Outcome 1 - Bacteria Flashcards
Transformation describes the process by which
extracellular DNA is uptaken by a bacteria cell from its enviroment
In order to be trasnformed a cell must
Be competent (changes in permeability and receptors for DNA)
In transformation when dsDNA enters the cell
nucleases break the hydrogen bonds and convert the dsDNA into ssdDNA to be inserted into the genome via homologous recombination. Otherwise the ssDNA is digested by nucleases
What processes render bacteria artificially competent
Chemical transformation and electroporation
Describe chemically induced competence
CaCl2 treatment in order to increase permeability of membrane, heat shock at 42C for 50seconds before return to ice bath. Recovery - enriched nutrient broth and incubation. Selection - spread onto selective agar
Describe electroporation
DNA, bacteria and glycerol are mixed + ice bath
Transferred to the electroporator and exposed to high voltage electric impulses
Enriched nutrient broth and incubation
Spread on selective agar
Define conjugation
A method of bacterial horizontal gene transfer between an F+ donor cell and a F- recipient cell via sex pili and cell to cell contact
F status of a bacteria is conferred by
Fertility factors aka Tra genes for pili assembly and production, transferosome and relaxosome assembly, and genes for DNA transfer
Describe the process of conjugation
Conjugation involves passage of genes between two mating types: “male”, F+ cells and “female”, F- types. The “F” status is conferred by F (fertility) factor that is carried on a plasmid. These encode the DNA replication enzymes and structural proteins for the sex
pilus- the tube which allows cell to cell contact and formation of the conjugation channel which is the route by which DNA can be transferred to an F- bacterium. After contact, and extension of the sex pili, the conjugation channel forms. The F factor plasmid replicates by the rolling circle method and a single DNA strand passes through the channel. Once
inside the other cell, a complementary strand is synthesised and a new plasmid created.
Only plasmid–encoded genes are transferred. The former F- bacterium now becomes an
F+ bacterium and contains the F+ plasmid. Only plasmid genes have passed over.
What method can be employed to quantify the expression of proteins that are
produced as a result of transformation?
Western blot
Define HFR
High frequency transfer in which chromosomal DNA is transferred with an F plasmid. However, the recipient does not become Hfr and does not become a donor.
What allows F plasmids to integrate into the chromosome for HFR
F plasmids are episomal and can integrate into the bacterial chromsome via an insertion point
Define sexduction
If the F plasmid is excised from the chromsome the Hfr cell becomes F+
When this happens sometimes bits of chromosomal DNA are taken too.
When the F plasmid is passed on to a F- recipient, which become F` (F prime) the chromosomal DNA transfers as well. This is called sexduction and the
resulting cell will have two gene copies (diploid)
What are some applications of conjugation
HFR is used to determin chromosomal gene order
Conjugation can be used to tranfer large DNA molecules without damaging the recipient cell
