using the NEB HiFi Assembly Cloning Kit (catalog # E5520S) according to the manufacturer's instructions with an approximate insert:backbone ratio of 3:1. A typical 20µL reaction was set up as indicated below and incubated in a thermocycler at 50°C for 15 minutes.

After assembly, the reaction was transformed into NEB 5-alpha cells as recommended. Resulting colonies were screened by restriction digest. To do so, miniprep DNA was prepared from 3ml overnight cultures by NEB Monarch miniprep kit (catalog #T1010S) according to the manufacturer's instructions. Restriction digests were performed as indicated in the “Restriction Digest” methods.

Transformation

1. Chemically competent cells (MG1655 or NEB5 alpha) were thawed on ice.
2. Two microliters of the chilled assembled HiFI cloning reaction were added to the competent cells. Tubes were mixed gently by pipetting up and down or by flicking the tube 4–5 times. (Tubes were not vortexed.)
3. The mixture was placed on ice for 30 minutes.
4. Samples were heat shocked at 42°C for 30 seconds.
5. Tubes were placed on ice for 2 minutes.
6. 950 μl of room-temperature SOC media was added to each tube.
7. Tubes were incubated at 37°C for 60 minutes with 250 rpm rotation.
8. Agar plates were pre-warmed to 37°C.
9. 200 μl of the cells were spread on LB agar plates with the appropriate antibiotic. (LB-Kanamycin plates were used for all pSB3K3 plasmids described in this project.) The remainder was spun down, the media dumped off , and the pellet was resuspended in the small amount of LB remaining in the tube and spread on a second plate. This yielded 2 plates, one with ~20% of the bacteria and the second with ~80%, increasing the chances of obtaining isolated colonies for subsequent testing.
10. Plates were incubated overnight at 37°C and viewed the next day.

Resulting colonies were screened by restriction digest to confirm insertion of the desired geneblock (pmntR-riboswitch-deGFP, pmntR-riboswitch-NanoLuc, and T7-riboswitch-sfGFP) as indicated in the “Restriction Digest”methods. To do so, miniprep DNA was prepared from 3 mL overnight cultures by NEB Monarch miniprep kit (catalog #T1010S) according to the manufacturer's instructions. The colonies were screened from HiFi cloning as described above using double digestion with EcoRI and SpeI-HF.

Whole-Cell Assay

Whole-cell assay of manganese sensor plasmids

The standard whole-cell assay utilized by our 2022 iGEM team was performed for the pSB3K3-pmntP-riboswitch-sfGFP, pSB3K3-pmntP-riboswitch-deGFP, pSB3K3-T7-riboswitch-sfGFP, and pSB3K3-pmntP-riboswitch-NanoLuc. This assay was performed as follows:

1. A 5ml overnight culture LB with 50µg/ml Kanamycin was set up from glycerol stock of the plasmid(s) of interest in wild-type MG1655 E. coli (for all plasmids with a pmntP promoter) or BL21(DE3) E.coli (for plasmids with a T7 promoter) and grown in a shaker at 250 RPM overnight at 37oC.
2. This cµLture was back-diluted into a larger working culture (20-100ml) of media with antibiotics and grown to an OD₆₀₀ of 0.5.
3. Cultures were then aliquoted into 1-5 ml volumes in culture tubes for treatment with manganese chloride.
4. At the desired timepoints, generally 2hr, 4hr, 6hr and 8hr, 100µl volumes were added to shielded 96-well microwell plates for A₆₀₀ and Fluor_485/515 readings. Each sample was read in triplicate using a BioTek Synergy H1 plate reader.
5. All readings were blanked against an LB control. Density-corrected fluorescence values were calculated by dividing the Fluor_485/515 by A₆₀₀ values, and then the fold-change in fluorescence was calcµLated versus the control sample with no manganese treatment (i.e. 0mM MnCl₂ control).

Whole-cell assay of the pSB3K3-pmntP-riboswitch-NanoLuc sensor

1. Cultures were grown as in the standard whole cell assay described above (i.e. overnight cultures diluted and grown to OD600 of 0.5) and incubated with or without manganese for the desired time (2hr - overnight).
2. At the desired endpoint, generally 24hr post addition of manganese, 10µL of each culture was transferred to a 96-well microwell plate. LB broth was again used as a blank.
3. NanoLuc substrate was prepared by diluting the furimazine substrate stock solution 1:50 in buffer provided in the Nano-Glo Luciferase Assay Kit (Promega, catalog number N1110). 10μl of NanoLuc substrate was added to each well.
4. The plate was tapped lightly to mix and read on a BioTek Synergy H1 plate reader using luminescence fiber, gain 135, signal integration of 1 sec per read, reads taken every 1 minute for a 20 minute time-course. This allowed us to assess the stability of the luminescent output and provided a readout of substrate excess or deficiency in our reactions.

Cell-Free

The following protocol utilizes lysate from BL21(DE3) E. coli and buffer (“mix”) containing all of the required buffers, polymerases, nucleotides, tRNAs and energy sources. The specific buffer components are detailed in Silverman et al 2019 [1].

* Template DNA used in these reactions included plasmid DNA from the sensor being tested or relevant control plasmids. Sensor plasmids tested included pSB3K3-pmntP-riboswitch-sfGFP, pSB3K3-pmntP-riboswitch-deGFP, pSB3K3-T7-riboswitch-sfGFP, pSB3K3-pmntP-riboswitch-NanoLuc and pSB3K3-pmntP-riboswitch-eforRed. All plasmids were purified by Omega E.Z.N.A. maxiprep and then ethanol precipitated for use in cell-free assays to ensure template DNA was of sufficient purity and concentration.
** The master mix was prepared by combining 3.65µl of buffer (“mix”) with 3.35µl cell extract per reaction.

Assays performed using the homemade cell free extract were performed as follows:

1. The BioTek Synergy H1 plate reader was preheated to 29°C.
2. The cell extract, buffer (“mix”) and positive control plasmid (P70a(2)-deGFP) were thawed on ice until needed.
Note: To minimize freezing and thawing cycles, only the number of reagent tubes required to set up the desired number of cell-free reactions were thawed.
3. Cell-free Master Mix was prepared by combining 3.65µl of buffer (“mix”) with 3.35µl cell extract per reaction.
4. Directly before use, Master Mix was vortexed for 2-3 seconds and briefly spun down. If any precipitate was visible hereafter, the sample was gently resuspended by pipetting about 10 times to ensure homogeneity. CarefµL attention was paid to pipetting technique to avoid formation of bubbles and foam.
5. Each reaction was set up with a final volume of 12.5μL. On ice, components were combined in 0.6ml sterile PCR tubes according to the run needs. (Specific reaction setups varied by experiment and are detailed in the results page.)
6. The assembled Cell Free reactions were vortexed for 2-3 seconds and briefly centrifuged to collect the entire volume at the bottom of the tube.
7. For sensors with a fluorescent readout, reactions were transferred 6µL per well into 96-well V-bottom microwell plates, covered with a plate sealer, and placed in the BioTek Synergy H1 plate reader for measurement. For NanoLuc luminescence measurements, the tubes containing the 12.5µL reactions were transferred to an incubator set to 29°C. Fluorescent measurements (Fluor_485/515nm) were taken every 10 minutes for 24 hours.
8. For sensors with a luminescent readout, measurements were performed at the chosen endpoint by adding 0.5µl or 1.0µl of sample into a total 10µl water in appropriate wells in a V-bottom plate. 10µl of diluted NanoLuc substrate was added to each well. The plate was tapped lightly to mix and read on a BioTek Synergy H1 plate reader using luminescence fiber, gain 135, signal integration of 1 sec per read, reads taken every 1 minute for 20 minutes time-course.

Western Blot

1. 10-well, 10% SDS-PAGE gels were prepared according to standard laboratory protocols.
2. Protein samples from whole-cell and cell free reactions were prepared as follows:
   Whole-cell: Pellet of 1 OD 600 equivalent of bacteria was resuspended in lysis buffer, sonicated 3 times at 80% power using a Sonic Dismembrator 60 (Fisher Scientific), brought to 18µL with water and mixed with 2µL of 10X SDS-PAGE loading dye. Cell-free: The cell-free reaction was brought to x µL with nuclease free water and mixed with 2µL of 10X loading dye.
3. Samples were boiled for 5 minutes and then both samples and an appropriate protein ladder (NEB 1kB ladder, catalog # NO552S) were loaded into the appropriate lanes of the gel.
4. The SDS-PAGE gel was run at 115V for 30 minutes until the samples completed their migration through the stacking gel. The voltage was increased to 135V and the gel was run until the dye front reached the bottom of the gel.
5. The gel was then transferred onto a 0.45umPVDF (Polyvinylidene fluoride) membrane using standard laboratory protocols and transferring set to a constant 0.35A amperage for 60 minutes.
6. Once proteins had been transferred to the PVDF membrane, the PVDF blot was soaked in a 5% milk blocking buffer for 1 hour. The blot was then soaked in primary antibody (e.g. mouse monoclonal anti-GFP) at a 1:1000 dilution in blocking buffer overnight on a rocker platform at 4°C.
7. The blot was washed 3 times with 0.05% TTBS (PBS with 0.05% Tween) for 10-15 minutes per wash on the rocker and then transferred into secondary antibody (goat anti-mouse HRP antibody) diluted 1:5000 in 5% milk and incubated for 1 hour on the rocker.
8. The blot was then washed in three 0.5% TTBS washes of 15 minutes each.
9. The blot was then transferred into chemiluminescence solution (Western Lightning Plus-ECL, Perkin Elmer # NEL105001EA or SuperSignal West Fempto Substrate, Thermo Fisher # 34096) and imaged in the Fuji LAS 400.

References