Experiments
Standard protocols
The following kits were used according to the manufacturer’s recommendations. Variations from the recommended protocol are noted as appropriate.
- NEB Plasmid Monarch Miniprep (# T1010S)
- Monarch® DNA Gel Extraction Kit Protocol (NEB #T1020S)
- Omega Biotek E.Z.N.A Plasmid DNA Maxi kit (#D6922-02)li>
Deviation from protocol:
Two additional spins were included prior to elution to remove residual ethanol containing wash buffer. Elutions were made in 1 mL water heated to 80°C to improve yield.
- NEBuilder® HiFi DNA Assembly Cloning Kit (# E5520S)
- Q5® High-Fidelity 2X Master Mix (# M0492S)
Reagent Stock Solutions
Antibiotic Stocks:
- 100 mg/mL Ampicillin (1000x) in water, stored at -20°C
- 50 mg/mL Kanamycin (1000x) in water, stored at -20°C
Chemical and Reagent Stocks:
HiFi cloning of pSB3K3-pmntP-rs-sfGFP geneblock into pSB3K3
The pSB3K3 backbone and pmnTP-rs-sfGFP geneblock were assembled and transformed into NEB5α using the NEB HiFi Assembly Cloning Kit (catalog # E5520S) according to manufacturer’s instructions with an approximate vector:insert ratio of 1:2. A 20ul 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 manufacturer’s instructions. Restriction digests were performed as indicated in the “Restriction Digest” methods.
Restriction digest of pSB3K3-RFP and pSB3K3-pmntP-rs-sfGFP using EcoRI and SpeI.
Plasmid DNA was digested with EcoRI, SpeI-HF or both in order to (1) obtain the pSB3K3 plasmid backbone for HiFi cloning, and (2) to confirm insertion of the pmntP-rs-sfGFP sensor geneblock into pSB3K3. Digests were set up using 400ng - 3 µg of plasmid DNA and digested for 4hr at 37C in a thermocycler followed by heat-inactivation of restriction enzymes at 80°C for 20 min. The table below shows a typical digest setup using ~400ng plasmid DNA. (The amount of enzyme was adjusted based on the amount of template used.) Subsequently, 25 μl of each reaction were run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.
| Material |
EcoR1 |
SpeI-HF |
Double |
Concentration / Activity |
|---|---|---|---|---|
| Plasmid |
9.3uL |
9.3uL |
9.3uL |
41.6ng/uL |
| 10x Cutsmart |
5uL |
5uL |
5uL |
10x |
| EcoRI |
0.25uL |
0uL |
0.25uL |
20U/uL |
| SpeI-HF |
0uL |
0.25uL |
0.25uL |
20U/uL |
| Water |
35.45uL |
35.45uL |
35.2uL |
- |
| Total |
50uL |
50uL |
50uL |
- |
Restriction digest of pTrc source plasmid (pTrc_VvTs_trGppsGB from WSU iGEM 2021) and pTrc-6X-HIS-mntR using EcoRI and SpeI.
Plasmid DNA was digested with EcoRI, SpeI-HF or both in order to (1) obtain the pSB3K3 plasmid backbone for HiFi cloning, and (2) to confirm insertion of the pmntP-rs-sfGFP sensor geneblock into pSB3K3. Digests were set up using 400ng - 3µg of plasmid DNA and digested for 4hr at 37C in a thermocycler followed by heat-inactivation of restriction enzymes at 80°C for 20 min. The table below shows a typical digest setup using ~400ng plasmid DNA. (The amount of enzyme was adjusted based on the amount of template used.) Subsequently, 25ul of each reaction were run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.
PCR of pTRC-6X-HIS-mntR insert to verify presence of insert
PCR was used in conjunction with restriction digest and sequencing as a confirmation of 6X-HIS-mntR insert in the pTrc plasmid backbone. The reaction was set up using the following primers:
Forward: 5’-cggttctggcaaatattctg-3’ (45% GC, Tm 51 °C)
Reverse: 5’-ctgatttaatctgtatcaggctg-3’ (39% GC, Tm 51°C)
PCR was set up in thin-walled PCR tubes using Q5 High-Fidelity 2X Master Mix (catalog # M0492S) according to manufacturer’s recommendations as follows:
98°C 30sec
98°C 10sec <--|
54°C 15sec 35 cycles
72°C 30sec <--|
72°C 2min
4°C hold
The PCR products were run on a 0.8% agarose gel for 1 hour. 5ul of 1kb DNA ladder was loaded, and the entire PCR sample for each reaction.
Preparation of chemically competent MG1655 ΔmntR E.coli
The following solutions were made:
- 100 mL of 0.1M CaCl2
- 60 mL of 0.1M CaCl2 + 15% glycerol
Protocol:
- 1 mL of an overnight culture of ΔmntR E. coli in LB medium (no antibiotic) was diluted in 99 mL of LB and incubated at 37°C, 200rpm until the OD600 reached 0.4.
- CaCl2 solutions and pre-labeled ΔmntR tubes were placed on ice before starting.
- The culture was separated evenly into 3 Oakridge tubes and centrifuged at 4°C, 4000rpm for 10 minutes.
- The supernatants were aspirated, and pellets were resuspended in 0.1 M CaCl2 and incubated on ice for 30 min.
- The samples were centrifuged for 10 min at 4°C, 4000rpm.
- Supernatant was dumped into bleach containers.
- Pellets were resuspended in 1 mL of 0.1M CaCl2 + 15% glycerol and combined into one tube along with another 2mL of CaCl2 + glycerol.
- One at a time, pre-chilled, pre-labeled 1.7 mL tubes were filled with 25uL of bacteria, snap frozen in liquid nitrogen, and stored on dry ice.
- 61 tubes of competent cells were stored at -80°C.
- Competency of cells was confirmed by transformation of pUC19.
Functional Testing of sensor pSB3K3-GB
The general outline of the sensor assay is shown below. The basic assay was initiated from a glycerol stock of the plasmid(s) of interest in wild-type MG1655 E. coli. A 5 mL culture was set up in LB with 50 µg/mL Kanamycin and grown in a shaker at 250 RPM overnight at 37°C. This culture was back-diluted into a larger working culture (20-100 mL) of media with antibiotic and grown to an OD600 of 0.5. Typically, cultures were aliquoted into 1-5 mL volumes in culture tubes for treatment with manganese chloride. After incubation with MnCl2M/sub>, 100 µl volumes were added to shielded microwell plates for A600 and Fuor485/515 readings.
