A Model Organism,
Cellular Slime Mold
Experimental Guide
for NBRP Training Course1Table of Contents

I. Introduction
II. Culture and preservation of cellular slime molds
III. Observation of cellular slime molds
IV. Transformation of cellular slime molds
V. PCR Gene Amplification
VI. Preparation of genomic DNA
VII. Isolation of cellular slime molds from soil
VIII. Methods for providing cellular slime mold strainsfrom NBRP
Ⅸ. Reference sites for cellular slime molds
X．Reference books and bibliography.
XI

Composition of culture media

2

Ⅰ．Introduction
Cellular slime molds are amoeboid eukaryotic microorganisms inthe soil that normally divide and multiply while feeding on bacteria.

..
. . .. .

daUpon starvation, they undergo a simple developmental process andwithin 24 hours complete multicellular bodies (zygotes) with only two cdifferent cell differentiation patterns (right figure).
Multicellularity in cellular slime molds is due to chemotacticb

集合movement in response to cAMP secreted by the cells of the assembly

細胞性粘 菌の生活史

center. Subsequently, slug-shaped migratory bodies

a: 増 殖 期 （単 細 胞 期）

The slug-shaped migratory body is then formed.

b-d: 形 態 形 成 期 （ 多 細 胞 期 ）At this stage, the cells that will become spores andthe cells that will become their supporting scapes
have already been determined (lower right figure).
The following is a list of experimental characteristics
of cellular slime molds-Simple cell division in culture medium-Bacterial predation and digestion-Directed amoebic movements such as chemotaxis-Easy induction of morphogenesis-Haploid through its life cycle.
-Easy transformation-Various expression vectors-Completion of genome sequencing-Enrichment of gene, strain, and publication databases-High homologous recombination efficiency-Ease of storage-A lot of cell lines etc.
This manual is intended to help newcomers to the field of cellular slime molds to handle
Dictyostelium discoideum as an experimental standard strain for their research. The contents ofthe book focus on cellular slime mold culture and the isolation of transformants using molecularbiological techniques and include a description of methods for isolating cellular slime molds
from soil.
It is my hope that this book will help to utilize the model organism cellular slime molds inmany research settings and lead to useful biological discoveries.
December 2022
Hidekazu Kuwayama,
Department of Life and Environmental Sciences, University of Tsukuba
3

Ⅱ．Culture and preservation methods for cellularslime molds
Wild strains of cellular slime molds are basically grown on agar or in liquidmedium, using bacteria as a food source. However, if a sterile culture strain isused, it is possible to culture only pure slime mold cells in a bacteria-free
medium.
Methods for culturing cellular slime molds(1) Co-member culture method with bacteria (observation of developmental
process)(Preparation) Standard strain of Dictyostelium discoideum (strain AX2)
Bacteria (food): Escherichia coli B/r or Klebsiella aerogenes
5LP agar (lactose 5.0 g, Bacto Peptone (BD REF No.; 211677) 5.0 g, Bacto
Agar 15.0 g / 1L Distilled H20; autoclaved) x 20 mL per 9 cm diameter petri
dish,Incubator (21°C)
1. Incubate the bacteria (food) (Escherichia coli B/r or Klebsiella aerogenes) in
5LP liquid medium (5LP agar medium without Agar) at 37°C overnight, thendrop onto 0.5 mL 5LP agar medium. Spread evenly on the agar and air-dry
on a clean bench until the moisture is absorbed.
2. Inoculate a standard strain of Dictyostelium discoideum (ex. strain AX2) in thecenter.
3. Incubate in an incubator maintained at 21 ºC for 4 to 5 days.
4. bacteria feed and form a plaque at the center. Near the center, starvation is inprogress and fruiting bodies are observed. Slug bodies, aggregates, andproliferating amoebae are observed as one approaches the periphery.

<Memo>

4

(2) Cell preparation by two-member culture method with bacteria on agar
medium (Under Water Culture method)(Preparation) Standard strain of Dictyostelium discoideum (AX2 strain)
Bacteria (food): Escherichia coli B/r or Klebsiella aerogenes
N agar medium (glucose 10.0 g, Bacto Peptone (BD REF No.; 211677) 10.0g, Na2HPO4-12H20 0.96 g, KH2PO4 1.44 g, Bacto Agar 15 g / 1 L Distilled
H20; (Autoclave sterilization)
Sterile phosphate buffer (Na2HPO4-12H20 1.07 g, KH2PO4 0.96 g / 1 L;
autoclaved)
Sterile distilled water, sterile test tubes, centrifuge tubes
Incubator (21°C)
1. Scrape an appropriate amount of bacteria (Escherichia coli B/r or Klebsiellaaerogenes) cultured on N agar medium and suspend in 3 mL of sterile waterin a sterile test tube.
2. Scrape an appropriate amount of Dictyostelium discoideum standard strain(strain AX2) amoeba or spores and suspend in sterile water with the bacteria.
3. Transfer the suspension onto N agar.
4. Incubate in an incubator at 21 °C for 4 to 5 days.
5. Transfer the cell suspension to a centrifuge tube by gently scraping thesurface with an alcohol-sterilized glass rod (spreader).
Centrifuge at 300-350 g (1,500 - 2,000 rpm) for 2 min.
Discard the supernatant and resuspend the cells in cold sterile phosphatebuffer.
8. Repeat steps 6 and 7 two more times (total of three cell washes).
9. Suspend the cellular slime mold cells in appropriate buffer and use forexperiments (keep on ice).
<Memo>

5

(3) Cell conditioning by two-member culture method with bacteria (liquid shaking
culture method)(Preparation) Standard strain of Dictyostelium discoideum (AX2 strain)
Bacteria (food): Escherichia coli B/r or Klebsiella aerogenes
5LP liquid medium (lactose 5.0 g, Bacto Peptone (BD REF No.; 211677) 5.0
g / 1L Distilled H20; autoclaved)
Sterile phosphate buffer (Na2HPO4-12H20 1.07 g, KH2PO4 0.96 g / 1 L;
autoclaved)
Sterile distilled water, sterile triangular flasks, centrifuge tubes
Incubator (21°C), shaker
1. Scrape an appropriate amount of bacteria (food) (Escherichia coli B/r or
Klebsiella aerogenes) cultured on N agar medium and suspend in 5LP liquidmedium transferred into a triangular flask (the volume of 5LP liquid medium
is (The volume of 5LP liquid medium is about 20-30% of the volume of theflask).
2. 2.(The volume of 5LP liquid medium is about 20~30% of the volume of theflask. 2) Scrape an appropriate amount of Dictyostelium discoideum standard
strain (strain AX2) amoeba or spores and suspend them in 5LP medium.
3. 3. Shake the medium (125 rpm) in an incubator maintained at 21 °C for 4 to 5
days.
4. 4. Collect cells in the early logarithmic growth phase (cell density 1.0-3.0 ×
106 cells/mL) in a centrifuge tube.
5. Centrifuge at 300-350 g (1,500-2,000 rpm) for 2 min.
6. Discard the supernatant and resuspend the cells in cold sterile phosphatebuffer.
7. Repeat steps 5 and 6 two more times (total of three cell washes).
8. Suspend the cellular slime mold cells in appropriate buffer and use forexperiments (keep on ice).
<Memo>

6

(4) Cell preparation by aseptic culture (bacteria-free method)(Preparation) Standard strain of Dictyostelium discoideum (AX2 strain)*Since wild strains of cellular slime mold are not necessarily asepticallyculturable, confirm in advance whether the strain to be used can be cultured
aseptically.
(Strains derived from strains marked as "AX" can basically be cultured
aseptically.)
HL5 medium (Glucose 14.3 g, Bacto Proteose Peptone (BD REF No.;
211684) 14.3 g, Bacto Yeast Extract (BD REF No.; 212750) 7.15 g, Na2HPO4-12H20 1.28 g, KH2PO4 0.485 g / 1 L; autoclaved)
Antibiotic solution (1000x; streptomycin sulfate 100 mg, Benzylpenicillin
potassium 70 mg / mL) filter sterilized and added to autoclaved HL5 medium.
Commercially available solutions are also acceptable.
Folic acid (folic acid) and vitamin B12 (cyanocobalamin) solution (10,000 x;
2.0 mg folic acid, 6 mg vitamin B12 / mL) Neutralize with NaOH solution,
check that the solution is dissolved, scalp, filter sterilize, and add toautoclaved HL5 medium. Then, filter sterilize and add to autoclaved HL5
medium.
Sterile phosphate buffer (Na2HPO4-12H20 1.07 g, KH2PO4 0.96 g / 1 L;
autoclaved)
Sterile petri dish (e.g., 9 cm diameter) or sterile triangular flask, centrifuge
tube
Incubator (21°C), shaker
1. Fill a sterile petri dish with 20 mL of HL5 medium (+ antibiotics, + folic acid, +vitamin solution; if HL5 is indicated below, it has already been added).
2. Scrape and suspend cellular slime mold spores (or amoeba cells) on agar (orin other liquid medium) with a platinum ear.
3. Incubate in an incubator at 21 °C for 4 to 5 days.
4. For liquid shaking culture, scrape off the cells with a Pasteur pipette, transfer
into a sterilized triangular flask, and incubate at 21 ºC with shaking at 125 rpm.
In this case, the volume of liquid medium should be 20% of the volume of theflask. 5.
5. Collect cells in the early growth phase (cell density of 1.0 - 3.0 × 106 cells /mL) in a centrifuge tube.
6. Centrifuge at 300-350 g (1,500-2,000 rpm) for 2 min.
7. Discard the supernatant and resuspend the cells in cold sterile phosphatebuffer.
8. Repeat steps 6 and 7 two more times (total of three cell washes).
79. Suspend the cellular slime mold cells in appropriate buffer and use forexperiments (keep on ice).
<Memo>

8

Methods of preserving cellular slime molds(1) Spore preservation method using silica gel(Preparation) Cellular slime mold that has formed fruiting bodies
Silica gel (white small particles; sterilized by dry heat at 180°C for about 2hours)
Heat-sensitive sterilized glass vials (lids dried after autoclaving)
Alternatively, sterile microtubes are acceptable (must be sterile and sealed)
0.5% dry milk solution (autoclaved and refrigerated)
1. Refrigerate the newly prepared cellular slime mold stock plates (plates on
which the fruiting bodies have formed).
2. Collect the spore masses of the fruiting bodies with a sterilized platinum ear
and take a small amount (about 0.5 mL) of the dry milk solution. Repeat thisprocedure to collect as many spores as possible. (Spores can also be directly
suspended in silica gel without dry milk solution for storage.)
3. Chill the dry milk solution containing spores with ice and drop the solution intoa pre-chilled silica gel container (approximately 0.2 mL / 1 g silica gel).
4. Seal the container (with lid) and shake well to make the contents uniform, thenplace this container in another container with desiccant and keep refrigerated.
(Method of returning the product to its original state)
To restore the spore to amoeboid cells(Preparation) Spores stored in silica gel
Bacteria (food): Escherichia coli B/r or Klebsiella aerogenes
5LP agar medium (lactose 5.0 g, Bacto Peptone (BD REF No.; 211677) 5.0 g,
Bacto Agar 15.0 g / 1L Distilled H20) *20 mL per 9 cm diameter petri dishIncubator (21°C)
1. 0.5 mL 5LP agar medium (Escherichia coli B/r or Klebsiella aerogenes)
incubated overnight at 37°C in 5LP liquid medium (5LP agar medium without
Agar) Spread evenly on the agar and air-dry on a clean bench until the
moisture is removed.
2. Add silica gel stock containing the spores (or add silica gel stock to thebacterial solution) and spread the spores evenly on the agar. (Or, add a few
grains of silica gel to the bacterial solution, stir well, and spread on 5LP agar
medium.
3. Keep at 21°C for 3 to 7 days.
4. After observing the fruiting bodies, transfer to a new 5LP bacterial medium or
sterile medium (e.g., HL5).
(e.g., HL5).
9

(2) Preservation of amoeba cells using DMSO
Some mutant strains that do not produce spores cannot be preserved
using the silica gel method. Such strains should be cryopreserved asamoeboid cells in an ultra-low temperature freezer (-80 °C or lower) or in
liquid nitrogen.
(Preparation) Cellular slime mold amoebae (suspended in HL5 medium or
phosphate)
Culture grade DMSO (e.g., Hybrimax; sigma, HPLC grade can also be used),
Sterile distilled water, Sterile cryovials (sterile phosphate buffer
Na2HPO4-12H20 1.07 g, KH2PO4 0.96 g / 1 L; autoclaved), -80 °C freezer,(liquid nitrogen)1. make a 20 % DMSO solution with sterile water and store refrigerated (orfrozen) (no filter sterilization). Cool on ice until immediately before use.
2. Prepare cell suspension. Cell suspensions can be either sterile cultures ofcells in HL5 or bicellular cultures of cells suspended in sterile phosphatebuffer after centrifugation to remove bacteria. The cell density should be 1.0
x 108 cells/mL.
3. Transfer 0.5 mL of the 20 % DMSO solution cooled on ice to a cryovials.
4. Add 0.5 mL of the suspended cells and pipet gently on ice.
5.Place in a cell-freezing container (e.g., BICELL) and place in a -80 C freezer
for at least several hours. (*The goal is to slowly cool the cells to asupercooled state.) If no container is available, insert the tubes into a box
with cotton folded in between, and place the box in a -80 C freezer forseveral hours.
6.Transfer the tubes to a cryobox or similar container and store at -80 °C or in
liquid nitrogen.
<Memo>

10

（Defrozen of cells）(Preparation) Frozen cell stock stored in cryo tubes
HL5 medium or 5LP liquid medium
Bacteria (food) (Escherichia coli B/r or Klebsiella aerogenes)
5LP agar (5.0 g lactose, 5.0 g Bacto Peptone (BD REF No.; 211677), 15.0 g
Bacto Agar / 1L Distilled H20; autoclaved) *20 mL per 9 cm diameter petri
dishIncubator (21°C)
1. Remove the tube from the freezer and allow it to stand at room temperature.
2. When the entire tube is dissolved, slowly add an appropriate amount of HL5
medium or 5LP medium and transfer to a 15 mL centrifuge tube. Transfer to a
15 mL centrifuge tube.
3.Add HL5 medium or 5LP medium until the volume reaches 15 mL. Centrifuge
at 300-350 g (1,500-2,00 rpm) for 2 min.
4.Discard the supernatant, add an appropriate volume of HL5, transfer to asterile petri dish, and add an appropriate volume of HL5 medium. 5LP When
adding 5LP medium for two-member cultures, add 1 to 2 mL of the
5LP-enriched bacterial suspension to the precipitated cells and transfer 0.5
mL of the suspension to 5LP agar medium. Seed the cells onto 5LP agar
medium.
5. Incubate at 21°C for 3 to 7 days.
When returning to HL5 liquid medium, transfer the lysed cell solution directly to
15 mL of liquid medium and allow DMSO to dilute to the point that it does notinhibit cell growth. DMSO will be diluted to a level that does not inhibit cellproliferation, so it is safe to do so.
In addition, it is possible to return cells from one tube to the freezer manytimes by placing the tube in a chilled container that will never melt frozen stock,
scraping off a little of the surface of the frozen stock with a sterilized thin spatula
on a clean bench, placing the tube in fresh nutrient medium in a petri dish, andreturning it to the freezer before the tube melts. Cells can be returned from thetube as many times as desired.
<Memo>

11

Ⅲ．Observation of cellular slime molds
Cellular slime molds grow as amoeboid cells, remove nutrients, and underappropriate moisture conditions, such as on agar plates, multicellular bodyformation occurs, forming a fruiting body consisting of a spore and a stalk.
Observation of the developmental process of cellular slime molds(1) Observation of development process by two-member culture with bacteria(Preparation) Standard strain of Dictyostelium discoideum (strain AX2)
Bacteria (food): Escherichia coli B/r or Klebsiella aerogenes
5LP agar (lactose 5.0 g, Bacto Peptone (BD REF No.; 211677) 5.0 g, Bacto
Agar 15.0 g / 1L Distilled H20) *20 mL per 9 cm diameter petri dishIncubator (21°C)
1. 0.5 mL 5LP agar medium (Escherichia coli B/r or Klebsiella aerogenes)
incubated overnight at 37°C in 5LP liquid medium (5LP agar medium
without Agar) Spread evenly on the agar and air-dry on a clean bench
until the moisture is absorbed.
2. Inoculate a standard strain of Dictyostelium discoideum (strain AX2) in thecenter.
3. Incubate in an incubator maintained at 21 ºC for 4 to 5 days.
4. bacteria feed and form a plaque at the center. Near the center, starvation isin progress and fruiting bodies are observed. Slug bodies, aggregates,and proliferating amoebae are observed as one approaches theperiphery.
<Memo>

12

(2) Longitudinal observation of cellular slime mold development process (onagar)(Preparation) Standard strain of Dictyostelium discoideum (AX2 strain)
Bacteria (food): Escherichia coli B/r or Klebsiella aerogenes
5LP liquid medium (lactose 5.0 g, Bacto Peptone (BD REF No.; 211677) 5.0
g, Bacto / 1L Distilled H20)
Sterile phosphate buffer (Na2HPO4-12H20 1.07 g, KH2PO4 0.96 g / 1 L;
autoclaved)
Sterile BSS (NaCl 0.6 g, KCl 0.75 g, CaCl2-2H20 0.4 g / 1 L; autoclaved)
Incubator (21°C)
Non-nutrient agar (Bacto Agar 15.0 g / 1 L Distilled H20)
1. pure culture in HL5 at early logarithmic growth phase (1.0 - 3.0 × 106 cells /mL) or 5LP 1. Collect cellular slime molds in centrifuge tubes after
two-member culture in HL5 at early logarithmic growth stage (1.0 - 3.0 × 106
cells/mL).
2. 2.Centrifuge at 300 - 350 g (1,500 - 2,000 rpm) for 2 min.
3. Discard the supernatant and resuspend in cold sterile phosphate buffer.
4. Repeat steps 2 and 3 two more times (total of three cell washes).
5. Resuspend the cells in cold phosphate buffer or cold BSS to 1.0 x 107 cells /mL (approximate volume).
6. Pipette 5 µL of the suspension onto non-nutrient agar. At this time, be careful
not to puncture the agar.
7. Air-dry the agar until it becomes dry, and incubate it in an incubator
maintained at 21 °C.
8. Observe the agar over time under a stereomicroscope.
<Memo>

13

(3) Cellular slime mold development process over time (in phosphate buffer
solution)
Cellular slime molds do not form fruiting bodies in water but can aggregate toaggregation. Cellular level observations of cell aggregation can be madeon a cover glass and under submerged conditions.
(Preparation) Standard strain of Dictyostelium discoideum (AX2 strain)
Bacteria (food): Escherichia coli B/r or Klebsiella aerogenes
5LP agar medium (lactose 5.0 g, Bacto Peptone (BD REF No.; 211677) 5.0 g,
Bacto Agar 15.0 g / 1L Distilled H20) *20 mL per 9 cm diameter petri
dish
Sterile phosphate buffer
Incubator (21°C)
Non-nutrient agar (Bacto Agar 15.0 g / 1L Distilled H20)
1. in pure culture or 5LP in HL5 in the early log phase (1.0 - 3.0 × 106 cells/mL) 1.
Collect cellular slime molds in a centrifuge tube after two-member
culture in HL5 or 5LP liquid medium (5LP agar medium without Agar) in
the early logarithmic growth phase (1.0 to 3.0 × 106 cells/mL).
2.Centrifuge at 300 - 350 g (1,500 - 2,000 rpm) for 2 min.
3.Discard the supernatant and resuspend in cold sterile phosphate buffer.
4. Repeat steps 2 and 3 two more times (total of three cell washes). 5.
5. Resuspend the cells in cold phosphate buffer to 1.0 x 10 7 cells / mL.
6. Transfer the cells to a glass bottom dish, etc. to reach 1.0 x 10 6 cells/cm2(approximate volume).
7. Incubate the cells in an incubator at 21 °C.
8.Observe the cells under an inverted microscope.
<Memo>

14

Ⅳ．Transformation methods for cellular slime molds
The research standard strain, Dictyostelium discoideum (AX strain), has anestablished transformation method. Various vectors necessary fortransformation are also in place.
Transformation of cellular slime mold AX strain (electroporation method)(Preparation) Standard strain of Dictyostelium discoideum (strain AX2)
HL5 medium (Glucose 14.3 g, Bacto Proteose Peptone (BD REF No.;
211684) 14.3 g, Bacto Yeast Extract (BD REF No.; 212750) 7.15 g,
Na2HPO4 -12H20 1.28 g, KH2PO4 0.485 g / 1 L)
Antibiotic solution (1000x; streptomycin sulfate 100 mg, benzylpenicillin
potassium 70 mg / mL) filter sterilized and added to autoclaved HL5
medium.
Folic acid (folic acid) and vitamin B12 (cyanocobalamin) solution (10,000 x;
2.0 mg folic acid, 6 mg vitamin B12 / mL) Neutralize with NaOH solution,
check that the solution is dissolved, scalp, filter sterilize, and add toautoclaved HL5 medium. Then, filter sterilize and add to autoclaved
HL5 medium.
Electroporation buffer (Na2HPO4-12H20 0.32 g, NaH2PO4 1.40 g, Sucrose
17.12 g / 1 L; filter sterilized)
DNA for transformation
2 mm wide electroporation cuvette
Sterile healing solution (100 mM CaCl2, 100 mM MgCl2; autoclaved)
Sterile petri dish (e.g., 9 cm in diameter)
Centrifuge tube
Incubator (21°C)
1. Prepare electroporation buffer. Sterilize electroporation buffer by filter
sterilization. 2.
2. Collect pure cultures of cellular slime molds in HL5 at early logarithmic
growth stage (1.0 - 3.0 × 106 cells/mL) in a centrifuge tube.
3.Centrifuge at 300 - 350 g (1,500 - 2,000 rpm) for 2 min.
4. Discard the supernatant, add cold electroporation solution to 5.0 × 107
cells/mL, suspend gently, and place on ice.
5. Transfer 400 μL to a cuvette, add 10 to 20 μg of DNA, and suspend gently.
6. Place the cuvette on ice for 30 minutes.
15

7. Apply electrical stimulation (e.g., BTX EC). (e.g., BTX ECM830, 500 V, 100
μsec, ×10: Biorad Gene Pulser, 0.85 kV, 25 μF, ×2: The lethality ofelectrical stimulation should not be less than 25%. (Usually about
50 %.) 8.
8. Allow to stand on ice for 5 minutes.
9. During this time, drop 4 µL of the healing solution onto a sterile petri dish.
10. Transfer the electrically stimulated cell suspension to a sterile petri dishand lightly suspend it in the healing solution.
11. Incubate at 21°C for 10 to 15 minutes.
12. Slowly add 15 mL of HL5 medium and incubate at 21 °C. If you wish to
separate the cells by colonies, do so. If you wish to separate the cellsby colony, dispense into a 96-well sterile plate at this point. In this case,add 40 mL of HL5 medium and dispense 100 µL / well.
13. After 12 to 24 hours, add 15 μL of 1,000-fold concentration of the selecteddrug (G418; 20 mg / mL, Blasticidin S 10 mg / mL: filter-sterilized); if thecells have been aliquoted into 96 wells, the Final concentration (G 418;
20 μg / mL, Blasticidin S 10 μg / mL).
14. After 4-5 days, replace the medium with the selective agent.
If transformation is successful, colonies will be observed after 10-14
days for G418 and 7-10 days for Blasticidin S.
<Memo>

16

Transformation of cellular slime mold AX strain (calcium phosphate
method)(Preparation) Standard strain of Dictyostelium discoideum (strain AX2)
HL5 medium (Glucose 14.3 g, Bacto Proteose Peptone (BD REF No.;
211684) 14.3 g, Bacto Yeast Extract (BD REF No.; 212750) 7.15 g,
Na2HPO4 -12H20 1.28 g, KH2PO4 0.485 g / 1 L)
Antibiotic solution (1000x; streptomycin sulfate 100 mg, benzylpenicillin
potassium 70 mg / mL) filter sterilized and added to autoclaved HL5
medium.
Folic acid (folic acid) and vitamin B12 (cyanocobalamin) solution (10,000 x;
2.0 mg folic acid, 6 mg vitamin B12 / mL) Neutralize with NaOH solution,
check that the solution is dissolved, scalp, filter sterilize, and add toautoclaved HL5 medium. Then, filter sterilize the solution and add it toautoclaved HL5 medium.
Bis-Tris HL5 medium (2.1 g Bis-Tris in HL5, pH 7.1 with HCl, filter
sterilized)
1.25 M CaCl2 solution (filter sterilized)
18 % glycerol solution in 1 x HBS
2 x HBS solution (4.0 g NaCl, 0.18 g KCl, 0.05 g Na2HPO4, 2.5 g HEPES,
0.5 g glucose / 1 L distilled water, pH to 7.1 with NaOH: filter sterile.)
DNA for transformation
Sterile petri dish (e.g., 9 cm in diameter)
Centrifuge tube
Incubator (21°C)
1. Sow 10 mL of HL5 pure culture of cellular slime molds in early logarithmic
growth phase (1.0 - 2.0 × 106 cells / mL) in a sterile petri dish.
2. Incubate at 21°C for 30 minutes.
3. Carefully remove the HL5, leaving the cells, and gently add 12.5 mL of
Bis-Tris HL5 and incubate at 21°C for 30 minutes.
4. Prepare 540 μL of 1x HBS solution containing 10-20 μg DNA.
5. Add 60 μL of 1.25 M CaCl2 to make 600 μL.
6. Gently remove Bis-Tris HL5 from the petri dish and slowly add a drop of DNA
solution from the center of the petri dish.
7. Cover the petri dish and incubate at 21°C for 30 minutes.
8. Gently add 12.5 mL of Bis-Tris HL5 and incubate at 21°C for 4 hours.
9. Gently remove Bis-Tris HL5 from the petri dish and slowly add 4 mL of 18%
glycerol solution in 1x HBS.
10. Incubate at 21°C for exactly 5 minutes.
1711. Aspirate off the glycerol solution in 1x HBS and add 15 mL of HL5 medium.
12. After 12 to 24 hours, add 15 μL of 1,000-fold concentration of the selecteddrug (G418; 20 mg/mL, Blasticidin S 10 mg/mL: filter sterilized). 418; 20 μg /mL, Blasticidin S 10 μg / mL).
13. After 4-5 days, replace the medium with the selective agent.
If transformation is successful, colonies will be observed after 10-14 days forG418 and 7-10 days for Blasticidin S.
<Memo>

18

Ⅴ．Gene amplification by PCR method
It is possible to amplify cellular slime mold genes by PCR using the generalgenome as a template, but the cellular slime mold genome is AT-rich andrequires slight modification of reaction conditions.
(Preparation) Dictyostelium discoideum standard strain (AX strain)
genomic DNA
Two oligo DNA
Heat-resistant polymerase (e.g. TOYOBO, KOD plus)
1.5 mL microtubes, microtubes dedicated for PCR
1. prepare DNA as template for PCR (genomic DNA, plasmid DNA, etc.)(genomic DNA, plasmid DNA, etc.) 2.
2. Use a high-fidelity thermostable enzyme (e.g., TOYOBO, KOD plus).
(e.g., TOYOBO KOD series, Takara Prime Star, Finnzyme Phusion, etc.)
10 × KOD plus ver2 buffer
3.0 μLdNTP mix (2.0 mM)
3.0 μLMgSO4 (25 mM)
3.0 μL
Primers （10μM）
5'-end3'-endTemplate(plasmid or genomeDNA)

1.5 μL
1.5 μL0.1 ng (plasmid)

KOD plus

10 ng(genome)
0.3 μL30.0 μL with dH20

up to

Thermal cycler conditions; 94°C, (94°C - 2 min, 50-60°C - 20 sec, 65°C - fulllength kbp x 1 min) x 25, 65°C - full length kbp x 1 min, Hold
Electrophoresis of 3.5 μL of the reaction solution is performed to confirm thatfragments of the desired length have been amplified. If necessary, purify andextract the genomic fragments.
If amplification is not successful, first perform a control experiment to confirm
that the reagents have not deteriorated. In the case of amplification using
genomic DNA as the template, adding 5% DMSO (final concentration) to thereaction solution may stabilize and amplify the reaction.

19

Ⅵ．Preparation of genomic DNA(Preparation) Standard strain of Dictyostelium discoideum (AX strain)
Centrifuge tube
Centrifuge, tabletop cooling centrifuge
Incubator (60°C and 37°C)
Sterile phosphate buffer (Na2HPO4-12H20 1.07 g, KH2PO4 0.96 g / 1 L)
STE buffer (10 mM Tris-HCl, 10 mM EDTA, 400 mM NaCl, pH 8.0)
Proteinase K (10 mg / mL)
10% SDS solution
Sterile distilled water
1.5 mL micro tube
TE-saturated phenol
Chloroform
99.5 % ethanol
70.0 % ethanol
Ribonuclease A solution (10 mg / mL)
1. collect 2.0 - 5.0 × 107 cells of acellular slime mold cells in a centrifuge tube.
2. Centrifuge at 300 - 350 g (1,500 - 2,000 rpm) for 2 min.
3. Discard the supernatant and add 450 μL of STE buffer to suspend the cells in
1.5 mL microtubes.
4. Add 50 µL of 10 % SDS solution, cover with a lid, and invert up and down 5-6
times.
5. Add 10 μL of Proteinase K solution and invert in the same manner.
6. Incubate at 60 °C for 1-2 hours, followed by incubation at 37 °C for 2 hours toovernight.
7. Add 500 μL of TE-saturated phenol/chloroform (1:1) solution, cover with a lid,
invert up and down 5-6 times, and centrifuge at 15 krpm for 10 minutes.
8. Carefully transfer 400 µL of the supernatant to a new micro tube without
removing the turbid white intermediate layer.
9. Repeat steps 7 and 8 up to a total of three times (if necessary).
10. Add 1.0 mL of cold 99.5% ethanol and cool at -20 °C for 30 minutes.
11. Centrifuge at 15 krpm for 10 minutes. 12.
12. Add 1.0 mL of 70% ethanol to the precipitate and rinse the precipitate.
13. Centrifuge gently and discard the supernatant.
14. Repeat the procedure of 12.13.
15. Air dry for 10-15 minutes, add 100 μL of sterile distilled water, and suspend
by tapping.
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16. Add 1 μL of Ribonuclease A solution and incubate at 37°C for 1 to 2 hours.
17. Store the genomic DNA in a cool place because it will be fragmented byfreezing and thawing.
<Memo>

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Ⅶ．Methods for isolating cellular slime molds from soil
Isolate cellular slime molds from the soil in moist areas under shady trees orfallen leaves.
(Preparation) Agar medium for separation (Glucose 1.0 g, Bacto Peptone (BDREF No.; 211677) 1.0 g, K2HPO4, 1.0 g, KH2PO4, 1.5 g, MgSO4 1.0 g, Agar
15.0 g / 1.0 L Distilled H2O) 20 mL per petri dish (same as below)
N agar medium (10.0 g glucose, 10.0 g Bacto Peptone (BD REF No.; 211677),
0.96 g Na2HPO4-12H20, 1.44 g KH2PO4, 15 g Bacto Agar / 1 L Distilled H20)
5LP agar medium (lactose 5.0 g, Bacto Peptone (BD REF No.; 211677) 5.0 g,
Bacto Agar / 1 L Distilled H20)
Bacteria (food) (Escherichia coli B/r or Klebsiella aerogenes)
Sterile distilled water
Sterile gauze
1. Collect the surface layer of soil.
2. Add 90 mL of sterile distilled water to 10 g of soil and stir well.
3. Strain through two layers of sterile gauze and add 7.5 mL of sterile distilledwater to 5 mL of filtrate.
4. Add 0.4 mL of bacteria-suspended sterile distilled water (scrape bacteria off
the agar plate with a platinum ear to suspend them) to 0.5 mL of the diluted
filtrate from 3. Add 0.4 mL of sterile distilled water (scrape the bacteria fromthe agar plate using a platinum ear, etc.) and spread evenly on the isolation
medium.
5. Incubate at 21 °C.
6. After 3 to 7 days, inoculate the fruiting bodies emerging on the medium onto
5LP agar coated with the bacterial suspension and incubate at 21 °C.
<Memo>

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Ⅷ．How to provide cellular slime mold strains from
NBRP
Cellular slime mold strains are collected and stored at the National
BioResource Project (NBRP) Cellular Slime Molds and are available for a fee(although they can be provided free of charge for educational purposes atsecondary schools, etc.).
How to apply for provision of cellular slime mold strains
https://nenkin.nbrp.jp/

Click distribution

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Information required for application
E-mail address
Credit card (for payment)
Delivery method (by FEDEX only)<Memo>

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Ⅸ．Reference sites on cellular slime molds
１．http://dictybase.org/
International site for cellular slime molds. A community site focusing on
Dictyostelium discoideum, a research standard strain of cellular slime molds.
Genome and gene information search, cellular slime mold laboratory, andinformation on the International Society for Cellular Slime mold Research canalso be obtained from this site.
2. http://dicty.jp/
The homepage of the Japanese Society for Cellular Mycology, where you canobtain information on the Japanese Society for Cellular Mycology, which wasestablished in 2011, as well as information on its annual meetings. Membershipis accepted at any time. There is also a link to the homepage of Japanesecellular slime mold researchers.
3. http://www.biol.tsukuba.ac.jp/hidekuwayama/index.html
This is the homepage of the Cellular Mycobacteria Laboratory at the Universityof Tsukuba. It introduces research contents and recruits graduate students.

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Ⅹ．Reference books, bibliography
1. cellular slime molds: new developments in research~Model Organisms, Drug Discovery Resources, Bio~.
Editors Tomoaki Abe and Yasuo Maeda, IPC (2012)
Revised edition of Model Organisms: Cellular slime molds published in
2000. Includes a chapter on experimental treatment of cellular slime molds.
2. Survival of Cellular Slime Fungi: Skilled Response to Environmental Stress(New Life Science Library: Rediscovering Organisms), H. Urushihara,
Science Inc.
An introductory book on cellular slime molds that explains the ecology andresearch on cellular slime molds in an easy-to-understand manner for
beginners.
3. Powerful Slime Fungi, by Yasuo Maeda, Tohoku University Press
An easy-to-understand explanation of the development of cellular slime
molds based on his own research.
4. Dictyostelium.
Richard H. Kessin.
Cambridge University Press, 2001
A review of cellular slime mold research written in English.
5. Differentiation in social amoebae.
John T Bonner.
Sci. Am. 201, 152-162, 1959
A review of cellular slime molds for the general public written in old English.
6. Transformation of Dictyostelium discoideum with plasmid DNA.
Gaudet P, Pilcher KE, Fey P, Chisholm RL.
Nat Protocols. 2(6):1317-1324, 2007.
7. A new set of small, extrachromosomal expression vectors for Dictyostelium
discoideum.
Veltman DM, Akar G, Bosgraaf L, Van Haastert PJ.
Plasmid. 61(2):110-118, 2009.

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A paper on various gene expression vectors of cellular slime molds. Vectors
are available at NBRP Cellular Slime molds.
7. A versatile set of tagged expression vectors to monitor protein localizationand function in Dictyostelium.
Dubin M, Nellen W.
Gene. 465(1-2):1-8, 2010.
Article on various gene expression vectors, especially fluorescent protein
fusion expression vectors, in cellular slime molds. The vectors are available
at NBRP Cellular Slime molds.
8. A user's guide to restriction enzyme-mediated integration in Dictyostelium.
Guerin NA, Larochelle DA.
J. Muscle Res. Cell Motil. 2002;23(7-8):597-604.
Review of methods for generation, isolation and genetic analysis of mutant
strains by gene insertion. The necessary vectors are available at NBRP
Cellular Motil. 2002;23(7-8):597-604.
10. PCR-mediated generation of a gene disruption construct without the use of
DNA ligase and plasmid vectors.
Hidekazu Kuwayama, Shinji Obara, Takahiro Morio, Mariko Katoh, Hideko
Urushihara and Yoshimasa Tanaka.
Nucleic Acids Research. 30 (2):e2, 2002
A paper on the production of gene disruption vectors by the PCR method.

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ⅩⅠ．Medium and others composition table
HL5 medium

Proteose Peptone（sterlized by autoclave）(BD REF No.; 211684)

Yeast Extract

14.3 g7.15 gBD REF No.; 212750

GlucoseKH2PO4
Na2HPO4・12H20
pure water
5LP medium（sterlized by autoclave）(for 5LP agar medium.
+ 15.0 g Bacto agar)

LactoseBacto Peptone(for N agar medium.
+ 15.0 g Bacto agar)

Separation medium（sterlized by autoclave）

5.0 g
5.0 g

(BD REF No.; 211677)

to 1 liter

pure water
N medium
バイ内（sterlized by autoclave）

14.3 g0.485gg
1.28g
to 1 liter

GlucoseBacto Peptone

10.0 g10.0 g(BD REFNo.; 211677)

KH2PO4
Na2HPO4・12H20
pure water
GlucoseBacto Peptone

1.44 g0.96 gto 1 liter
1.0 g
1.0 g

(BD REFNo.; 211677)

KH2PO4
Na2HPO4
MgSO4
Bacto agar
pure water
phosphate buffer（sterlized by autoclave）

Na2HPO4·12H20
KH2PO4

autoclave）(for N agar medium.+ 15.0
g Bacto agar)

1.07 g0.96 gto 1 liter

蒸留水
BSS （sterlized by

1.0 g
1.5 g
1.0 g
15.0 gto 1 liter

0.60 g

NaCl
KCl
CaCl2・2H2O
pure water28

0.75 g0.40 gto 1 literElectroporation Buffer(sterilized through filter)

Healing solution（sterlized by autoclave）

NaH2PO4·12H20

NaH2PO4
Sucrosepure water

CaCl2・2H2O

MgCl2
pure water

0.32 g1.40 g17.12 gto 1 liter1.51 g0.952 gto100 mL〓 caution 〓Please refrain from eating, drinking, and smoking in therecombination laboratory.
<memo>Name© 2022 H. Kuwayama

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