Transformation Methods

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Due to Clostridium's natural resistance to foreign DNA uptake and its anaerobic physiology, transformation protocols require careful optimization of many factors such as growth phase, electroporation settings, and recovery conditions. This page summarizes established electrotransformation and conjugation procedures across Clostridium strains, highlighting differences in the procedures across lab groups.

In all electroporation procedures, the grow-up culture used for transformation is started with OD 0.04-0.05 from an overnight culture of OD 0.4-0.8. Cells are made electrocompetent using electroporation buffer (EPB), which consists of sucrose and salts, through harvest and wash steps. Electrocompetent cells are mixed with plasmid DNA and electroporated, and immediately recovered for varying times until plating and incubating. Additionally, all work is performed anaerobically, with the exception of centrifugation which is typically conducted outside of an anaerobic chamber.

Summary of Electrotransformation Procedures
Organism Overnight culture OD Growth Phase (OD600) Growth Media Harvest/Wash Procedure plasmid DNA EPB kV Time constant Recovery Transformation Efficiency Reference
C. acetobutylicum 0.6 1.2 RCM Cells harvested by centrifugation at room temp, washed and suspended in 1.8 ml ice cold EPB. 0.6 ml samples of cells chilled in ice for 5 minutes in a sterile cuvette. Plasmid DNA added, followed by 2 minute incubation in ice. 10-500 ng[1]; 5-10 ug[2] 270 mM sucrose, 5 mM NaH2PO4, pH 7.4 2 25 - 13 ms Cells immediatley added to 10 mL pre-warmed RCM and incubated for 4 hours before plating ~5.0x10^4 transformants/ug DNA[1] [1],[2]
0.4-0.8 0.6-0.8 2xYTG Cells centrifuged for 10 min at 6000xg and 4C, washed and suspended with EPB. 0.5-2 ug 272 mM sucrose, 5 mM NaH2PO4 2 25 inf. Cells immediately added to 10 mL 2xYTG pre-warmed to 37 C and incubated for 4 hours before plating - [3]
- 0.4-0.8 2xYTG Cells harvested at 6500xg for 5 min. Washed in 10 mL ice-cold EPB. Suspended in 2 mL ice-cold EPB. 0.5 mL cells transferred to a 4 mm electroporation cuvette, followed by addition plasmid DNA. 3 min incubation on ice. 1-5 ug 270 mM sucrose, 5 mM NaH2PO4, pH 7.4 2 25 inf. - Cells immediately transferred to 4 mL of 2xYTG and incubated 4-6 hours. Cells harvested by centrifugation 6500xg for 5 min and plated - [4]
- 0.5 CGM Cells centrifuged for 10 minutes at 7000xg and 4C. Cells washed three times with EPB and resuspended in 2 mL of the same buffer. 0.5-2 ug 270 mM sucrose, 686 mM NaH2PO4, pH 7.4 2.5 25 inf. - 1 mL 2xYTG added to mixture and incubated for 4 hours. - [5]
OD = 1.2 Cells harvested and washed with EPB. Pellets suspended in 1/20 of a volume of EPB. 0.8 mL of competent cells mixed with DNA 10 ug 10% PEG 8000 in distilled water 2.5 25 inf 30-40 ms Postelectroporation incubation -10 minutes on ice. Cells diluted into 9 volumes TGY and incubated 7 hours then plated. [6]
C. pasteurianum - 0.6-0.8 Reduced 2xYTG (sucrose and glycine add at OD = 0.3-0.4) Cells centrifuged for 20 minutes at 8500xg and 4C, washed and suspended with SMP buffer. 0.5 ug suspended in 20 uL of 2mM Tris-HCL SMP buffer (270 mM sucrose, 1 mM MgCl2, 5 mM NaH2PO4), pH 6.5 1.8 25 inf. 12-14 ms Cuvette flooded with 1 mL 2xYTG containing 0.2M sucrose, entire suspension transferred to 9 mL of same medium, incubated 4-6 hours 7.5x10^4 transformants/ug DNA [7]
0.4-0.8 0.6-0.8 2xYTG (sucrose and glycine added at OD = 0.3-0.4) Cells centrifuged for 10 minutes at 4000xg and 4C, washed and suspended with SMP buffer. 0.5-2 ug and 30 uL of 100% ethanol SMP buffer (270 mM sucrose, 1 mM MgCl2, 5 mM NaH2PO4) 1.8 25 inf. Cells immediately added to 10 mL pre-warmed recovery media (2xYPG with 0.2 M sucrose) and incubated for 16 h before plating - [3]
0.8-1.2 0.6-0.8 2xYTG Cells centrifuged for 10 minutes at 8,500xg and 4C, washed in 5 mL ice col SMP buffer, and finally resuspended in 600 uL ice cold SMP buffer. 580 uL transferred to pre-chilled cuvette containing 30 uL of 96% ethanol and plasmid DNA. Incubated on ice 5 minute. 0.5-5 ug SMP buffer (270 mM sucrose, 1 mM MgCl2, 5 mM NaH2PO4), pH 6.5 1.8 25 inf. 10-18 ms Cells transferred to pre-warmed 2xYTG and recovered 6 to 16 hours. -1.8x10^2 transformants/ug DNA

-2.6x10^5 transformants/ug DNA (mutant strain)

[8]
C. ljungdahlii Frozen cultures inoculated then transferred twice in PETC. 15-16 hours before preparing electrocompetent cells, mid to late log culture transferred into two serum bottles with PETC and DL-threonine then grown overnight. 0.2-0.3 PETC (or YTF) Cells centrifuged for 10 minutes at 10,000xg and 4C, washed twice with 200 mL SMP and resuspended in SMP at a final concentration of 10^10 to 10^11 cells/ml. Antifreezing buffer (60% DMSO-40% SMP) can be added to competent cells; competence of frozen cells stable for about 1 month. For electroporation, 25uL cells thawed for on ice 1 minute, then mixed with DNA and transferred to pre-chilled cuvette. 1-5 ug SMP buffer 0.625 25 600 - Cuvette flooded with 0.5 mL fresh PETC medium, then transferred to 10 mL PETC. Electroporated cells recovered until their cell densities were higher than immediately after the electroporation (9-12 hours). Cultures mixed with 20 mL RCM molten agar and poured. 1.1-1.7 transformants/ug plasmid DNA [9][10]
Early exponential growth 8-12 hour incubation, OD 0.3-0.7 PETC medium with DL-threonine Cells harvested, washed twice, and suspended in ice-cold EPB. Cell suspension chilled on ice for 5 minutes in cuvette. 0.1-1.5 ug SMP buffer 2.5 25 600 - Cells transferred to prewarmed medium and incubated until growth was visible. - [11]
C. beijerinckii *200 uL Cb spores heat-shocked at 75C for 10 min, cooled on ice for 2 min, then inoculated into 10 mL TGY. Culture grown at 35C until reaching an OD 0.9-1.1, then plated onto TGY agar and incubated until single colonies appeared. *A single colony was inoculated into 10 mL TGY and incubated 10 hours. Cells then transferred into 90 mL fresh TGY media and incubated until OD 0.6-0.8. TGY Cells centrifuged for 6 minutes at 4,000xg and 4C. Washed once with 50 mL EPB, then resuspended in 2 mL EPB before incubation on ice for 5 minutes. DNA gently mixed with 400 uL electrocompetent cells in a pre-chilled cuvette. 10 ug 5mM KH2PO4, 270 mM sucrose, 1mM MgCl2, 10% PEG-8000 2.5 25 inf. 2.9-4.2 ms Cells diluted in 4 mL TGY and incubated 6 hours. Recovered cells pelleted at 3,000xg for 5 minutes, mixed with semi-solid TGY agar, and incubated 48-72 hours. - [12]
starter culture grown for 12 hours late exponential phase (OD=1.2) TGY 10 mL of cells harvested, washed resuspended with EPB. 400uL competent cells mixed with plasmid DNA and electroporated. 10 ug 10% (w/v) polyethylene glycol 8000 in distilled water 2.5 25 inf. - Cells quickly transferred to 5mL TGY broth and recovered 8 hours before plating. - [13]
C. botulinum
C. perfringens OD = 1.2 Cells harvested and washed with EPB. Pellets suspended in 1/20 of a volume of EPB. 0.8 mL of competent cells mixed with DNA 1 ug 10% PEG 8000 in distilled water 2.5 25 inf 30-40 ms Postelectroporation incubation -10 minutes on ice. Cells diluted into 9 volumes TGY and incubated 1 hour aerobically then plated. [6]
C. cellulolyticum Grown 17-24 hours to late exponential phase (OD 0.5-1.0) GS Cells harvested for 10 minutes at 6000xg and 4C, washed twice and resuspended with EPB. Plasmid DNA added to pre-chilled electroporation cuvettes followed by cell suspension, then incubated on ice 10 minutes. 1-2 ug 270 mM sucrose, 1 mM MgCl2, 5 mM sodium phosphate buffer, pH 7.4 1.5 25 100 1.9-2.0 ms GS medium added to cuvette immediately after electroporation and incubated overnight before plating. 10^2 transformants/ug [14]
Cells grown until mid-log phase synthetic medium Washed and resuspended with ice-cold EPB. Cell suspension transferred to cuvette with DNA 0.5-1 ug 270 mM sucrose, 5 mM K2HPO4, pH 6.5 2.0 25 1000 - Cell suspension incubated in prewarmed synthetic medium for 6 hours before plating. - [15]
Cells grown until OD 0.4-0.6. Ampicillin, threonine, or glycine added and cultures grown for 2-3 more hours GS-2 with cellubiose Washed and resuspended with ice-cold EPB. Cell suspension transferred to cuvette with DNA 2.0 275 mM sucrose, 5 mM K2HPO4, pH 6.5 8.0-9.5 ms Cell suspension incubated in prewarmed synthetic medium for 6 hours before plating. 3x10^3 transformants/ug (with glycine) [16]
C. tyrobutyricum Overnight growth, late log phase Cells grown for 4 hours, OD around 0.8 CGM with DL-threonine Cells harvested, washed twice, and suspended in ice-cold EPB. Cell suspension chilled on ice for 5 minutes in cuvette. 10-15 ug SMP buffer (270 mM sucrose, 1 mM MgCl2, 7 mM NaH2PO4), pH 7.4 2.5 25 600 - Cells transferred to prewarmed media and incubated 3 hours prior to plating. - [17]

In conjugation procedures, a donor strain, typically E. coli, is transformed with the desired plasmid. The strain is grown overnight before harvesting and adding to an overnight culture of the Clostridium strain. The mating mix is spotted onto an agar plate and incubated anaerobically.

Summary of Conjugation Procedures
Organism Donor Strain Donor Preparation Recipient Preparation Mating Mating Time Harvesting Efficiency Notes Reference
Clostridioides difficile strain 630 HB101(pK24) or CA434 Late to early stationary phase starter culture OR overnight culture Late log starter culture OR overnight culture 8-16 hr
C. difficile CA434 Donor transformed with plasmid to be mobilized, then grown overnight. A 1 ml aliquot centrifuged at 5K for 1 min, then resuspended in PBS. Centrifugation repeated and harvested cells resuspended in recipient strain. Overnight culture, 100-200mL used to resuspended harvested donor cells. Mating mix spotted onto well-dried BHI agar plate. 7 hours E coli counter selected for and antibiotic added to select for plasmid uptake. Plates incubated 24-72 hours. 1.2x10^-6 to 5.5x10^-5 transconjugants/donor cell -same procedure successful for C. sporogenes[18] [19]
CA434 Donor transformed with plasmid to be mobilized, then grown overnight. A 1 ml aliquot centrifuged at 4000g for 2 minutes then resuspended in PBS. Centrifugation repeated and harvested cells resuspended in recipient strain. Heat treated to 44-52C for 15 minutes prior to conjugation with donor. Mating mix spotted onto well-dried BHI agar plate. 8-24 hours Harvested using TY broth, serially diluted and spread on plates to select for transconjugants 10^-7 to 10^-4 transconjugant CFU/total C.diff CFU (changed with heat treatment temp) -used clinical strain known for low efficiency

-heat treatment increased efficiency but decreased cell viability

[20]
CA434 Overnight culture with plasmid grown overnight and washed in PBS. Overnight culture Pellet from 1 mL of donor resuspended in 150 uL of recipient. Spotted onto BHIS agar. 24 hours Harvested into 500 uL PBS and plated to select for transconjugants. -4.48 x 10^-7 transconjugant colonies/CFU donor[21]

-3.1x10^-9 to 2.7x10^-8 transconjugants/CFU donor[22]

- [21]
C. autoethanogenum CA434 and sExpress Donor with shuttle vector inoculated and incubated 14-16 hours. Four hours prior to conjugation, subcultured and incubated until OD 0.2-0.4. 1 ml centrifuged at 3000g for three minutes, then resuspended in PBS. Starter culture initiated from glycerol stock over 72 hours until late exponential phase. Subcultured and incubated for 16-20 hours, until OD 0.1-0.2. 200 ul used for resuspending the donor. Combined cultures transferred to YTF agar plate. 20 hours PBS used to flood surface of agar plate, spreader used to dislodge and re-suspend the bacterial growth. Bacterial slurry transferred to selective YTF agar plate and incubated 72 hours. 10^-7 to 10^-5 transconjugants/donor cell -developed new donor strain [23]
HB101 (R702) Donor with plasmid grown to OD 0.5 and washed with PBS. Grown to OD 0.5. 2 mL donor culture mixed with 0.2 mL recipient. Mixture spotted onto agar plates. 24 hours Counterselection of e. coli and selection for plasmid construction. - - [24]
C. acetobutylicum Donor: HB101

Recipient: C. ac NCBI 8052

Donor strain grown until OD > 4.0 in BHIB. Grown overnight in TYG. Exponentially growing cultures, OD 0.6-1.2 used for matings. Donor and recipient mixed in a 1000:1 ratio. Bacteria from mating mixture were deposited by filtration on a cellulose nitrate membrane. Incubated overnight on plates with RCM. Harvested from the filter by vortex mixing in holding buffer. Recipient and donor counter-selected by aerobic incubation and antibiotic addition. 2.0x10^-6 - 6.0x10^-7 transconjugants/donor cell - did not work for C. ac strains P262, DSM 1731 and ATCC 824 [25]
C. sporogenes Recipient: ATCC 13732 and M-55 Overnight culture washed in PBS. Overnight culture grown in TYG. 1 mL donor and 200 uL recipient mixed and spotted onto agar plate. 7 hours Mixture resuspended in PBS before plating onto selective agar. 1.5x10^-7 to 8.86x10^-5 transconjugants/recipient - [26]
C. novyi-NT - Overnight culture washed in PBS. Overnight culture grown in TYG. 1 mL donor and 200 uL recipient mixed and spotted onto agar plate. 7 hours Mixture resuspended in PBS before plating onto selective agar. 1.5x10^-7 to 8.86x10^-5 transconjugants/recipient - [26]
C. beijerinckii CA434 Overnight culture Donor strain transformed with plasmid to be mobilized and grown overnight. 1 mL aliquot centrifuged and resuspended in PBS. Centrifugation repeated and cells resuspended in 100-200 mL of an overnight culture of C. beijerinckii in TYG medium. Mating mix spotted onto plate. Bacterial growth harvested and procedure repeated to ensure good recovery of transconjugants. 7 hours [19]
C. tyrobutyricum CA434 Donor strain transformed with plasmid, cultured overnight to OD 1.5-2.0. Cells centrifuged and washed, then resuspended into recipient cells. Overnight growth, OD 2.0-3.0 in RCM. 24 hours; 12 hours[27] [28]

To compare methods for Clostridium transformation to other bacteria strains, the following table summarizes procedures for Bacillus subtilis, another firmicute, and Eubacterium limosum, another anaerobe.

Organism Day-Before Cell Preparation Day-Of Cell Preparation Growth Media Harvest/Wash Procedure plasmid DNA EPB kV Time constant Recovery Transformation Efficiency Reference
Bacillus subtilus Prewarmed medium is inoculated by cell patches from an overnight plate to an OD of 0.5. Prewarmed medium is inoculated with stationary phase culture and incubated for 90 minutes. SpC medium Cells centrifuged at 8,000xg for 5 minutes and at room temperature. Resuspend in saved supernatant. Add glucose to freeze. For transformation, thaw competent cells and add one volume SpII + EGTA^f. Add competent cells to the DNA solution and incubate in a roller drum. DNA solution < 1ml - - - - - - [29]
Streak recipient strain and incubate 18 hours. Inoculate so that slight turbidity is visible. Incubate with aeration for 2 hours. MW Distribute competent culture into tubes, add 0.1 mL or less DNA and incubate for 1 hour. Add SC and centrifuge for 10 minutes at 8000xg and room temperature. Resuspend in SC and plate. [29]
Prewarmed medium is inoculated by cell patches from an overnight plate to an OD of 0.5-0.7. Prewarmed medium is inoculated with stationary phase culture (75 minutes after logarithmic growth ends) and incubated for 110 minutes. SpII Distribute competent culture into tubes, add 2ug quantity of DNA and incubate for 80 minutes. Plate on agar with antibiotic. [30]
Eubacterium limosum Frozen culture inoculated and grown up for 18 hours until early exponential phase (0.3-0.5). DSMZ Chilled on ice 20 minutes then centrifuged at 15,000xg for 10 min at 4C, washed and resuspended in sucrose buffer then glycerol/sucrose buffer. Competent cells aliquoted and stored at -80C. For transformation, thaw competent cells and add DNA. 0.1-2 ug 270 mM sucrose 2500 25 600 950 uL DSMZ added following pulse, cuvettes placed in incubator for 6 hours before plating. 2.5x10^4 CFU/ug [31]
100 mL culture grown until OD = 0.5 DSM135 Centrifuged at 15,500xg for 15 minutes at 4C. Pellet washed three times with EPB. After third wash, pellet transferred to 1.5 mL tube and centrifuged at 17,000xg for 15 minutes at 4C, then resuspended in EPB and prepared plasmid added. 0.05-3 ug 270 mM sucrose 20 kV/cm 25 600 1 mL RCM added to cuvette. Cells incubated for 16 hours then diluted and plated. 2.5x10^5 CFU/ug [32]
Cells grown until early exponential growth phase (OD 0.3-0.5) DSM135 with methanol and DL-Threonine Cells harvested at 7690xg for 10 minutes at 4C, then washed two times with EPB. Pellet resuspended in EPB and DMSO, stored in -80C until use. 25 uL of competent added with DNA in cuvette. 3-5 ug SMP Buffer (270 mM sucrose, 1 mM MgCl2, 7 mM NaH2PO4, pH 6) 0.625 25 600 - Recovered in DSM 135 with methanol. After one to two doublings, antibiotic added to select for recombinant strains. After further increase of OD, cells transferred two more times into fresh medium with antibiotic. - [33]
Overnight preculture Inoculated with overnight culture, grown to OD 0.2-0.5. Pacaud medium Harvested and washed twice at 7000xg and 4C, resuspended in ice-cold EPB. Following wash steps, pellets resuspended in EPB with DMSO and stored at -80C for later use. Plasmid DNA mixed with thawed competent cells and incubated on ice 5 minutes. 1 ug 270 mM sucrose, 5 mM Na2HPO4, pH 6.8) 1.7 - - 6 ms Cells recovered in medium 2-4 hours before plating. 2.09x10^6 CFU/ug (shuttle vector)

1x10^2 CFU/ug (suicide vectors)

[34]


  1. ↑ 1.0 1.1 1.2 Mermelstein, Lee D., et al. “Expression of Cloned Homologous Fermentative Genes in Clostridium Acetobutylicum ATCC 824.” Nature Biotechnology, vol. 10, no. 2, Feb. 1992, pp. 190–195, pubmed.ncbi.nlm.nih.gov/1368230/, https://doi.org/10.1038/nbt0292-190. Accessed 23 Sept. 2021.
  2. ↑ 2.0 2.1 Al-Hinai, Mohab A., et al. “Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration.” Applied and Environmental Microbiology, vol. 78, no. 22, 15 Nov. 2012, pp. 8112–8121, pubmed.ncbi.nlm.nih.gov/22983967/, https://doi.org/10.1128/aem.02214-12. Accessed 3 Dec. 2022.
  3. ↑ 3.0 3.1 Joseph, Rochelle Carla, and Nicholas R. Sandoval. “Single and Multiplexed Gene Repression in Solventogenic Clostridium via Cas12a-Based CRISPR Interference.” Synthetic and Systems Biotechnology, vol. 8, no. 1, 24 Dec. 2022, pp. 148–156, www.sciencedirect.com/science/article/pii/S2405805X22001247, https://doi.org/10.1016/j.synbio.2022.12.005.
  4. ↑ Seo, Hyeongmin, et al. “Butyrate as a Growth Factor of Clostridium Acetobutylicum.” Metabolic Engineering, vol. 86, no. 1096-7176, Oct. 2024, www.sciencedirect.com/science/article/pii/S1096717624001332, https://doi.org/10.1016/j.ymben.2024.10.005.
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  30. ↑ Westbrook, Adam W., et al. “Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus Subtilis.” Applied and Environmental Microbiology, vol. 82, no. 16, 3 June 2016, pp. 4876–4895, journals.asm.org/doi/full/10.1128/aem.01159-16, https://doi.org/10.1128/aem.01159-16.
  31. ↑ Sanford, Patrick A, and Benjamin M Woolston. “Expanding the Genetic Engineering Toolbox for the Metabolically Flexible Acetogen Eubacterium Limosum.” Journal of Industrial Microbiology and Biotechnology, vol. 49, no. 5, 26 July 2022, academic.oup.com/jimb/article/49/5/kuac019/6650221, https://doi.org/10.1093/jimb/kuac019. Accessed 13 Mar. 2023.
  32. ↑ Song, Yoseb, et al. “Development of Highly Characterized Genetic Bioparts for Efficient Gene Expression in CO2-Fixing Eubacterium Limosum.” Metabolic Engineering, vol. 72, 29 Mar. 2022, pp. 215–226, www.sciencedirect.com/science/article/pii/S1096717622000520?via%3Dihub#appsec1, https://doi.org/10.1016/j.ymben.2022.03.016.
  33. ↑ Flaiz, Maximilian, et al. “Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-Tagged Proteins Using Metabolically Engineered Strains of Eubacterium Limosum.” Biotechnology for Biofuels, vol. 14, no. 1, 10 May 2021, https://doi.org/10.1186/s13068-021-01966-2. Accessed 4 Oct. 2025.
  34. ↑ Millard, James, et al. “Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen Eubacterium Limosum.” Microorganisms, vol. 11, no. 5, 10 May 2023, pp. 1256–1256, www.mdpi.com/2076-2607/11/5/1256, https://doi.org/10.3390/microorganisms11051256. Accessed 5 Nov. 2025.
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