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Table 2 Comparison of published electrotransformation procedures used in Acetobacterium woodii and a new procedure for Acetobacterium strains

From: Developing a genetic engineering method for Acetobacterium wieringae to expand one-carbon valorization pathways

 

Strätz, 1994

Leang, 2013

Straub, 2014

Hoffmeister, 2016

Weitz, 2021

Baker, 2022

New protocol

Protocol

1

2

3

4

5

6

7

References

[29]

[22, 31, 33,34,35,36,37,38]

[39]

[19, 20, 40, 41]

[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)

  1. aNI not indicated, b1SMP 270 mM sucrose, 1 mM MgCl2, 7 mM NaH2PO4, b2SMP 270 mM sucrose, 1 mM MgCl2, 1 mM NaH2PO4, cDMSO dimethyl sulfoxide, dDMF dimethylformamide, eDcm cytosine methyltransferase, fDam adenine methyltransferase, T type strain