Biotecnologia

Priyanka Singh et al. / Journal of Pharmacy Research 2012,5(5),2778-2782

Research Article
ISSN: 0974-6943

Available online through
www.jpronline.info

Identification and cloning of oil degrading genes of Pseudomonas aeruginosa in E.coli.
1

*1. Priyanka Singh, 2 and Manoj Kumar Singh
Department of Biotechnology, Madhav Institute of Technology & Science, Gwalior, Madhya Pradesh,India
2
Department of Training and Research & Development, Clonegen Biotechnology Pvt. Ltd,
D-34, Sector-2, Noida, Uttar Pradesh - 201 301, India

Received on:11-01-2012; Revised on: 17-02-2012; Accepted on:19-04-2012
ABSTRACT
Environmental pollution has formed a great concern the world over and oil pollution forms and important issue among all the types of pollutions due to it
large levelimpact on the health of the ecosystem. Our work aimed at isolation of oil degrading Pseudomonas species from soil and to clone the oil degrading
gene from it into the E.coli. In this soil samples from different sources were collected in a sterile polybag and then brought to lab, where many isolates of
the Pseudomonas were isolated and their oil degrading ability was checked in a synthetic media.The isolate showing oil degrading ability was identified as
Pseudomonas aeruginosa by growing on selective media cetrimide and through various biochemical tests. Oil degradation studies were done with the isolated
strain at varying pH conditions of 5,6 and 7, in crude oil and pH7 was the found to be the optimum pH for the growth and biodegradation activity. The oil
degrading gene was firstamplified using the PCR and Random primer and the amplified products were cloned into the E.coli DH5a, using the vector pBR322 .
KEY WORDS: Gene Cloning, E.coli, Oil Degrading, Pseudomonas aeruginosa
INTRODUCTION
Pseudomonas aeruginosa is an opportunistic bacterium that lives in soil,
water, and even in environments like hot tubs. For most healthy people, this
bacterium seldom poses a problem.Occasionally people will develop conditions like hot tub rash, and swimmer’s ear, which may be due to contact with
these germs. These conditions can sometimes resolve without treatment, or
with minimal treatment, like antibiotic drops for swimmer’s ear. Unfortunately, Pseudomonas aeruginosa is much more dangerous to certain populations, including those who have weak immune systems, the elderly, andthose
who have been hospitalized for long periods of time. People with cystic
fibrosis and with full-blown AIDs frequently die from infections created by
the bacteria. Those who have undergone chemotherapy, have had transplants,
or undergoing any kind of immunosuppressed conditions are far more at risk
for developing bacterial infections due to Pseudomonas aeruginosa, and
because thisbacteria is relatively resistant to most antibacterial medications,
infection can be deadly, particularly when it becomes infection of the lungs or
bloodstream. Doctors and medical researchers often refer to Pseudomonas
aeruginosa as a blue-green pus bacterium, and/or a gram-negative bacterium.
The first reference is to the pus, which can show blue to green colours, and
the second refers to theGram method for staining bacteria to determine what
type it is. When samples of gram-negative bacteria, particularly those that
are considered aerobic, are stained, they resist colour and typically show up
in slides under the microscope as a pink colour.

However, the bioavailability of weekly soluble hydrophobic compounds for
microbial conversion is usually low and thus limits theirdegradation rate in
aqueous medium. The use of surfactants has been found to enhance degradation of crude oil [2, 3] or other hydrocarbons [4]. Among various surfactants,
rhamnolipids are considered to be the most in degrading hydrocarbons [5]. For
example, the biodegradation of long-chain alkanes was stimulated by addition
of rhamnolipid [6]. This facilitated biodegradation is probably due to the...