Rapid and multiplex preparation of engineered Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing and sequencing†

Acknowledging its unique conical lumen structure, Mycobacterium smegmatis porin A (MspA) was the first type of nanopore that has successfully sequenced DNA. Recent developments of nanopore single molecule chemistry have also suggested MspA to be an optimum single molecule reactor. However, further investigations with this approach require heavy mutagenesis which is labor intensive and requires high end instruments for purifications. We here demonstrate an efficient and economic protocol which performs rapid and multiplex preparation of a variety of MspA mutants. The prepared MspA mutants were demonstrated in operations such as nanopore insertion, sequencing, optical single channel recording (oSCR), nanopore single molecule chemistry and nanopore rectification. The performance is no different from that of pores however prepared by other means. The time of all human operations and the cost for a single batch of preparation have been minimized to 40 min and 0.4$, respectively. This method is extremely useful in the screening of new MspA mutants, which has an urgent requirement in further investigations of new MspA nanoreactors. Its low cost and simplicity also enable efficient preparations of MspA nanopores for both industrial manufacturing and academic research.

imidazole, 0.5% (w/v) Genapol X-80, pH=8.0) was added to the beads. The beads were resuspended and shaken on a rotary mixer at room temperature (RT) for 5 min. After magnetic separation, the supernatant was discarded. Then 100 µL eluting buffer B1 (0.5 M NaCl, 20 mM HEPES, 219.5 mM imidazole, 0.5% (w/v) Genapol X-80, pH=8.0) was added to the beads. The beads were resuspended and shaken on a rotary mixer at room temperature (RT) for another 5 min. After magnetic separation, the supernatant was discarded. Eventually, 100 µL eluting buffer B2 (0.5 M NaCl, 20 mM HEPES, 500 mM imidazole, 0.5% (w/v) Genapol X-80, pH=8.0) was added to the beads. The tube was shaken on a rotary mixer at room temperature (RT) for 5 min. After magnetic separation, the supernatant was collected. Based on results of gel electrophoresis characterization ( Figure S3A), the collected supernatant contains the desired M2 MspA in an octameric form, ready for all downstream nanopore measurements without any further purifications.

Electrophysiology recordings and data analysis.
All electrophysiology measurements were performed as previously reported 4 . Briefly, a measurement chamber is consisted of two compartments separated by a Teflon film (30 µm thick) with an orifice (~100 µm in diameter).
Before the measurement, the compartments were first cleaned with Milli-Q water and ethanol. They were then blown dry with compressed nitrogen gas. The orifice on the film was pretreated with pentane containing 0.5% (v/v) hexadecane and air dried to evaporate the remaining solvent. The measurement device was placed in a Faraday cage, which serves to shield external electromagnetic noises. The Faraday cage was mounted on a floating table, which effectively minimizes mechanic vibrations. Prior to the measurement, approximately a 0.5 mL of electrolyte buffer was added to both compartments. A pair of Ag/AgCl electrodes were placed in each compartment, in contact with the buffer to form a closed circuit. By convention, the compartment that is electrically grounded is defined as the cis and the opposing side was defined as the trans. 100 µL 1,2-Diphytanoyl-snglycero-3-phosphocholine (DPhPC) was added to both compartments. To form a self-assembled phospholipid bilayer, the buffer in one of the compartments was pipetted up and down till the two compartments were electrically sealed. MspA nanopores were added to cis to initiate spontaneous pore insertions. To avoid further channel insertions, the buffer in the cis compartment was immediately exchanged with fresh buffer upon a single nanopore insertion.
All electrophysiology signals were amplified by an Axopatch 200B patch clamp amplifier and digitized by a Digidata 1550 B digital-to-analog converter (Molecular Devices, UK) with a 25 kHz sampling rate and filtered at a 1 kHz angular frequency. All single molecule sensing events were detected by the built-in "single-channel search" function of Clampfit 10.7 (Molecular Devices, UK) and further analyzed (histogram generation, fitting and plotting) by Origin Pro 2016. pH=8.0) to the beads and vortex for 30 seconds and discard the supernatant after magnetic separation. The beads were further washed with LE buffer for another 3 times and the LE buffer was eventually discarded. After pre-treatment, the beads are ready to be used.

Regeneration of Ni-charged magbeads.
After each use, the magbeads can be regenerated for multiple further uses. Briefly, 400 µL cleaning buffer Ⅰ (20 mM Tris-HCl, 100 mM EDTA, pH=8.0) was added to the magbeads and vortexed for 30 min. After magnetic separation, the supernatant was discarded. Afterwards, 400 µL Milli-Q water was added to the beads and vortexed for 10-second. After magnetic separation, the supernatant was discarded. Repeat the above procedure of Milli-Q water washing for three times. Afterwards, 400 µL cleaning buffer Ⅱ (1 M NaOH, 2 M NaCl) was added to the beads and vortexed for 30 min. After magnetic separation, the supernatant was discarded. Next, wash the beads with Milli-Q water till the pH of the liquid was changed back to neutral. Then discard the supernatant. 400 µL 100 mM NiSO 4 was added to the beads. Mix the beads with the buffer for 30 min at room temperature on a rotary mixer. Finally, wash the beads with Milli-Q water for another 6 times. Exchange the liquid to 20% ethanol and store the re-charged Ni-charged magbeads at 4 ℃. The regenerated beads can be re-used for more than five times without noticeable loss of performance.
6. Cost estimation of MspA preparation. The entire pore preparation process does not require any high-end instruments. The major cost of consumables per MspA preparation was roughly estimated based on a 15 mL culturing volume for one type of MspA. To be specific, these consumables include 0.76 g LB agar ($ 0.04 per use), 0.12 g LB broth ($ 0.006 per use) and 100 µL magnetic beads. The magnetic beads can be reused for 5 times so the cost per single used was estimated accordingly ($ 0.34 per use). Though the actual cost may slightly vary due to different suppliers of consumables, the overall cost should be ~0.4 $ per single preparation, highly affordable by most academic groups and attractive for large scale industrial manufacturing.
Briefly, to prepare the lipid oil solution, 25 mg 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (Avanti Polar Lipids) was dissolved in pentane in a 5 mL glass vial. It is then air dried with compressed nitrogen gas till a thin lipid film was formed on the inner wall of the glass bottle.
To prepare a 5 mg/mL lipid/oil solution, a 5 mL mixture of hexadecane and silicone oil with a 1:1 volume ratio and thoroughly vortexed to reach a homogeneous distribution. MspA nanopores, EDTA (400 µM) and Fluo-8H sodium salt (33.3 µM) were placed in the potassium chloride buffer    1. * marks the stop codon.
2. The hexa-histidine tag placed on the C terminus of each gene is designed for nickel affinity chromatography purification.