Summer Training PresentationsDepartment of Molecular and Human Genetics

Understanding of the life cycle of Dictyostelium

discoideum and cloning of putative gene

Mentor:

Dr. Shweta Saran

Developmental Biology Lab

SLS JNU, New Delhi.

Presented By:

Bhupender Verma

M.Sc. (F) MHG BHU

Introduction Dictyostelium discoideum are single

cellular organisms

Live on decaying logs and feed on

bacteria

Dictyostelium discoideum follow asexual

cycle-

• when there is plenty of food available

• Live solitary

• Are haploid

• Reproduce by binary fission

Also called as “Social amoebae”

During starvation organisms aggregate

and migrating slug is formed that

culminates in a fruiting body to release

spores

Figure: Scott F. Gilbert-Developmental Biology 8th edition

Domain – Eukarya

Kingdom- Amoebozoa

Superphylum- Conosa

Phylum- Mycetozoa

Class- Dictyostelia

Order- dictyosteliida

Family- Dictyosteliidae

Genus- Dictyostelium

Species- D. discoideum

Classification:

Life cycle

In lab Dicty are grown in HL5 liquid media at 22ºC.

In nutrient rich conditions Dicty divide by binary fission

and remain solitary.

For studying their development a concentrated solution

is transferred to the NNA plates.

In the absence of nutrients, these myxomoebae join

together to form moving streams of cells converging at a

central point.

Different developmental stages that can be observed:

• Tight aggregate

• Migrating slug (pseudoplasmodium or grex) – slimy

sheath encased

• Fruiting body

Vegetative loose aggregate mound migratory slug

Early culminant fruiting body

Cloning of the putative gene:

Strategy:

• Design the primers

• Gradient PCR for optimizing annealing conditions

• Standard PCR for obtaining amplified insert

• Digestion of vector and insert with HindIII and PstI

• Liagtion of insert to the vector

•Transform bacteria with recombinant vector

• Isolate cloned plasmid and digest for checking if insert is

desirable

Gradient PCR for optimizing annealing conditions:

5 kb

1.5 kb

500 bp

5 kb

1.5 kb

500 bp

Design the primers

• Gradient PCR

• Standard PCR

• Digestion with HindIII

and PstI

• Liagtion

• Bacterial

transformaton

• Isolate cloned plasmid

and digest for checking

if insert is desirable

1 2 3 4 5 6 7 8 9 10 M 11 12 13 14 15 16 17 18 19 20

21 22 23 24 25 26 27 28 29 30 M 31 32 33 34 35 36

Standard PCR for obtaining amplified insert:

5 kb

1.5 kb

500 bp

Design the primers

• Gradient PCR

• Standard PCR

• Digestion with HindIII

and PstI

• Liagtion

• Bacterial

transformaton

• Isolate cloned plasmid

and digest for checking

if insert is desirable

pDrive cloning vector-

Design the primers

• Gradient PCR

• Standard PCR

• Digestion with HindIII

and PstI

• Liagtion

• Bacterial

transformaton

• Isolate cloned plasmid

and digest for checking

if insert is desirable

Digestion of vector and insert with HindIII and PstI

5 kb

1.5 kb

500 bp

Design the primers

• Gradient PCR

• Standard PCR

• Digestion with HindIII

and PstI

• Liagtion

• Bacterial

transformaton

• Isolate cloned plasmid

and digest for checking

if insert is desirable

Checking insert from fallout size:

5 kb

1.5 kb

500 bp

5 kb

1.5 kb

500 bp

Design the primers

• Gradient PCR

• Standard PCR

• Digestion with HindIII

and PstI

• Liagtion

• Bacterial

transformaton

• Isolate cloned plasmid

and digest for checking

if insert is desirable

Cloning two segments of a putative gene

in pDrive vector for making Knockout-

1. Insert 1 – obtained from digestion (HindIII & NotI) of cloning

vector as pop out

2. pDrive vector – already had other insert so digest with (HindIII &

NotI) & obtain vector-BSR

3. Ligate insert to vector

4. Transform bacteria

5. Grow bacteria and isolate plasmid

6. Digest plasmid for checking proper ligation

7. Proceed with the colony that had proper insert

8. PCR of insert 2

9. Digest insert 2

10.Ligation

11.Transformation

12.Isolate plasmid and check

1. Insert 1-as pop out

2. pDrive -had other

insert

3. Ligation of insert 1

4. Transform E.coli

5. Grow E.coli &

isolate plasmid

6. Digest plasmid for

check

7. Proceed with sample

having proper insert

8. PCR of insert 2

9. Digest insert 2

10.Ligation

11.Transformation

12.Isolate plasmid and

check

Digestion with HindIII & NotI

+BSR

5 kb

1.5 kb

500 bp

Check after elution from

agarose gel:

1. Insert 1-as pop out

2. pDrive -had other

insert

3. Ligation of insert 1

4. Transform E.coli

5. Grow E.coli &

isolate plasmid

6. Digest plasmid for

check

7. Proceed with sample

having proper insert

8. PCR of insert 2

9. Digest insert 2

10.Ligation

11.Transformation

12.Isolate plasmid and

check

1. Insert 1-as pop out

2. pDrive -had other

insert

3. Ligation of insert 1

4. Transform E.coli

5. Grow E.coli &

isolate plasmid

6. Digest plasmid for

check

7. Proceed with sample

having proper insert

8. PCR of insert 2

9. Digest insert 2

10.Ligation

11.Transformation

12.Isolate plasmid and

check

For checking again digest

with HindII & NotI

5 kb

1.5 kb

500 bp

5 kb

1.5 kb

500 bp

1. Insert 1-as pop out

2. pDrive -had other

insert

3. Ligation of insert 1

4. Transform E.coli

5. Grow E.coli &

isolate plasmid

6. Digest plasmid for

check

7. Proceed with sample

having proper insert

8. PCR of insert 2

9. Digest insert 2

10.Ligation

11.Transformation

12.Isolate plasmid and

check

For checking again digest

with HindII & NotI

5 kb

1.5 kb

500 bp

5 kb

1.5 kb

500 bp

After insert1 what do we have?

pDrive-Insert1-BSR

1. Insert 1-as pop out

2. pDrive -had other

insert

3. Ligation of insert 1

4. Transform E.coli

5. Grow E.coli &

isolate plasmid

6. Digest plasmid for

check

7. Proceed with sample

having proper insert

8. PCR of insert 2

9. Digestion of insert 2

10.Ligation

11.Transformation

12.Isolate plasmid and

check

5 kb

1.5 kb

500 bp

Digestion with HindIII &

BamhI:

1. Insert 1-as pop out

2. pDrive -had other

insert

3. Ligation of insert 1

4. Transform E.coli

5. Grow E.coli &

isolate plasmid

6. Digest plasmid for

check

7. Proceed with sample

having proper insert

8. PCR of insert 2

9. Digestion of insert 2

10.Ligation

11.Transformation

12.Isolate plasmid and

check

Now we are ready with a DNA segment that is “insert1-BSR-insert2”This insert can be used for knocking out this putative genesAnd my insert is still under study

Result:

pDrive Insert1-BSR-Insert2

RICARDO ESCALANTE and JUAN J. VICENTE- Dictyostelium

discoideum: a model system for differentiation and patterning(2000)

L. Eichinger, J. A. Pachebat, G. Glockner- The genome of the social

amoeba Dictyostelium discoideum (2005)

Developmental biology- Scott F. Gilbert- 8th edition.

References:

Thank you!