8. BACTERIAL GENETICS
GENE MANIPULATION
 Genetic information – in nucleotide
sequence of DNA (rarely RNA)
 Bacteria – most genes in chromosome,
some can be in plasmid(s)
 Genome – a set of genes in the cell,
bacteria – 1 set (N, haploid), eucaryotes
– 2 sets (2N, diploids) with the exception
of sexual cells
 Mutation
The changes in genome, which are
reflected in phenotype.
They are heritable, spontaneous (= error
in DNA replication), influencing one cell
and one base pair (point mutation)
Mutagens – factors that increase the
probability of mutations (UV-light, γrays, etc.) – induced mutagenesis
 DNA repair
The cell ability to detect DNA change, to
mark it and to repair changed DNA =
cell homeostasis
 No sexual processes in bacteria “parasexual” exchange of genetic
information: conjugation, transduction,
transformation
 Conjugation
The transfer of genetic information from
donor cell to the acceptor cell using cellto-cell contact.
Conditions – sex pilli, presence of F
plasmid in donor cell (free or in
chromosome = Hfr cell)
 DNA transformation
The uptake of naked DNA (fragment) by
acceptor (competent) cell and its
integration into chromosome
Also plasmids can use this way to enter
the cell
 Transduction
= Gene transfer with vector (virus)
Bacterial genes are incorporated into a
phage because of error in its lytic cycle
and these genes are transported into
acceptor cell
Important terms – temperate phage,
lysogeny, prophage
Prophage (phage incorporated in
bacterial chromosome) during leaving
host chromosome can took the host
genes (= specialized transduction)
 GENE MANIPULATION
= aimed transfer of genes based moustly
on transduction
 Isolation of genes
(1) from donor cell using restrinction
endonuclease („restrinctase“)
(2) from m-RNA using reverse
transcriptase
(3) gene synthesis according to gene map
(4) gene buying
 Recombinant vector construction
Vector (responsible for gene transfer)
= virus, plasmid (R, Ti for plants),
cosmid;
Characteristics of acceptable vector –
markers, restrinction map
Steps in construction – vector opening
with restrinctase, genes incorporation
with ligase
Result – mixture of recombinant and
original vectors
 Recombinant cell construction
Recombinant vectors (plasmids) are
mixed with acceptor cells and different
means are used to increase probability
of plasmid entering into cell.
Result – mixture of acceptor cells:
original – with original plasmid – with
recombinant plasmid
 Selection of recombinant cell
Recombinant cell is selected according
to plasmid marker(s)
 Recombinant cell cloning
Selected cell are cultivated to get more
cells with new genetic information, the
heritability is tested
 Expression of new genes
The optimisation of media to get asked
product
Another possibilities of gene transfer
Gene gun – gene(s) is/are trapped on the
metal particle and with high energy
introduced into the acceptor
Injection (micromanipulation) – gene is
directly inserted into the nucleus of
ovum (esp. mammals)
Examples of gene manipulation results:
- tomato – one time ripening
- tomato – hyaluronidase blocking
- bacteria – production of insulin
interferons, “human”
derivates, hormones, etc.
- soya, maize – herbicide resistance
- etc.