Question

1. How would one isolate and culture Pseudomonads putida from soil sample contaminated with hydrocarbons

2. Describe two physical properties that will enable one to isolate TOL plasmid from the chromosomal DNA of a bacteria.

3. Describe a method to restrict or digest the TOL plasmid.

4. Explain two methods to be used to increase the ligation efficiency of restriction products with blunt ends.

5. How can one ascertain if one's restriction digestion is successful.

Answer 1

1. Ans- Isolation and culture of pseudomonas putida from samples contaminated with hydrocarbons methodology includes:-

Sample collection- surface sedimented sample collected over a period of 12 months. The collected samples were pooled and transferred to pre-sterilized, labeled, and maintained until analysis.

THB population- THB population is enumerated by pour plate method . 1 ml of the aliquots from each of the dilution was inoculated by pour plate method into NA in duplicates. The plates were incubated at 37 degrees Celsius for 24-48 hours.

HDB population- this is done by spread plate techniques by inoculating aliquot into sterile BHA.

Identification of THB and HDB population- the enumerated bacteria were isolated and stored in a NA on the basis of colony and morphology and further performing a series of biochemical tests. The isolates which grow on both the agar plates were confirmed as hydrocarbons.

P.putida grows perfectly on LB medium on agar plates in shake flasks and bioreactors. Composition per liter is, 5g yeast extract and 10g NaCl. 2g/L glucose to accelerate growth. Using 230 rpm, 30 degrees Celsius, and baffled shake flasks. And it has been shown that flagella motion is not crucial for survival under lab conditions as chemotaxis is usually not necessary in rich medium. The study monitored the isolation and culture formation of the hydrocarbon containing P.putida as the efficient among the various strains isolated towards spill treatment.

**Dear students as per rules I have to do only one question. If you want to get all the questions answer please post them separately. Thank you!