protocol
Transferring whole genomes from bacteria to yeast
spheroplasts using entire bacterial cells to reduce
DNA shearing
Bogumil J Karas1, Jelena Jablanovic1, Edward Irvine1, Lijie Sun1, Li Ma2, Philip D Weyman1, Daniel G Gibson1,
John I Glass2, J Craig Venter1,2, Clyde A Hutchison III1, Hamilton O Smith1 & Yo Suzuki1
1Department of
Synthetic Biology and Bioenergy, J. Craig Venter Institute, La Jolla, California, USA. 2Department of Synthetic Biology and Bioenergy, J. Craig Venter
Institute, Rockville, Maryland, USA. Correspondence should be addressed to B.J.K. ([email protected]) or Y.S. ([email protected]).
© 2014 Nature America, Inc. All rights reserved.
Published online 6 March 2014; doi:10.1038/nprot.2014.045
Direct cell-to-cell transfer of genomes from bacteria to yeast facilitates genome engineering for bacteria that are not amenable
to genetic manipulation by allowing instead for the utilization of the powerful yeast genetic tools. Here we describe a protocol
for transferring whole genomes from bacterial cells to yeast spheroplasts without any DNA purification process. The method
is dependent on the treatment of the bacterial and yeast cellular mixture with PEG, which induces cell fusion, engulfment,
aggregation or lysis. Over 80% of the bacterial genomes transferred in this way are complete, on the basis of structural and
functional tests. Excluding the time required for preparing starting cultures and for incubating cells to form final colonies, the
protocol can be completed in 3 h.
INTRODUCTION
Genome-wide engineering is often essential for generating
microbes that are suitable for laboratory experiments or industrial
applications. However, the two general approaches for bacterial
genome engineering both suffer from the lack of a precise understanding of underlying biological processes and from the resulting
shortage of available methods. The first approach is to start with
an organism that has the desired properties and to enhance these
properties via genetic manipulation within the native organism1.
Unfortunately, tools for genome engineering are limited in many
organisms. The second, alternative approach is to transfer the
genome or a genome-scale fragment from an organism of interest
into a model organism such as yeast, and to perform engineering of the transferred genome in the model organism 2–4. Yeast
cells are capable of accommodating complete genomes derived
from bacteria as plasmids5–7. It is also possible to combine parts
of these genomes with ‘synthetic’ pieces that are built by using
a hierarchical assembly strategy that involves the incorporation
of fragments assembled from oligonucleotides3. The appeal of
genome-scale synthesis is that the streamlined process is readily automated to liberate experimentalists from the complicated
logistics of generating desired genomes by using molecular biology techniques. Therefore, cloning of bacterial genomes in yeast
enables facile and precise engineering, including the integration of designer sequences in genomes of organisms that are not
amenable to genetic manipulation.
One of the technical challenges in genome cloning is the handling of the megabase-sized DNA that is prone to physical breakage before transfer into yeast. To minimize this problem, DNA
can be purified from a cell embedded in an agarose matrix so
that DNA is confined to the hollow previously occupied by the
cell during the decomposition of the cellular material. However,
damage can still occur when purified DNA is released from the
protecting agarose matrix, through the action of heat and digestive enzymes, before it is mixed with yeast spheroplasts.
Here we present a protocol for cell-to-cell transfer of bacterial
genomes to yeast spheroplasts. This involves the preparation of
a bacterial culture and the generation of yeast spheroplasts. The
cells are then combined in the presence of PEG and plated along
with the top agar of a yeast medium (Figs. 1 and 2). Recently,
we have shown that one solution to the problem of DNA breakage is to introduce bacterial cells into a mixture containing yeast
spheroplasts and PEG to directly transfer bacterial genomes to
yeast (Fig. 1; ref. 8). The bacterial cells provide protection against
physical damage until they contact yeast spheroplasts. The addition of PEG to populations of cells has been noted to cause aggregation or fusion of cells, as well as engulfment of smaller cells by
larger cells9–15. The PEG-mediated process has been also shown to
result in transfer of DNA materials of various types (for example,
yeast artificial chromosomes) from bacterial protoplasts to
mammalian cells16, from bacterial protoplasts to bacterial protoplasts17,18, from bacterial mini cells to yeast spheroplasts19 and
from yeast spheroplasts to mammalian cells20.
This direct cell-to-cell transfer method was first applied to the
genomes of the cell wall–less bacteria Mycoplasma mycoides and
M. capricolum. It was then used for the genome of the Gramnegative bacterium Haemophilus influenzae (1.8 Mb) (refs. 8,21).
These genomes are also clonable with the approach using purified
DNA material, but the direct transfer approach may be advantageous with larger genome sizes. Notably, by using our method,
we were able to transfer H. influenzae genomes without a specific
pro­cedure to remove the cell wall from the bacterial cells8. The
cloned genome was stable; we have not detected any instance of
unwanted recombination between repeat sequences within the
genome or of integration of the genome into a yeast chromosome subsequent to cloning of the bacterial genome. In addition
to genomes, we have found that the same method is effective for
transferring bacterial artificial chromosomes from Escherichia coli
to yeast (B.J.K. et al., unpublished data).
Some sequences are required for the maintenance of a bacterial
genome in yeast, such as the AT-rich sequences that act as yeast
replication origins, termed autonomous replicating sequences
(ARS). These sequences are abundant in AT-rich genomes, but
nature protocols | VOL.9 NO.4 | 2014 | 743
protocol
© 2014 Nature America, Inc. All rights reserved.
Yeast
spheroplasts
1
2
3
Mix
PEG
or
or
Select
additional copies must be introduced for stable replication of a
GC-rich genome in yeast22. A centromere and a yeast selection
marker are also required in the bacterial genome. These elements
can be conveniently introduced together into the bacterial genome
as a yeast vector. Our current standard procedure is to introduce a
DNA cassette containing an ARS, the CEN6 centromere and the
yeast selection marker HIS3 (Supplementary Note) along with a
bacterial antibiotic selection marker, and any additional required
sequences, into the genome of the donor bacterium. The choice
of the bacterial selection marker depends on which antibiotic is
effective in the bacterium of interest. HIS3 can be replaced with
a different marker (for example, URA3) as long as the recipient
yeast strain has the matching auxotrophic mutation complemented by the marker gene. We have so far verified that the yeast
strains VL6-48, BY4741 and W303a (see MATERIALS) are compatible with our method.
Suitable methods for introducing the yeast vector cassette into
the genome in a bacterium are dependent on the bacterial species
in question. We generally approach this problem by introducing
the yeast vector cassette either randomly or in a targeted manner. For the random approach, we use the Tn5 transposome kit
(Epicenter). We attach the required 19-bp Tn5 ends to a fragment
containing the yeast cassette and a bacterial antibiotic resistance
marker by PCR; we then load the transposase protein in vitro and
introduce the transposome via electroporation or PEG-mediated
transformation. The site of insertion can be determined later
by using inverse PCR followed by Sanger sequencing 23. For the
targeted approach, we use homologous recombination: we prepare a plasmid (including any features specific to the method for
transformation) in which upstream and downstream sequences
homologous to the targeted sequences flank the cassette and a
bacterial selection marker. This plasmid can be constructed by
combining PCR fragments using Gibson assembly24. Available
approaches for introducing the generated cassette into bacteria
include electroporation, natural transformation8,25 and conjugation. When the bacterial genome contains a sequence that is
toxic6 to yeast, the yeast vector should be targeted to that site to
replace the sequence.
Bacterial
cell
Transfer
Figure 1 | Diagram of direct cell-to-cell genome transfer from bacteria
to yeast spheroplasts. Bacterial cells (blue) containing a yeast selectable
marker and the yeast centromere within their genomes (bacterium genomes
are represented as green circles) are prepared and mixed with yeast
spheroplasts in the presence of PEG. Genome transfer from the bacterium to
yeast may happen via engulfment of the bacterial cell (1, asterisk) and/or
via lysis of bacterial cells in the PEG solution, resulting in the release of
genomic DNA, which is taken up by yeast spheroplasts (2, asterisk) and/or
via cell fusion (3, asterisk). Yeast cells that acquired a bacterial genome are
then selected on appropriate agar plates.
To clone a bacterial chromosome with high GC content, multiple copies of ARS need to be inserted into the genome, as described
above, before genome transfer to ensure stable transmission in
yeast. An effective approach is to use assembly of a complete
chromo­some5 from TAR-cloned overlapping genomic fragments26
after inserting an ARS in the middle of each fragment22,27.
We observed in all three species tested that the transfer efficiency of a genome increases by fivefold or more when donor
cells do not contain restriction enzyme genes 8. The genetargeting methods described above can be used to knock out
these genes before genome transfer. Heat treatment increases the
genome transfer rate about fivefold for the M. mycoides strain
YCPMmyc1.1, from which restriction enzyme systems were not
removed, and about twofold for a strain lacking endogenous
restriction enzymes. For M. capricolum subsp. capricolum strain
California Kid, from which the sole restriction enzyme system
was not removed, genome transfer efficiency increased about
twofold and no increase was noticed for the strain lacking the
endogenous restriction enzyme (unpublished data). Whether
the effect of heat is due to inactivation of restriction enzymes is
still undetermined (B.J.K. et al., unpublished data). Additional
factors that affect the transfer efficiency are being studied by using
numerous deletion mutants of M. mycoides, and further knowledge of the transfer mechanism may lead to future improvements
of the method (B.J.K. et al., unpublished data).
The final step in this process is to bring the genome back to
life. To do this, the genome cloned and engineered into yeast
would need to be transferred to an organism where the genome
Day 1
Day 2
Days 2–10
Figure 2 | Timeline for tasks in the direct
genome transfer protocol. Tasks related to
0h
7h
22 h 23 h 24 h 25 h 26 h 27 h 28 h 29 h
the preparation of yeast cells are indicated in
orange, including starting the yeast culture and
Yeast
1
1
3–5
6–10
diluting the yeast culture (1; numbers indicate
11–15
16–18
PROCEDURE steps), as well as washing the yeast
Bacterium
2
2
2
cells in 1 M d-sorbitol when appropriate OD600
A(i)
B(i)
A(ii–vi) or B(ii–v)
is reached, storing them at 4 °C (3–5) and
preparing yeast spheroplasts (6–10). Tasks related to the preparation of bacterial cells are marked in blue, including starting the bacterial culture, adding an
appropriate antibiotic to inhibit the initiation of DNA replication (enriching for complete genomes) and collecting cells 1 h later (for cell wall–less bacteria)
or treating cells with lysozyme (for bacteria that have cell walls such as H. influenzae). Tasks related to genome transfer are indicated in green, including
treating the cell mixture with PEG and observing yeast colonies.
744 | VOL.9 NO.4 | 2014 | nature protocols
protocol
functions28,29. This organism may be a cell of a closely related
species or of the identical species. This downstream transfer
procedure may also be facilitated by direct cell-to-cell
transfer, as shown in the recent example of transferring a yeast
artificial chromosome from yeast to mouse embryonic stem cells
via cell fusion20. Detailed study of the mechanisms of genome
transfer is expected to yield ideas for enhancing the utility of
the technology.
© 2014 Nature America, Inc. All rights reserved.
MATERIALS
REAGENTS
Yeast strains
• Saccharomyces cerevisiae VL6-48 (ATCC MYA-3666: MATα his3-∆200
trp1-∆1 ura3-52 lys2 ade2-1 met14 cir0)30. The BY4741 strain (MATa his3∆1
leu2∆0 met15∆0 ura3∆0)31 produced similar results as VL6-48.
The W303-1A strain (MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his311,15 ybp1-1)5 had a roughly five-times-lower genome transfer efficiency
than VL6-48
Bacterial strains
• M. mycoides strain YCpMmyc1.1 (ref. 28), M. capricolum subsp. capricolum (strain
California KidT, ATCC 27343) (ref. 28) and H. influenzae Rd (strain KW20
ATCC 51907). Basic handling of these bacteria has been described in refs. 32,33
Chemicals
• Adenine hemisulfate salt (Sigma-Aldrich, cat. no. A9126)
• EDTA, 0.5 M, Ambion molecular biology grade, pH 8 (Life Technologies,
cat. no. AM9261)
• Ammonium sulfate solution, 25% (wt/vol) (Teknova, cat. no. A0515)
• Bacto agar (Becton Dickinson, cat. no. 214030)
• Bacto peptone (Becton Dickinson, cat. no. 211677)
• Bacto tryptone (Becton Dickinson, cat. no. 211705)
• BBL brain heart infusion (BHI; Becton Dickinson, cat. no. 299070)
• BBL mycoplasma broth base (Becton Dickinson, cat. no. 211458)
• Bacto yeastolate (Becton Dickinson, cat. no. 255772)
• β-Mercaptoethanol (Sigma-Aldrich, cat. no. M3148) ! CAUTION This
compound is very hazardous if inhaled, ingested or if it comes in contact
with skin.
• β-Nicotinamide adenine dinucleotide hydrate (Sigma-Aldrich,
cat. no. N6522-1G)
• Calbiochem glycerol, molecular biology grade (Millipore, cat. no. 356352)
• Calcium chloride (CaCl2; Sigma-Aldrich, cat. no. C5080)
• Chloramphenicol (Sigma-Aldrich, cat. no. C0378) ! CAUTION This
compound is very hazardous if ingested. It is hazardous on skin contact.
• Dextrose (Sigma-Aldrich, cat. no. D9434)
• Difco yeast extract-peptone-dextrose (YPD; Becton Dickinson,
cat. no. 242810)
• d-Sorbitol (Sigma-Aldrich, cat. no. S1876)
• Heat-inactivated (HI)-FBS (Life Technologies cat. no. 10082-147)
• Yeast extract solution (Life Technologies, cat. no. 18180-059)
• Connaught Medical Research Laboratories (CMRL)-1066 medium (10×)
without l-glutamine, without sodium bicarbonate (Life Technologies,
formula no. 01-0127DJ)
• Hemin (Sigma-Aldrich, cat. no. H9039-100G)
• l-Glutamine (Sigma-Aldrich, cat. no. G3126-100G)
• Lysozyme, from chicken egg white (Sigma-Aldrich, cat. no. L6876)
• Magnesium chloride (MgCl2; Sigma-Aldrich, cat. no. M8266)
• Multiplex PCR kit (Qiagen, cat. no. 206143)
• Penicillin G sodium salt (Sigma-Aldrich, cat. no. P3032)
• Phenol red solution (Sigma-Aldrich, cat. no. P0290)
• PEG-8000 (USB, cat. no. 19959)
• Puromycin dihydrochloride (A. G. Scientific, cat. no. P-1033)
• Sodium bicarbonate (NaHCO3; Sigma-Aldrich, cat. no. SS8875)
• Sodium hydroxide (NaOH; 1 N, Thermo Fisher Scientific, cat. no. SS277)
! CAUTION This compound is very hazardous if inhaled, ingested or if it
comes in contact with skin.
• Sodium phosphate dibasic heptahydrate (Na2HPO4.7H2O; Sigma-Aldrich,
cat. no. S9390)
• Sodium phosphate monobasic monohydrate (NaH2PO4.1H2O;
Sigma-Aldrich, cat. no. 71504)
• Sucrose (Sigma-Aldrich, cat. no. S0389)
• Synthetic complete medium lacking histidine (Teknova, cat. no. C7112)
• Tetracycline hydrochloride (Sigma-Aldrich, cat. no. T7660) ! CAUTION It is
harmful to the reproductive system and liver.
• Triethanolamine (Sigma-Aldrich, cat. no. 90278-100ML)
• UltraPure 1 M Tris-HCl (Life Technologies, cat. no. 15568-025)
• X-gal solution, 40 mg ml–1 (Teknova, cat. no. X1220)
• Zymolyase-20T (from Arthrobacter luteus, AMS Biotechnology, cat. no. 120491-1)
EQUIPMENT
• Balance (Mettler Toledo Classic Plus model PB3002-S/FACT 3,
Thermo Fisher Scientific, cat. no. 01916263)
• Beaker, 1 liter (Corning, cat. no. 7000-1L)
• Centrifuge (Avanti J-26 XP, Beckman Coulter, cat. no. 393124)
• Centrifuge rotor (JA-25.50, Beckman Coulter, cat. no. 363058)
• Centrifuge tubes, 15 ml (Denville Scientific, cat. no. C1018-P)
• Centrifuge tubes, 50 ml (Denville Scientific, cat. no. C1060-P)
• Disposable plastic cuvettes (Thermo Fisher Scientific, cat. no. 14-955-127)
• Erlenmeyer flasks with baffles, 250 ml (Corning, cat. no. 4450-250)
• Glass bottles, 1 liter (Corning, cat. no. 1396-1L)
• Hot plate stirrer (VWR International, cat. no. 12365-476)
• Inoculating loop, 10 µl (VWR International, cat. no. 12000-810)
• Incubator shaker (New Brunswick Scientific Excella E25, Thermo Fisher
Scientific, cat. no. 14-285-734)
• Incubator, set to 30 °C (Thermo Fisher Scientific, cat. no. 11-690-637D)
• Incubator, set to 37 °C (Thermo Fisher Scientific, cat. no. 11-690-637D)
• Microtube centrifuge (Micro CL 17, Thermo Fisher Scientific,
cat. no. 75002450)
• Microwave oven
• Microcentrifuge tubes, 1.7 ml (Denville Scientific, cat. no. C-2170)
• Microcentrifuge tube rack (USA Scientific, cat. no. 2380-1008)
• Membrane syringe filter, 0.2 µm (Life Technologies, cat. no. 4612)
• pH meter (Oakton Instruments, cat. no. 35614-22)
• Pipette controller (VWR International, cat. no. 46102-256)
• Bottle-top vacuum filter, 0.22-µm-pore, 1 liter (Corning, cat. no. 431174)
• Standard stir bar with spinning ring (Cole-Parmer, cat. no. EW-04612-30)
• Spectrophotometer (model Genesys 10S Vis, Thermo Fisher Scientific,
cat. no. 840-207900)
• Sterile 100-mm Petri dishes (Denville Scientific, cat. no. M5300)
• Syringes, 10 ml (Becton Dickinson, cat. no. 301030)
• Serological pipettes, 25 ml (Denville Scientific, cat. no. P7135)
• Thermal cycler (C1000 Touch, Bio-Rad Laboratories, cat. no. 185-1148EDU)
• ThermoGrid rigid strip, 0.2 ml PCR tubes (Denville Scientific, cat. no. C18064)
• Water bath, set to 37 °C (VWR International, cat. no. 89032-216)
• Water bath, set to 50 °C (VWR International, cat. no. 89032-216)
• Vacuum pump (Welch Vacuum Technology, model no. 2522B-01, Fisher
Scientific, cat. no. 01-051-1A)
• Vortex mixer (VWR International, cat. no. 58816-121)
REAGENT SETUP
YPD medium, 2× In a 1-liter beaker, combine 100 g of YPD powder and
160 mg of adenine hemisulfate salt, and then add deionized (DI) H2O to a
final volume of 1 liter. Mix for 20 min with the use of a magnetic stir bar on
a hot-plate stirrer set to 50 °C. Sterilize the medium by passing it through a
0.22-µm-pore, 1-liter bottle-top vacuum filter connected to a vacuum pump
(spheroplasts of yeast grown in autoclaved medium produced less-consistent
results). Store the medium at room temperature (~22 °C) or at 4 °C for no
longer than 12 months.
Dextrose, 20% (wt/vol) In a 1-liter beaker, add 200 g of dextrose; fill the
beaker with DI H2O to 1 liter. Dissolve the dextrose with the use of a stirring
bar on a hot-plate stirrer. Sterilize the medium by passing it through a
0.22-µm-pore, 1-liter bottle-top vacuum filter connected to a vacuum pump.
Store the solution at 4 °C for no longer than 12 months.
Bacto yeastolate, 2% (wt/vol) Add 20 g of Bacto yeastolate to a 1-liter beaker
and fill it with DI H2O to a final volume of 1 liter. Dissolve Bacto yeastolate
with the use of a stirring bar on a hot-plate stirrer. Autoclave the solution for
15 min at 120 °C. Store it at 4 °C for no longer than 3 months.
nature protocols | VOL.9 NO.4 | 2014 | 745
© 2014 Nature America, Inc. All rights reserved.
protocol
Tetracycline hydrochloride, 0.4% (wt/vol) In a 15-ml centrifuge tube,
combine 40 mg of tetracycline hydrochloride and 10 ml of H2O. Dissolve by
vortexing. Sterilize by drawing the solution in a 10-ml syringe and passing it
through a 0.2-µm membrane syringe filter. Put 850-µl aliquots into 1.7-ml
tubes and store them at −20 °C for no longer than 1 month.
SP-4 liquid medium Combine 3.5 g of BBL mycoplasma broth base, 10 g of
Bacto tryptone, 5.3 g of Bacto peptone, 50 ml of CMRL-1066 medium
(10×), 25 ml of 20% (wt/vol) dextrose, 35 ml of yeast extract solution,
100 ml of 2% (wt/vol) Bacto yeastolate, 170 ml of HI-FBS, 2.5 ml of
penicillin G (40,000 U ml−1), 14.6 ml of 7.5% (wt/vol) NaHCO3, 5 ml of
l-glutamine (200 mM) and 4 ml of 0.5% (wt/vol) phenol red solution; add
DI H2O to bring the volume of the pH-adjusted solution to 1 liter (ref. 32).
Mix by using a magnetic stirring bar on a hot-plate stirrer. Adjust the pH to
a range of 7.6–7.8 by adding an appropriate volume of 1 N NaOH. Sterilize
the medium by passing it through a 0.22-µm-pore, 1-liter bottle-top vacuum
filter connected to a vacuum pump. Store the medium at 4 °C for no longer
than 6 months.
l-Glutamine, 200 mM In a 100-ml beaker, add 7.3 g of l-glutamine and
fill the beaker with DI H2O to a final volume of 50 ml. Mix for 10 min
with the use of a magnetic stirring bar on a hot plate. Sterilize the solution
by passing it through a 0.22-µm-pore, 1-liter bottle-top vacuum filter
connected to a vacuum pump. Keep the solution at −20 °C for no longer
than a few years.
Nicotinamide solution, 1% (wt/vol) Dissolve 500 mg of β-nicotinamide
adenine dinucleotide hydrate in DI H2O. Adjust the volume to 50 ml with DI
H2O and filter-sterilize the solution. Store the solution at 4 °C for no longer
than 1 month or at −20 °C for no longer than 6 months.
Hemin solution, 0.1% (wt/vol) In a 250-ml bottle, combine 200 mg of
hemin, 4 ml of triethanolamine and 192 ml of DI H2O. Incubate the bottle
at 65 °C for 30 min. After the hemin dissolves, adjust the volume to 200 ml
by using DI H2O. Sterilize the solution by passing it through a 0.22-µm-pore,
1-liter bottle-top vacuum filter connected to a vacuum pump. Store the
solution at 4 °C for no longer than a few months.
Supplemented BHI liquid medium In a 1-liter beaker, add 38 g of BHI
medium powder and fill the beaker with DI H2O to 980 ml (ref. 33).
Autoclave for 10 min at 121 °C. This solution can be stored at 4 °C for
many months. Next, to prepare supplemented BHI liquid medium,
add 400 µl of nicotinamide solution and 20 ml of hemin solution just
before use.
Puromycin, 5% (wt/vol) In a 15-ml tube, add 10 ml of DI H2O, and then
add 0.5 g of puromycin. Dissolve the puromycin by vortexing. Sterilize by
drawing the solution in a 10-ml syringe and by passing it through a 0.2-µm
membrane syringe filter. Put 1-ml aliquots into 1.7-ml tubes and store them
at −20 °C for no longer than 12 months.
Resuspension buffer Resuspension buffer is 0.5 M sucrose, 10 mM Tris-HCl,
10 mM CaCl2 and 2.25 mM MgCl2 (pH 7.5). Combine 171.15 g of sucrose,
10 ml of 1 M Tris-HCl solution at pH 7.5, 0.147 g of CaCl2 and 0.238 g of
MgCl2, and then add DI H2O to bring the volume of the pH-adjusted
solution to 1 liter. Adjust the pH to 7.5 with NaOH. Sterilize the solution by
using a 0.22-µm-pore, 1-liter bottle-top vacuum filter, and then store it at
room temperature for no longer than 12 months.
Lysozyme, 2.5% (wt/vol) Resuspend 0.25 g of lysozyme in 10 ml of DI H2O.
Sterilize by drawing the solution in a 10-ml syringe and passing it through
a 0.2-µm membrane syringe filter. Put 1-ml aliquots into 1.7-ml tubes and
store them at −20 °C for no longer than 12 months.
d-Sorbitol, 1 M In a 1-liter beaker, add 182 g of d-sorbitol and add DI H2O
to a final volume of 1 liter. Dissolve with the use of a magnetic stirring bar
on a stirring plate. Autoclave the solution for 15 min at 120 °C and store it at
room temperature for no longer than 12 months.
SPEM solution SPEM solution is 1 M d-sorbitol, 10 mM EDTA (pH 8) and
10 mM sodium phosphate. Combine 182 g of d-sorbitol, 20 ml of 0.5 M
EDTA (pH 8), 2.08 g of Na2HPO4·7H2O and 0.32 g of NaH2PO4·1H2O;
add DI H2O to a final volume of 1 liter. Mix with a magnetic stirring bar
on a hot-plate stirrer until dissolved. Sterilize the solution by using a
0.22-µm-pore, 1-liter bottle-top vacuum filter connected to a vacuum pump.
Store the solution at room temperature for no longer than 12 months.
Glycerol, 50% (vol/vol) Add 500 ml of glycerol in a 1-liter glass bottle and
fill the bottle with DI H2O to a final volume of 1 liter. Autoclave the solution
for 15 min at 120 °C and store it at room temperature for no longer than
12 months.
Zymolyase-20T solution Zymolyase-20T solution is Zymolyase-20T, 1 M
Tris-HCl and 50% (vol/vol) glycerol. Combine 200 mg of Zymolyase-20T,
1 ml of 1 M Tris-HCl, 10 ml of 50% (vol/vol) glycerol and 9 ml of sterile DI
H2O in a sterile 15-ml centrifuge tube. Make 45-µl aliquots in PCR tubes
to avoid repetitive freeze-thaw cycles, and then store them at −20 °C for no
longer than 12 months.
STC solution STC solution is 1 M d-sorbitol, 10 mM Tris-HCl, 10 mM
CaCl2 and 2.5 mM MgCl2. Combine 182.17 g of d-sorbitol, 10 ml of 1 M
Tris-HCl solution at pH 7.5, 0.147 g of CaCl2 and 0.238 g of MgCl2; add DI
H2O to a final volume of 1 liter. Sterilize the solution with a 0.22-µm-pore,
1-liter bottle-top vacuum filter, and then store it at room temperature for no
longer than 12 months.
PEG solution, 20% (wt/vol) PEG solution (20% (wt/vol)) is PEG-8000,
10 mM Tris-HCl, 10 mM CaCl2 and 2.5 mM MgCl2 (pH 8). Combine
200 g of PEG-8000, 10 ml of 1 M Tris-HCl solution at pH 8.0, 0.147 g of
CaCl2 and 0.238 g of MgCl2; add DI H2O to bring the volume of the pHadjusted solution to 1 liter. Adjust the pH to 8.0 with NaOH. Sterilize by
passing the solution through a 0.22-µm-pore, 1-liter bottle-top vacuum
filter connected to a vacuum pump, and store it at 4 °C for no longer than
6 months. Equilibrate the solution to 37 °C before use. Check the pH once a
month to ensure that it stays at 8.0.
SOS medium SOS medium is 1 M d-sorbitol, 6 mM CaCl2, 2.5 g per liter
yeast extract and 5 g per liter Bacto peptone. In a 1-liter beaker, combine
182.17 g of d-sorbitol, 0.67 g of CaCl2, 2.5 g of yeast extract and 5 g of Bacto
peptone; add DI H2O to a final volume of 1 liter. Mix on a hot-plate stirrer
with the use of a magnetic stirring bar, and filter-sterilize the medium in a
0.22-µm-pore, 1-liter bottle-top vacuum filter. Store the solution at room
temperature for no longer than 12 months.
Synthetic complete sorbitol medium lacking histidine (−His) agar plates In a 1-liter bottle, add 182 g of d-sorbitol, 20 g of Bacto agar, 23.5 g of
synthetic complete medium lacking histidine and 160 mg of adenine
hemisulfate salt and add DI H2O to a final volume of 980 ml. Autoclave the
mixture for 20 min at 120 °C. Then add 20 ml of 25% (wt/vol) ammonium
sulfate solution by using sterile technique.
Top agar Prepare synthetic complete sorbitol medium lacking histidine agar
solution, as described above. Keep the autoclaved bottle at room temperature for no longer than 12 months. Before spheroplast preparation, melt the
medium in a microwave oven and prepare 8-ml aliquots in 15-ml centrifuge
tubes. Equilibrate the aliquots to 50 °C in a water bath until use. Melted
medium can be kept at 50 °C for up to 24 h.
PROCEDURE
Yeast culture preparation ● TIMING 24 h
1| In the morning on the day before constructing spheroplasts (Fig. 2), inoculate 20 ml of 2× YPD medium with the
S. cerevisiae strain VL6-48. Grow the culture at 30 °C with constant shaking at 225 r.p.m. At the end of the day, measure
the OD600 of the culture and make appropriate dilution in a fresh 50 ml of 2× YPD medium in a 250-ml Erlenmeyer flask with
baffles so that the fresh culture reaches an OD600 value of 2.5–3.0 at the desired harvest time on the following day. The
doubling time of VL6-48 is ~90 min.
746 | VOL.9 NO.4 | 2014 | nature protocols
© 2014 Nature America, Inc. All rights reserved.
protocol
Bacterial culture preparation ● TIMING 5–20 h
2| While yeast culturing is ongoing, prepare bacterial cultures via option A or B, depending on the bacterial strain used.
Use option A for cell wall–less, slow-growing bacteria such as M. mycoides strain YCpMmyc1.1 and M. capricolum subsp.
capricolum strain California Kid, with the doubling time of ~1–2 h. Use option B for fast-growing, Gram-negative bacteria
such as H. influenzae Rd strain KW20, with the doubling time of ~30 min.
(A) Culture of cell wall–less, slow-growing bacteria
(i) In the evening of the day before combining with yeast spheroplasts (Fig. 2), inoculate 50 ml of SP-4 medium with
an appropriate number of cells to obtain a culture of cells in the exponential phase (pH ~7.0) the following morning.
Grow the culture at 37 °C without shaking.
 CRITICAL STEP Collection of mycoplasmas at an early exponential phase produces the highest rate of complete
genomes transferred to yeast.
(ii) Add chloramphenicol to the culture at a final concentration of 100 mg per liter and incubate it for an additional 1 h.
 CRITICAL STEP If a mycoplasma strain is resistant to chloramphenicol, use other antibiotics that inhibit protein
synthesis, such as tetracycline (20 µg ml–1) or puromycin (100 µg ml–1).
(iii) Centrifuge the cells at 10,000g for 5 min at 10 °C (in a 50-ml centrifuge tube). Decant the supernatant.
(iv) Resuspend the cells in 50 ml of resuspension buffer. Centrifuge the suspension at 10,000g for 5 min at 10 °C.
Decant the supernatant.
(v) Resuspend the cells in 500 µl of resuspension buffer.
(vi) Incubate M. mycoides strain YCpMmyc1.1 cells for 15 min at 49 °C or M. capricolum subsp. capricolum strain California
Kid for 15 min at 47 °C before mixing with yeast spheroplasts.
 CRITICAL STEP The time and temperature should be optimized for each bacterial strain.
(B) Culture of fast-growing, Gram-negative bacteria
(i) In the morning on the day of the transfer (Fig. 2), inoculate 50 ml of BHI medium supplemented with 4 mg per liter
nicotinamide adenine dinucleotide and 10 mg per liter hemin (sBHI) with a sufficient number of bacterial cells to
obtain in a few hours a culture with an OD600 of 0.4 at 37 °C with constant shaking at 200 r.p.m. Once an OD600 value
of 0.4 has been reached, add 100 µl of puromycin (50 g per liter) and incubate the culture for an additional 30 min
with constant shaking at 200 r.p.m. Centrifuge the culture at 7,000g for 5 min at 10 °C. Decant the supernatant.
 CRITICAL STEP If your bacterial strain is resistant to puromycin, use other antibiotics that inhibit protein
synthesis, such as tetracycline or chloramphenicol.
(ii) Resuspend the cells in 9.0 ml of resuspension buffer, and then add 500 µl of 0.5 M EDTA (pH 8) and 500 µl of
25 mg m1−1 lysozyme; incubate the cells for 30 min in a 37 °C bath.
(iii) Centrifuge the cell suspension at 7,000g for 4 min at 10 °C. Decant the supernatant.
(iv) Gently resuspend the pellet in 1 ml of resuspension buffer by gently pipetting up and down by using a wide-orifice tip.
Add 39 ml of bacterial resuspension buffer and mix by inversion. Centrifuge the suspension at 7,000g for 4 min at
10 °C. Decant the supernatant.
(v) Resuspend the pellet in resuspension buffer for a final volume of 500 µl.
Preparation of yeast spheroplasts ● TIMING 75 min
3| When the yeast culture of Step 1 reaches an OD600 of 2.5–3.0 (Fig. 2), centrifuge the cells at 2,500g for 5 min at 10 °C.
Decant the supernatant.
4| Resuspend the pellet in 20 ml of sterile H2O and vortex. Add an additional 30 ml of sterile H2O and mix by inversion.
Centrifuge the suspension at 2,500g for 5 min at 10 °C and decant the supernatant.
5| Resuspend the pellet in 20 ml of 1 M d-sorbitol and vortex. Add an additional 30 ml of 1 M d-sorbitol and mix it by
inversion. Centrifuge the suspension at 2,500g for 5 min at 10 °C, and decant the supernatant.
 PAUSE POINT The cell pellet can be kept at 4 °C for at least 16 h without any noticeable effect on genome transfer.
6| Resuspend the pellet thoroughly in 20 ml of SPEM solution by vortexing. Add 30 µl of β-mercaptoethanol and 40 µl of
Zymolyase-20T. Incubate for 30 min at 30 °C with constant shaking at 75 r.p.m.
 CRITICAL STEP Shaking more vigorously than 75 r.p.m. can destroy yeast cells during Zymolyase-20T treatment.
7| After 30 min of Zymolyase-20T treatment, measure the OD600 for 0.2 ml of Zymolyase-20T–treated culture mixed with
0.8 ml of 1 M d-sorbitol (mixture a) and 0.2 ml of Zymolyase-20T–treated culture mixed with 0.8 ml of H2O (mixture b).
nature protocols | VOL.9 NO.4 | 2014 | 747
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If the OD600 ratio (mixture a/mixture b) is between 1.8 and 2.0, the spheroplasts are ready. If the mixture a/mixture b value
is below 1.8, incubate the mixture further while checking the OD600 ratio every 5 min.
 CRITICAL STEP Over-spheroplasting of yeast results in a drastic decrease in surviving yeast colonies.
8| To the prepared spheroplasts, add 30 ml of 1 M d-sorbitol and mix by gentle inversion. Centrifuge the mixture at 1,000g
for 5 min at 10 °C. Decant the supernatant.
 CRITICAL STEP The inversion must be gentle and centrifugation must be done as instructed because yeast spheroplasts are
fragile.
9| Gently resuspend the pellet in 10 ml of 1 M d-sorbitol by passing it 5–10 times through a 10-ml pipette. Add an
additional 30 ml of 1 M d-sorbitol and gently mix by inversion. Centrifuge the suspension at 1,000g for 5 min at 10 °C and
decant the supernatant.
10| Resuspend the pellet in 2 ml of STC solution; incubate the suspension at room temperature for 10–20 min.
© 2014 Nature America, Inc. All rights reserved.
PEG treatment of the cellular mixture ● TIMING 75 min
11| Preincubate one agar plate per experiment at 37 °C, to be used for plating transformed yeast cells.
12| Combine 200 µl of yeast suspended in STC (Step 10) and 50 µl of bacterial suspension (Step 2A(vi) or 2B(v)). Mix by
gently flicking the tube and incubate it at room temperature for 5 min.
13| Add 1 ml of 20% (wt/vol) PEG equilibrated at 37 °C and mix by gently flicking the tube and inverting it 6–10 times.
Incubate the tube at room temperature for 15–20 min and centrifuge it at 1,500g for 7 min at room temperature. Remove
the supernatant by using a 1,000-µl pipette.
14| Resuspend the pellet in 800 µl of SOS medium and incubate it at 30 °C for 30 min. During the incubation, prepare 8-ml
aliquots of top agar and equilibrate them at 50 °C in a water bath.
 CRITICAL STEP Top agar must be equilibrated at 50 °C, because higher temperatures are harmful to yeast spheroplasts.
15| Combine the transformed spheroplasts from the previous step with top agar, mix by inverting 3–5 times and pour the
mixture on the agar plate equilibrated at 37 °C (Step 11). Allow the top agar to solidify for 5 min and transfer the plate to a
30 °C incubator.
 CRITICAL STEP When large numbers of transformants (for example, 5,000) are expected, it may be advantageous to
combine only a fraction of the 800 µl of transformed spheroplasts (for example, 100 µl or less).
Identification of colonies with complete genome transfer ● TIMING 4–9 d
16| Transformants will be visible as colonies in 2–6 d. Pick individual colonies by using a sterile toothpick and make a short
single streak on a fresh agar plate (single colony isolation is not necessary). Incubate the plate at 30 °C for 1–2 d.
? TROUBLESHOOTING
17| Repeat the transfer of growing yeast cells to eliminate nontransformed yeast cells, residual free bacterial cells and DNA
from lysed cells.
18| To begin analyzing the integrity of the transferred genome, test to see whether loci throughout the genome are present
by using the multiplex PCR kit. To analyze roughly 5 loci with amplicon sizes of 800 bp or smaller per reaction, directly
introduce yeast cells from a small part of the re-streaked patch into a 20-µl PCR as a template without any specific procedure
for lysis. The standard Qiagen multiplex PCR program takes care of lysis. To analyze up to ten loci per reaction, with sizes of
some amplicons exceeding 1,200 bp, yeast DNA must be purified as described in Karas et al.6
? TROUBLESHOOTING
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
748 | VOL.9 NO.4 | 2014 | nature protocols
protocol
Table 1 | Troubleshooting table.
Step
Problem
Possible reason
Solution
16
No or few transformant
colonies
Yeast medium for selecting transformants
was incorrect
Ensure that all required components are added
to the medium
Yeast culture was not in log phase (Step 3) Ensure that the culture is harvested at the OD600
of 2.5–3.0
© 2014 Nature America, Inc. All rights reserved.
18
Low frequency (<50%) of
intact transferred genomes
PEG (20% (wt/vol)) solution was old or at
an improper pH
Prepare fresh 20% (wt/vol) PEG solution and check its
pH regularly
PEG (20% (wt/vol)) solution was not
equilibrated to 37 °C before use
Equilibrate 20% (wt/vol) PEG solution to 37 °C
Bacterial cells in late exponential
phase (Step 2A(i) or 2B(i)) were used,
or no antibiotic was added (Step 2A(ii)
or 2B(i))
Follow the protocol to increase the fraction of fully
replicated genomes
● TIMING
Step 1, yeast culture preparation: 24 h
Step 2, bacterial culture preparation: 5–20 h
Steps 3–10, preparation of yeast spheroplasts: 75 min
Steps 11–15, PEG treatment of cellular mixture: 75 min
Steps 16–18, identification of colonies with complete genome transfer: 4–9 d
A schematic diagram of the timeline is shown in Figure 2.
ANTICIPATED RESULTS
The number of transformants varies, depending on the
bacterial strain (Table 2), the presence of restriction
enzyme system(s) and whether bacterial cell wall removal
was performed, if applicable. Experiments with wild-type
M. mycoides should yield about 5,000–10,000 transformant
colonies per transformation. Removal of restriction enzyme
systems (RE−) can increase the number of transformants to
25,000–35,000 colonies. Wild-type and RE− M. capricolum
experiments should yield 5,000–10,000 and 25,000–35,000
transformant colonies, respectively, per transformation.
Wild-type and RE− H. influenzae should yield 0–20 and
100–1,000 colonies, respectively.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments This work was supported by Synthetic Genomics, Inc.
B.J.K. was supported by the Natural Sciences and Engineering Research Council
of Canada Postdoctoral Fellowships Program and by Synthetic Genomics, Inc.
Y.S. was also supported by the US Defense Advanced Research Projects Agency
contract no. N66001-12-C-4039 and the US Department of Energy cooperative
agreement no. DE-EE0006109.
AUTHOR CONTRIBUTIONS B.J.K., J.J., P.D.W., D.G.G., J.I.G., J.C.V., C.A.H.,
H.O.S. and Y.S. designed the research. B.J.K., J.J., E.I., L.S., L.M., P.D.W., D.G.G.,
C.A.H. and Y.S. performed experiments. B.J.K., J.J., P.D.W. and Y.S. wrote
the paper.
COMPETING FINANCIAL INTERESTS The authors declare competing financial
interests: details are available in the online version of the paper.
Table 2 | Expected numbers of transformant yeast colonies.
Strain
Number of colonies
Wild-type M. mycoides
5,000–10,000
Restriction systems minus M. mycoides
25,000–35,000
Wild-type M. capricolum
5,000–10,000
Restriction system minus M. capricolum
25,000–35,000
Wild-type H. influenzae
0–20
Restriction systems minus H. influenzae
100–1,000
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com/reprints/index.html.
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