Itsa Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum

1. Development of an
electrotransformation
protocol for genetic manipulation
of Clostridium pasteurianum
Michael E Pyne1, Murray Moo-Young1, Duane A Chung1,2* and C
Perry Chou1*
Published: 9 April 2013
www.ncbi.nlm.nih.gov/pubmed/23570573

2. Abstract
• Industrial biofuels proliferation and co-integration with fossil
fuels. Reducing the production cost,increasing Revenues.
• Anaerobe Clostridium pasteurianum, the only microorganism
known to convert glycerol alone directly into butanol.
• The development of an electrotransformation protocol
permitting high-level DNA transfer to C. pasteurianum ATCC
6013.
• Key factors affecting-CpaAI restriction-modification system,
cell-wall-weakening using glycine,ethanol-mediated
membrane solubilization, field strength of the electric pulse,
and sucrose osmoprotection.

3. Background
• Restrictive feedstock
cost,accounting for up to 80% of
total biobutanol production-
limiting industrialization.
• Glycerol,generated as a waste
represents approx 10% (w/w) of
the unpurified biodiesel product.
• C.pasteurianum only species that
combines the capacity for
glycerol-butanol-producing
pathway.
• Type-II restriction endonucleases
have also been identified
,including CpaAI .

4. Protection of plasmid DNA from CpaAI
restriction
• Type-II restriction
endonuclease, designated
CpaAI with 5’-CGCG-3’
recognition.
• To electrotransform C.
pasteurianum, series of E.
coli-Clostridium shuttle
vectors.
• Expression of the M.FnuDII
methyltransferase (with 5’-
mCGCG-3’ methylation site
of both DNA strands) from
plasmid pFnuDIIMKn.

5. High-level electrotransformation of C.
pasteurianum
• Cell-wall-weakening-glycine, DL-threonine,lysozyme, and
penicillin G.
• Individually,were screened for the effect of glycine and DL-
threonine by supplying the additives in the presence of 0.25 M
sucrose.
• Lysozyme and penicillin G were screened by addition at the
wash stage.
• Only glycine and DL-threonine improved the
electrotransformation efficiency.
• Glycine regimen with respect to concentration and duration of
exposure.

6. High-level electrotransformation of C.
pasteurianum
• Osmoprotection-0.27 M sucrose
was adopted as the optimum
sucrose concentration.
• Cell membrane solubilization-
Ethanol added at 5 and 10%
provided a 1.6- and 1.3-fold
respective increase in
electrotransformation efficiency.
• Electric pulse parameters-low
voltages in the range of 1.8-2.0
kV,Pulse duration changes were
not predictive of the effects on
electrotransformation efficiency.

7. High-level electrotransformation of C.
pasteurianum
• DNA quantity and outgrowth
duration-number of
transformants increase
linearly between 0.5 and 5.0
μg of pMTL85141, the
greatest efficiency occurred
using 0.5 μg of plasmid DNA.
• The greatest
electrotransformation
efficiency was attained at 16
hours.
• 7.5 × 104 transformants μg-1
DNA, an increase of more
than three orders of
magnitude.

8. Preparation of protoplasts and assay of
CpaAI activity
C. pasteurianum 100 ml culture (OD600 of 0.4-0.6)
+
25 ml of protoplast buffer (25 mM potassium phosphate,pH 7.0+6 mM
MgSO4+15% lactose+200 μg/ml lysozyme)
Incubation for 45 minutes anaerobically at 37°C
25 ml of protoplasts (centrifugation-8,500×g for 20 minutes lysed in 20 ml of TEMK
buffer (4 mM Tris–HCl,pH 8.0+0 mM EDTA+6.6 mM 2-mercaptoethanol+25 mM
KCl),
Incubation at 37°C for 1 hour
centrifugation-20,000×g for 15 minutes supernatants containing protoplast
extracts stored at −80°C.
1.0 μg plasmid DNA+25% protoplast lysate+20 μl of 1× CpaAI reaction buffer (6 mM
Tris–HCl,pH 7.4+6 mM MgCl2+6 mM 2-mercaptoethanol).
At 37°C for 2–4 hours

9. DNA Isolation and manipulation
• Plasmid DNA was extracted and purified from C.pasteurianum
•
• 3–9 ml-late-exponential phase cells were collected by centrifugation and washed
twice in KET buffer(0.5 M KCl+0.1 M EDTA+0.05 M Tris–HCl, pH 8.0)+200 μl SET
buffer (25% sucrose, 0.05 M EDTA,and 0.05 M Tris–HCl, pH 8.0)+5 mg/ml lysozyme
• Incubated anaerobically at 37°C for 20mins
• RNase A in 100 μg/ml and cell lysis and plasmid purification+400 μl of alk.SDS sol
II.
• Colony PCR of C.pasteurianum by suspending single colonies in 50 μl colony lysis
buffer (20 mM Tris–HCl,pH 8.0+2 mM EDTA +1% Triton X-100).
• Microwave Heating for 2 minutes at maximum power setting+1μl of the cell
suspension+9μl PCR containing Std Taq DNA Polymerase. An initial denaturation of
5 minutes at 95°C was employed to further cell lysis.

10. Vector construction
• Plasmid pFnuDIIMKn was derived from pFnuDIIM to allow
methylation of E. coli-C. pasteurianum shuttle vectors.
• Firstly FRT-kan-FRT PCR cassette was amplified from plasmid
pKD4 and inserted into the MCS of BlpI/XhoI-digested pET-
20b(+) to generate pETKnFRT.
• Then FRT-kan-FRT cassette was digested out of pETKnFRT
using ScaI and EcoRI and subcloned into the corresponding
restriction sites within the catP gene of pFnuDIIM to yield
pFnuDIIMKn.

11. Preparation of electrocompetent cells and
electrotransformation
• Seed culture preparation-inoculation of 20 ml of reduced
2×YTG+0.2 ml of a thawed glycerol stock-20-2-dilution..
• overnight growth at 37°C
• 1 ml culture transferred to 125 ml Erlenmeyer flask(20 ml of
reduced 2×YTG). Cells grown to exponential phase Filter sterilized
solutions of sucrose and glycine 0.4 M and 1.25%.
• Growth until culture attained an OD600 of 0.6-0.8 (approx2–3 h)
and 20 ml culture transferred to 50 ml pre-chilled, screw-cap
centrifuge tube.
At this point, all manipulations at 4°C
• Cells removed from anaerobic chamber and collected by
centrifugation at 8,500×g-20 minutes.Pellet returned to anaerobic
chamber and washed in 5 ml of filter sterilized SMP buffer (270 mM
sucrose+1 mM MgCl2+5 mM sodium phosphate-pH 6.5)
centrifugation cell pellet resuspended in 0.6 ml SMP buffer.

12. Preparation of electrocompetent cells and
electrotransformation
• For transfer of plasmids to C.pas, E. coli-C.pas shuttle vectors co-
transformed with pFnuDIIMKn into E. coli ER1821 to methylate external
cytosine residue.
• Plasmid mixtures isolated and 0.5 μg 20 μl of 2 mMTris–HCl,pH 8.0
580 μl of C. pas competent cells.
• cell-DNA mixture transferred to pre-chilled electroporation cuvette with
0.4 cm gap+30 μl of cold 96% ethanol.
• Incubation on ice for 5 minutes
• Decay pulse using Gene Pulser 1.8 kV, 25 μF, at time constant of 12–14
ms.
• Following pulse delivery,cuvette flooded with 1 ml 2×YTG medium+0.2 M
sucrose+9 ml of the same medium.Recovery cultures incubated for 4–6
hours prior to plating 50–250 μl aliquots onto 2×YTG agar plates(15 μg/ml
thiamphenicol+20 μg/ml erythromycin). Plates were incubated for 2–4
days under secondary containment within 3.4 L Anaerobic Jars each with a
3.5 L Anaerobic Gas Generating sachet.

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