1. project ppt for mid review

8/7/2019 1. Project Ppt for Mid Review

1/19

PROJECT TOPIC:MOLECULAR

CHARACTERISATION OF

MICROBES FROM EFFLUENTS OFDYE INDUSTRY

-By

Akanksha Kalra

07BBT021

B.Tech Biotechnology

Guide:Dr. T.B Sridharan


2/19

OBJECTIVE

1. Cultivating microbes from the effluents of dyeindustry.

2. Isolation of plasmid and genomic DNA from

inoculated samples.

3. Microbial characterization & Biochemical tests.

4. Isolation of individual colonies from inoculatedcultures.

5. Establish the types of plasmids found byelectrophoresis.

6. Analysis of protein profile.


3/19

MATERIALS & METHODS

For Inoculation:a. Luria Bertani Hi Veg Broth

b. Sample for inoculation

For Isolation:

a. Solution A ( At pH-8.0 EDTA, Glucose and Tris HCl) (to beautoclaved and stored at 4C)

b. Solution B [Sodium Hydroxide (0.2 N), Sodium DodecylSulphate(1%) , freshly prepared]

c. Solution C ( Sodium potassium acetate, glacial acetic acid anddistilled water )

d. Ice chips

e. 100% Alcohol

f. 70 % Alcohol

g. Loading dye


4/19

For Gel Electrophoresis:

a. 1 X TAE bufferb. TE buffer

c. Agar (0.7 %)

d. Electrophoresis apparatus

e. Ethidium Bromide dye

For Petri Plate:

1. Sterilized plates

2. LB agar medium

For Dilutions:

a. LB medium

b. Inoculated Cultures


5/19

SAMPLES USED


6/19

PRINCIPLES

For isolation of plasmid

1. Glucose gives osmotic shock that leads to therupture of cell wall and membrane.

2. EDTA inhibits nucleases.

3. Solution B lyses the cell completely.

4. Alkaline pH of Solution B denatures

chromosomal DNA but not the covalentlyclosed circular plasmid DNA.

5. Alkaline Lysis method was used for isolation.


7/19

5. Solution C neutralizes the alkaline pH

6. Solution C precipitates protein and forms SDS-

protein complex

7. Centrifugation pellets the Protein DNA

aggregates


8/19

INOCULATIONOFCULTURE

Samples were inoculated in sterilized LB medium.


9/19

INOCULATIONOF PETRI PLATES


10/19

PROCEDUREFORISOLATION

1. Take 2ml of culture in an eppendorf andcentrifuge at 8,000 rpm (at 4C) for 10 mins.

2. Discard the supernatant and let the supernatantdry properly. If required with tissue

3. Store in an ice box for 5 mins

4. Add 200 l of Solution A. Tap the eppendorf tomix the solution.

5. Store in ice for 5 mins.6. Add 150 l of freshly prepared Solution B. Mix

by inverting the eppendorf slowly for 2 mins.Store in ice for 5 mins.


11/19

6. Add 200 l of Solution C and mix by inverting

the eppendorf slowly. Store in ice for 5 mins.

7. Spin in the centrifuge at 10,000 rpm for 10

mins at 4C.

8. Take the supernatant in a fresh eppendorfwithout disturbing the precipitate and add

equal amount of 100% alcohol to it.

9. Spin down at 14,000 rpm for 10 mins at 4C.10. Discard the supernatant. Add 150 l of 70%

alcohol to wash the precipitate.


12/19

11. Spin down at 14,000 rpm for 10 mins at 4C.

12. Discard the supernatant and let theprecipitate dry till the alcohol smell is fully

lost.

13. Add 20 l of TE buffer to the precipitate.14. Add 8 l of gel loading buffer.

15. Load 15 l of the sample to the gel.


13/19

PROCEDUREFOR GEL

ELECTROPHORESIS

1. To make the gel take 500 mg of agarose in aflask.

2. Add 50 ml of TAE buffer to it and heat it till the

agarose fully dissolves.3. Let the solution come down to almost room

temperature (40C) and then add 2 l ofethidium bromide dye to it.

4. Pour that solution into an electrophoresisapparatus and let it solidify.

5. After loading the samples run the apparatus at50 V for 45 mins.


14/19

Isolated Plasmids


15/19


16/19

RESULTS OBTAINED

Some organisms were obtained for the inoculated Petriplates.

Petri plates got contaminated many times.


17/19

Only plasmid obtained while isolation.

The 1st and the 3rd picture show similar kind of plasmid but

the 2nd and 4th picture shows different plasmids.

Only natural plasmids obtained. Functions are unknown.


18/19

On inoculation 8 colonies of organisms were

found.

Only 3 colonies showed plasmids.

Experiment was repeated for the other 5

colonies but no presence of plasmid DNA

found.

Concluding that only 3 colonies will show the

presence of plasmid DNA.

From them 3 plasmids were isolated.


19/19

WORKTOBEDONE

Plasmid is yet to be characterized.

Analysis of the protein profile of theorganisms.

Total cellular protein

Membrane protein

Proteins present in the effluents are

responsible for the survival of organisms.

SDS-PAGE to be performed to characterizethese protein.

1. project ppt for mid review

Documents