Fig. 1. The workflow of rapid and multiplex MspA preparation. Phase I: the plasmid DNAs encoding for MspA mutants (Table S2†) were heat-shock transformed into E. coli BL21(DE3) competent cells and cultured on an agar plate. Human operations: agar plate preparation and heat-shock transformation (10 min). Phase II: single colonies collected from phase I are grown in liquid LB medium, IPTG induced and cultured overnight. The cells were harvested by centrifugation. Human operations: single colony collection (4 min), IPTG induction (3 min) and bacteria pellet collection (3 min). Phase III: the collected pellets are resuspended in the lysis buffer and heated at 90 °C for 10 min in a heating block to thoroughly lyse the cells. A significant portion of non-specific proteins was fully denatured during heating. Centrifugation was performed to collect the supernatant which contains the desired proteins. Human operations include lysis buffer exchange and pellet resuspension (5 min) along with supernatant collection (3 min). Phase IV: the supernatant of the bacterial lysate is purified using Ni-charged magbeads (the orange dashed box). Briefly, the beads are incubated with the supernatant and sequentially washed with washing buffer A, eluting buffer B1 and eluting buffer B2. Human operations include adding the supernatant of bacterial lysate to tubes (3 min), adding washing buffer A to tubes (3 min), adding eluting buffer B1 to tubes (3 min) and adding eluting buffer B2 to tubes (3 min). Phase V: gel electrophoresis diagram for the preparation of 5 kinds of MspA nanopores simultaneously. Gel electrophoresis was carried out with a 4–20% Mini-PROTEAN TGX Gel (Cat. #4561083, Bio-Rad) and a 200 V bias was applied for 27 min. Lane M: precision plus protein standards (Bio-Rad). All procedures such as plasmid transformation, liquid culturing, lysate heating and magnetic separation can be carried out in parallel. The time of human operations was only 40 min in total. The preparation doesn't require any high-end instruments and the consumable costs are negligible.