TRANSFORMATION
OF COMPETENT CELLS WITH DNA ISOLATED FROM
COLIFORMS OBTAINED IN NAIROBI RIVERS.
A project submitted for examination in fulfillment of the requirements for Course Unit SBT
RESEARCH PROJECT in the BSC (Microbiology & Biotechnology) Degree Program.
Student's Name: LUMBASIO GAYNOR NOREH
Registration Number:
Sign
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123/31019/2009
Degree Program: BSC (Microbiology & Biotechnology)
Project Supervisor: Name
Ilrz.,. N. (, N
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SCHOOL OF BIOLOGICAL SCIENCES
CPBS
UNIVERSITY OF NAIROBI
MAY. 2013
'Z
Date: 13/05/2013
Abstract
Various techniques are used in the manipulation of DNA and cloning processes. The combining
of this techniques enables one produce genetically modified organisms in the laboratory.
Microbial culturing methods, DNA extraction, restriction enzymes, genetic markers, vectors and
electroporation techniques are of much importance in cloning. A cloning strategy is to be applied
to achieve the goal of producing a recombinant DNA and transformation of a competent cells.
The project involves isolating coliforms from different rivers. The DNA obtained from the cell
after culture underwent manipulation and resultant in formation of recombinant DNA. The
recombinants were of much importance as this can be used to add important traits into
organisms. As for our case the traits are to be random as the fragments produced from the
restriction of genomic bacterial DNA will not undergo hybridization to find cloned genes. The
project follows a simple methodology. It involves the bacterial isolation from two of Nairobi's
rivers through basic microbiology techniques and followed by cell culture techniques of water
microbiology. Genomic DNA isolation of the cultured samples using commercial kits,
subsequent DNA precipitation and Quantitative analysis follows. DNA manipulation involving
restriction of the extracted DNA and a cloning vector (PUe 19) using and endonuclease and
ligating the together to form recombinant DNA molecules. Recombinant DNA molecules are
used to transform competent JM 109 cell using an electroporator. A genetically modified
organism will result. Growth of the recombinants was to occur in the selective media containing
the screening reagents.
The results were both encouraging and disappointing, as it was easy to obtain colonies of
colonies from the samples from the rivers. Genomic extraction using kits yielded enough DNA
quantities but precipitating resulted. Restriction and ligation were the downstream processes
resulting in smear formation in gel electrophoresis photography. This could indicate fragment
formations at different recognition sequences. The transformation technique used is claimed to
be highly efficient and the culture of transformants cells containing recombinant DNA in
selective media resulted in no transformants growing in culture. No colonies were observed.
The transformation of competent cell may have not occur due to their death during storage hence
could not be transformed and hence could not be grown on the selective media. Proper storage of
cells is mandatory for better results.