Construction of a gene bank for a halotolerant bacteria micrococcus sp.(Strain G1) in E. coli.

number: 
582
إنجليزية
Degree: 
Imprint: 
Biotechnology
Author: 
Lamiaa Fingan Al-Malki
Supervisor: 
Dr. Jaladet M. S. Jubra'el
Dr. Falah Attawi
year: 
2002
Abstract:

This study included Isolation of chromosomal DNA from a Halotolerant bacteria Micrococcus Sp. Strain Gl (isolated from saltren soils in south of Iraq) by using two methods .DNA yields obtain from repeated experiments were different according to the method used. Average DNA yields obtained using the method based on modified Marmur's (1961), were in a rang (180 - 200) fig /100 m1- broth, with a purity of 1.6. Whereas using the salting - out procedure based on Pospiech and Nenman (1995),. The average DNA yields were 400 ug / lOOml-broth with purity 1.8 .The Molecular weight of all chromosomal DNA preparation was about 50 kb. Plasmid DNA pBR322 was also isolated from E-coli strain HB1O1 that was used as cloning vector in this study. For this by using two methods were also used. The first method was adapted from Holmes and Quigley (1981) the average yield of plasmid DNA was (0.3 - 0.4) ug with a purity of (1.6- 1.8) . The second method described by Maniatis et. al. (1982) produced average yields of Plasmid DNA from repeated experiments (0.4 - 0.5) fig with a purity of (1.6 - 1.8). Complete digestion of chromosomal DNA was performed using Bam HI restriction endonuclease. plasmid DNA was also digested with the same enzyme to produce compatible ends Next step in cloning experiment was ligation of chromosomal DNA fragments and Plasmid DNA with T4 DNA ligase .A reasonable Ligation efficiency was obtained from repeated experiments. Finally recombinant plasmid were introduced into the host cell E-coli strain MM294 via transformation experiments, two different method were employed. The first involved a method described by Hanhan (1983) and the second was based on the one described by Maniatis et.al. (1982) .The results of transformation using the two methods were somewhat similar. Transformation efficiency was calculated according to the formula reported by Mohan et.al. (l99l) .In this study transformation efficiency was about 2xl05 colony per fig DNA and about 1 x 10~ colony per jiig DNA respectively. Control experiment involving normal plasmid produced an efficiency of about 2.8x106 and 1.9xl06 respectively. Anther control (replica plating) on selective media containing antibiotic ampicillin and tetracycline. The results were colonies grow in media containing ampicillin and not grow in media containing tetracycline.