Strätz, 1994 | Leang, 2013 | Straub, 2014 | Hoffmeister, 2016 | Weitz, 2021 | Baker, 2022 | New protocol | |
---|---|---|---|---|---|---|---|
Protocol | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
References | [29] | [39] | [21] | [32] | This work | ||
Transformed organisms | A. woodiiT | A. woodiiT C. ljungdahliiT | A. woodiiT | A. woodiiT | A. woodiiT | A. woodiiT A. woodii lab-adapted strain | A. woodiiT A. wieringaeT A. wieringae JM |
Preparation of cells and plasmid | |||||||
Cell weakening agent on growth media | NIa | 40 mM DL-threonine | 20 mM DL-threonine | 40 mM DL-threonine | 40 mM DL-threonine | NIa | 40 mM D-threonine |
OD600 (Harvesting) | 0.5 | 0.2–0.3 | 0.4–0.6 | 0.3–0.5 | 0.3–0.7 | 0.2–0.4 | 0.3–0.6 |
Centrifugation steps | Outside of chamber | Outside of chamber | Inside of chamber | Outside of chamber | Inside of chamber | Inside of chamber | Inside of chamber |
Anaerobic chamber atmosphere | NI | 83% N2, 7% H2, 10% CO2 | NI | 95% N2, 5% H2 | 95% N2, 5% H2 | 80% N2, 10% H2, 10% CO2 | 95% N2, 5% H2 |
Wash-buffer | 270 mM sucrose | SMPb1 | SMPb1 | SMPb1 | SMPb1 | SMPb2 | SMPb1 |
pH wash-buffer | NI | 6.0 | 6.0 | 6.0 | 6.0 | 5.8 | 6.0 |
Washing steps | 2 | 2 | 2 | 2 | 2 | 2 | 1–2 |
Resuspension buffer | 270 mM sucrose | SMPb1 10% DMSOc | SMPb1 | SMPb1 10% DMSO | SMPb1 10% DMFd | SMPb2 15% DMSO | SMPb1 10% DMF |
pH of res. buffer | NI | 6.0 | 6.0 | 6.0 | 6.0 | 5.8 | 6.0 |
Cell density after resuspension [OD600] | 25 | 200–300 | 30–50 | 300–500 | 40–100 | 30 – 60 | 30 |
Freeze − 80 °C | No | Yes | No | Yes | Yes | Yes | Yes |
Plasmid methylation | Dcm+e Dam+f | Dcm− Dam+ | Dcm+ Dam+, pACYC184–methyltransferase gene of C. ljungdahlii | Dcm+ Dam+ | Dcm+ Dam+ | Dcm+ Dam+ | Dcm+ Dam+ |
E. coli strain for plasmid-prep | XL1-Blue (Stratagene) | B strain (NEB express) | XL1-Blue MRF' (Stratagene) pMCljS (CLJU_c03310) | XL1-Blue MRF' (Stratagene) | XL1-Blue MRF' (Stratagene) | HB101 (Promega) | TOP10 (Invitrogen) |
Electroporation process | |||||||
Preincubation with plasmid on ice | 5 min | No | No | No | 1 min | Yes | 1–2 min |
Volume of cells [µL] | 40 | 25 | 600 | 25 | 25 | 100 | 200 |
Plasmid amount [µg] | 0.40 | 1–5 | 1 | 1–5 | 3–5 | 1 | 1–3 |
Electroporation cuvettes gap [cm] | 0.2 | 0.1 | 0.4 | 0.1 | 0.1 | 0.2 | 0.2 |
Cell-plasmid ratio (OD600 × mL/µg) | 2.5 | 1–7.5 | 18–30 | 1.5–12.5 | 0.2–0.8 | 3–6 | 2–6 |
Electric pulse | 10 kV 400Ω 25μF | 0.625 kV 600Ω 25μF | 2.5 kV 600Ω 25μF | 0.625 kV 600Ω 25μF | 0.625 kV 600Ω 25μF | 1.0 kV 200Ω 50μF | 2.0 kV 20Ω 25μF |
Electric field strength [kV/cm] | 50 | 6.25 | 6.25 | 6.25 | 6.25 | 5 | 10 |
Electroporator | NI | Gene Pulser Xcell (Bio-Rad) | NI | Gene Pulser Xcell (Bio-Rad) | Gene Pulser Xcell (Bio-Rad) | Gene Pulser Xcell (Bio-Rad) | E. coli Pulser (Bio-Rad) |
Incubation after electroporation on ice | 5 min | – | – | – | – | – | – |
Cultivation after electroporation | 0.960 mL GPM broth | 10 mL PETC 1754 10 g/L fructose | 5 mL modified DSMZ 135 10 g/L fructose | 5 mL modified DSMZ 135 3 g/L fructose | 5 mL modified PETC 1754 7 g/L fructose | 10 mL modified DSMZ 135 3.6 g/L fructose | 3 mL basal media 10 g/L fructose |
Recovery cultivation time | 7 h | 9–12 h | 3 days | > 12 h | Till doubling then antibiotic was added until growth was observed | 5 h | 20 h |
Plating | Liquid culture on solid agar (Petri dish) | Liquid culture mixed with molten agar (Petri dish) | Liquid culture | Liquid culture | Liquid culture on solid agar (Petri dish) | Liquid culture on solid agar (Petri dish) | Liquid culture mixed with molten agar (serum bottle) |
Antibiotic [µg/mL] | Tetracycline 10 | Thiamphenicol 5 | Thiamphenicol 10 | Thiamphenicol 10 | Thiamphenicol 10 | Thiamphenicol 12.5 | Thiamphenicol 15 |
Incubation | NI | Inside of chamber | NI | Inside of chamber | Inside of chamber | Inside of chamber | Outside of chamber |
Plasmids (Gram+ replicon) | pMS3, pMS4 (pAMβ1) | pMTL_8312 (pCB102), pMTL84151 (pCD6) | pJIR750 (pIP404) | pJIR750 (pIP404), pMTL82151 (pBP1), pMTL83151 (pCB102), pMTL84151 (pCD6) | pMTL83151 (pCB102) | pMTL83141 (pCB102) | pMTL82151 (pBP1), pMTL83151 (pCB102), pMTL84151 (pCD6), pMTL85151 (pIM13) |