Soc medium

Chemical transformation of E. coli was performed by mixing 1–5 µl of ligation or Gibson reaction with 50 µl thawed, chemically competent stable E. coli (NEB) and incubating on ice for 5–30 min. Cells were subsequently heat-shocked at 42 °C for 30 s, followed by a 5 min incubation on ice, and, finally, 950 µl SOC-medium (NEB) was added to the cell suspension. After outgrowth for 10–30 min at 37 °C, cells were plated on agar plates containing appropriate antibiotics, followed by overnight incubation at 37 °C or 48 h incubation at room temperature.

A library of random mutants of fosmid G7 was generated using the EZ-Tn5^TM insertion kit (Lucigen Corporation) following the manufacturer's instructions. Briefly, an equimolar amount (0.007 pmol) of the EZ-Tn5^TM transposon and target DNA (G7 fosmid) were used to maximize the insertion efficiency while minimizing multiple insertion events. The 10-μl reaction was incubated at 37 °C for 2 h and stopped upon addition of 1 μl of EZ-Tn5 10× stop solution and incubation at 70 °C for 10 min. 1 μl of the reaction was added to 50 μl of thawed electrocompetent cells EC300110 (Lucigen Corporation) in a prechilled tube. Electroporation was performed with a Gene Pulser Xcell^TM electroporation system (Bio-Rad) following the manufacturer's instructions (1-mm gap, 1800 V, 25 microfarad, and 200 Ω). SOC medium (New England Biolabs) was added immediately after electroporation (950 μl), and the cells were transferred to a 15-ml tube for incubation at 37 °C with shaking for 1 h. Transformed cells were plated on LB + 12.5 μg/ml chloramphenicol and 50 μg/ml kanamycin. 192 random mutants were arrayed in two 96-well plates. Sialidase activity in both plates was investigated using the lysis-based assay with 4MU-α-Neu5Ac substrate as described above.

For electro-transformation, the GA reaction was diluted in three volumes of DNase and RNase-free dH2O (Thermo Scientific™, MA, USA) prior to starting the electroporation process. A volume of 2 microliters of the diluted GA reaction was transferred into a pre-cool cuvette (1 mm, Bio-Rad, Hercules, CA, USA) containing 50 µL of electrocompetent E. coli, BL21. The electroporation was performed using GenePulser^® (Bio-Rad, CA, USA) with the parameter set at 1700 kV, 25 µF, 200 Ω. Then, 950 µL of SOC medium (New England BioLabs, MA, USA) was immediately added into the transformant cuvette, which was further incubated at 37 °C for 16–18 h. A volume of 150 microliters of the transformants was plated onto LB agar plates containing 100 mg/mL ampicillin. The plates were incubated at 26 °C for 36–38 h. The positive colonies were selected for further culturing in LB broth. Both pKLS3_O189 and pKLS3_NP05 were isolated from the E. coli culture using GeneMark^® Plasmid Minipreps kit following the manufacturer’s protocol (GeneMark, Taipei City, Taiwan). The plasmids were submitted for nucleotide sequencing (Macrogen, Seoul, Republic of Korea).

The human Brunello CRISPR knockout pooled library was a gift from D. Root and J. Doench (Addgene, 73178)^{53 (link)}. In brief, the amplification of the plasmid DNA library was achieved by retransforming 100 µl ElectroMAX Stbl4 electrocompetent cells (Invitrogen, Thermo Fisher Scientific) with 400 ng of the plasmid library in four separate repetitions. For each repetition, the transformation was performed using 25 µl of cells and 100 ng of the library DNA with an ice-cold electroporation cuvette (0.1 cm gap size, Bio-Rad). Cells were shocked at 1.8 kV (preset settings, Ec1; Gene Pulser Xcell Electroporation Systems, Bio-Rad) and immediately mixed with 980 µl prewarmed (30 °C) SOC-medium (NEB). The electroporated cells in SOC-medium (4 ml in total) were combined in a ventilated falcon tube, and 6 ml of additional SOC-medium was added. Next, the 10 ml culture was shaken at 30 °C for 1 h, plated on 12 × 15 cm LB agar plates containing 100 µg ml⁻¹ carbenicillin and incubated overnight for 16 h at 30 °C. The bacteria were next scraped from the plate using a cell scraper after adding 10 ml ice-cold LB medium per plate. The cell suspension was centrifuged (4 × 30 ml) at 4,000 relative centrifugal force (r.c.f.) for 10 min. The plasmid DNA library was extracted from the pellet using the Plasmid Maxi Kit (QIAGEN).

E. coli host strain NEB 5-alpha F’ l^q (New England Biolabs) was exclusively used to generate, store, and amplify the E. coli/B. burgdorferi shuttle vectors listed in Table 1. The resulting strains were grown on LB agar plates or in Super Broth (35 g/L bacto-tryptone, 20 g/L yeast extract, 5 g/L NaCl, and 6 mM NaOH) liquid medium with shaking at 30°C [53 (link)]. Transformation was achieved by heat shock followed by recovery in SOC medium (New England Biolabs) for 1h at 30°C with shaking. Antibiotic selection was achieved using spectinomycin at 50 μg/mL or rifampin at 25 μg/mL in liquid culture or 50 μg/mL in plates.