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Genetics

Microbial Genetics

Plant Genetics

Microbial Genetics

Staphylococcus Aureus

Nocardis Otitidis

Pseudomonas Aeruginosa

Serratia Marcescens

Enterobacter Aerogenes

Klebsiella Pneumoniae

Microbial Identification
(16S rDNA)

Pseudomonas aeruginosa ATCC 10145


PROJECT REPORT

NAME OF SCIENTIST:
Mr. Mahendra Kumar Trivedi

SERVICE:
DNA FINGERPRINTING SERVICE (BY RAPD)

SERVICE NO.:
DF 54

Project Title	DNA Fingerprinting by RAPD Analysis
Cat#:	CSER 48
Name of Scientist:	Mr. Mahendra Kumar Trivedi
Institute:	Life Energies ltd, Mumbai.
Sample name:	Pseudomonas aeruginosa ATCC 10145

Objective:

The objective of this project is to Genotype five samples (CONTROL Versus TREATED- A, A1, B, B1) of the same line with five primers on Pseudomonas aeruginosa ATCC 10145.

Job Requirement:

Isolation of genomic DNA from Pseudomonas aeruginosa samples.
Generating RAPD-Fingerprinting profiles showing Polymorphic bands with five RAPD primers

Gram-positive material and primers

Three series of Inoculums (Control, Series A and Series B) were prepared for Pseudomonas aeruginosa. The Control series was procured from Bangalore Genei while the Series "A" and series 'B' were handed over to Mr. Mahendra Kumar Trivedi in sealed tubes on 23rd January 2008 during his visit to our R&D facility at Bangalore. On the same day, we provided one isolated room to Mr. Trivedi where he subjected the Series 'A' and Series 'B' to his treatment modality.

Whilst handing over these cultures to Mr. M. K. Trivedi for treatment purposes, optimum precautions were taken to avoid the contamination.

After the presumed treatment, Mr. Trivedi returned the samples to Ms. Bindu Damodaran (Designation - Executive Manager - Microbiology) in the same condition as handed over to him in an hour's time on the same day.

We did not know the treatment modality as it was kept confidential throughout the project.

MATERIALS & METHODS:

Sub culturing of treated samples.

1% Inoculum from two treated tubes (series A and series B) was taken and inoculated to fresh 5 ml medium and incubated at 37 C with 160 rpm for 18 hrs and labelled as series A1 and B1 respectively. The cultures were spun down and genomic DNA was isolated.

Genomic DNA was isolated using Ultrapure Genomic DNA prep Kit (Cat# KT 83).

Steps Involved:

Genomic DNA was isolated from Pseudomonas aeruginosa samples (Control, Treated A, Treated B provided by Mr. M. K. Trivedi and sub cultured samples Treated A1, Treated B1 provided by Bangalore Genei) using Ultrapure Genomic DNA prep Kit (Cat# KT 83).
Performed RAPD reactions using five RAPD primers (RBA Series used separately) to find polymorphism between control and treated samples under PCR conditions as mentioned:

Cycle condition:

94°C	94°C	35°C	72°C	94°C	38°C	72°C	72°C
7 min	60 sec	60 sec	120 sec	60 sec	60 sec	90 sec	7 min
	8 cycles			35 cycles

12 of PCR products were loaded on to 1.5 % agarose gel and resolved by electrophoresis. The gel was subsequently stained with ethidium bromide and viewed under UV-light. Photographs were documented subsequently (attached).
Polymorphic DNA bands are marked by arrows on the copy of the report.

Fig. 1. RAPD Profile of Pseudomonas aeruginosa samples Generated using Genei
RAPD primer RBA 8A, RBA 13A & RBA 20A.

Lane Description:

1: Control
2: Treated A
3: Treated A.1
4: Treated B
5: Treated B.1
M:100 bp Ladder (GeneiTM) Cat # RMBD19S;

Fig. 2. RAPD Profile of Pseudomonas aeruginosa samples Generated using Genei
RAPD primer RBA 23A & RBA 25A.

Lane Description:

1: Control
2: Treated A
3: Treated A.1
4: Treated B
5: Treated B.1
M:100 bp Ladder (GeneiTM) Cat # RMBD19S;

Calculation:
	A/ Bx100 = Percent polymorphism

Where,
	A= Polymorphic bands in treated plant B= No of bands in control

Sl. No	Bands scored	Description	Primer
1	16	Six bands are Common to Control and Treated samples; No band is unique to Control sample; One band is unique to Treated sample A; Three bands are unique to Treated sample A1; No band is unique to Treated sample B; No band is unique to Treated sample B1.	RBA 8A
2	16	Five bands are Common to Control and Treated samples; Two bands are unique to Control sample; Two bands are unique to Treated sample A; Two bands are unique to Treated sample A1; Two bands are unique to Treated sample B; Two bands are unique to Treated sample B1.	RBA 13A
3	12	Six bands are Common to Control and Treated samples; One band is unique to Control sample; Two bands are unique to Treated sample A; No band is unique to Treated sample A1; No band is unique to Treated sample B; No band is unique to Treated sample B1.	RBA 20A
4	14	Four bands are Common to Control and Treated samples; Two bands are unique to Control sample; Two bands are unique to Treated sample A; Three bands are unique to Treated sample A1; Two bands are unique to Treated sample B; Two bands are unique to Treated sample B1.	RBA 23A
5	7	Four bands are Common to Control and Treated samples; One band is unique to Control sample; Two bands are unique to Treated sample A; No band is unique to Treated sample A1; Two bands are unique to Treated sample B; No band is unique to Treated sample B1.	RBA 25A

RESULTS & DISCUSSION:

Control & Treated sample A

1. The level of true polymorphism between Control & Treated sample A was evaluated at 41 % with RBA 8A primer.

2. The level of true polymorphism between Control & Treated sample A was evaluated at 30 % with RBA 13A primer.

3. The level of true polymorphism between Control & Treated sample A was evaluated at 44 % with RBA 20A primer.

4. The level of true polymorphism between Control & Treated sample A was evaluated at 66 % with RBA 23A primer.

5. The level of true polymorphism between Control & Treated sample A was evaluated at 71 % with RBA 25A primer.

Control & Treated sample A1

6. The level of true polymorphism between Control & Treated sample A1 was evaluated at 75 % with RBA 8A primer.

7. The level of true polymorphism between Control & Treated sample A1 was evaluated at 30 % with RBA 13A primer.

8. The level of true polymorphism between Control & Treated sample A1 was evaluated at 0 % with RBA 20A primer.

9. The level of true polymorphism between Control & Treated sample A1 was evaluated at 77 % with RBA 23A primer.

10. The level of true polymorphism between Control & Treated sample A1 was evaluated at 0 % with RBA 25A primer.

Control & Treated sample B

11. The level of true polymorphism between Control & Treated sample B was evaluated at 41 % with RBA 8A primer.

12. The level of true polymorphism between Control & Treated sample B was evaluated at 30 % with RBA 13A primer.

13. The level of true polymorphism between Control & Treated sample B was evaluated at 0 % with RBA 20A primer.

14. The level of true polymorphism between Control & Treated sample B was evaluated at 55 % with RBA 23A primer.

15. The level of true polymorphism between Control & Treated sample B was evaluated at 28 % with RBA 25A primer.

Control & Treated sample B1

16. The level of true polymorphism between Control & Treated sample B1 was evaluated at 41 % with RBA 8A primer.

17. The level of true polymorphism between Control & Treated sample B1 was evaluated at 30 % with RBA 13A primer.

18. The level of true polymorphism between Control & Treated sample B1 was evaluated at 0 % with RBA 20A primer.

19. The level of true polymorphism between Control & Treated sample B1 was evaluated at 55 % with RBA 23A primer.

20. The level of true polymorphism between Control & Treated sample B1 was evaluated at 57 % with RBA 25A primer.

Treated sample A & Treated sample A1

21. The level of true polymorphism between Treated sample A & Treated sample A1 was evaluated at 0 % with RBA 8A primer.

22. The level of true polymorphism between Treated sample A & Treated sample A1 was evaluated at 40 % with RBA 13A primer.

23. The level of true polymorphism between Treated sample A & Treated sample A1 was evaluated at 25 % with RBA 20A primer.

24. The level of true polymorphism between Treated sample A & Treated sample A1 was evaluated at 77 % with RBA 23A primer.

25. The level of true polymorphism between Treated sample A & Treated sample A1 was evaluated at 0 % with RBA 25A primer.

Treated sample B & Treated sample B1

26. The level of true polymorphism between Treated sample B & Treated sample B1 was evaluated at 0 % with RBA 8A primer.

27. The level of true polymorphism between Treated sample B & Treated sample B1 was evaluated at 50 % with RBA 13A primer.

28. The level of true polymorphism between Treated sample B & Treated sample B1 was evaluated at 0 % with RBA 20A primer.

29. The level of true polymorphism between Treated sample B & Treated sample B1 was evaluated at 40 % with RBA 23A primer.

30. The level of true polymorphism between Treated sample B & Treated sample B1 was evaluated at 50 % with RBA 25A primer.

Treated sample A & Treated sample B

31. The level of true polymorphism between Treated sample A & Treated sample B was evaluated at 0 % with RBA 8A primer.

32. The level of true polymorphism between Treated sample A & Treated sample B was evaluated at 0 % with RBA 13A primer.

33. The level of true polymorphism between Treated sample A & Treated sample B was evaluated at 44 % with RBA 20A primer.

34. The level of true polymorphism between Treated sample A & Treated sample B was evaluated at 11 % with RBA 23A primer.

35. The level of true polymorphism between Treated sample A & Treated sample B was evaluated at 14 % with RBA 25A primer.

Treated sample A1 & Treated sample B1

36. The level of true polymorphism between Treated sample A1 & Treated sample B1 was evaluated at 34 % with RBA 8A primer.

37. The level of true polymorphism between Treated sample A1 & Treated sample B1 was evaluated at 0 % with RBA 13A primer.

38. The level of true polymorphism between Treated sample A1 & Treated sample B1 was evaluated at 0 % with RBA 20A primer.

39. The level of true polymorphism between Treated sample A1 & Treated sample B1 was evaluated at 22 % with RBA 23A primer.

40. The level of true polymorphism between Treated sample A1 & Treated sample B1 was evaluated at 28 % with RBA 25A primer.

CONCLUSION:

Polymorphism was detected between Control & Treated sample A. The percentage of true polymorphism observed between Control & Treated samples of Pseudomonas aeruginosa sample is on an average 50%.
Polymorphism was detected between Control & Treated sample A1. The percentage of true polymorphism observed between Control & Treated samples of Pseudomonas aeruginosa sample is on an average 36%.
Polymorphism was detected between Control & Treated sample B. The percentage of true polymorphism observed between Control & Treated samples of Pseudomonas aeruginosa sample is on an average 36%.
Polymorphism was detected between Control & Treated sample B1. The percentage of true polymorphism observed between Control & Treated samples of Pseudomonas aeruginosa sample is on an average 30%.
Polymorphism was detected between Treated sample A & Treated sample A1. The percentage of true polymorphism observed between Control & Treated samples of Pseudomonas aeruginosa sample is on an average 28%.
Polymorphism was detected between Treated sample B & Treated sample B1. The percentage of true polymorphism observed between Control & Treated samples of Pseudomonas aeruginosa sample is on an average 28%.
Polymorphism was detected between Treated sample A & Treated sample B. The percentage of true polymorphism observed between Control & Treated samples of Pseudomonas aeruginosa sample is on an average 17%.
Polymorphism was detected between Treated sample A1 & Treated sample B1. The percentage of true polymorphism observed between Control & Treated samples of Pseudomonas aeruginosa sample is on an average 16%.
The treatment modality of Mr. Mahendra Kumar Trivedi is not known to us, the company is only concerned about detecting DNA Polymorphism in Control & Treated lines of Pseudomonas aeruginosa samples.

Approved by: Dr. Avijit Roy

Manager R & D
Bangalore Genei.

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