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Class notes Biomedical Sciences (BSc) Basic Immunology, ISBN: 9781416046882
Lecture Notes BB2705 Genetic Engineering & Immunobiology at Brunel University
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BB2705 Genetic Engineering and ImmunobiologyWhy are bacteria useful?
- Bacteria can be grown in vast numbers quickly
- They are haploid cells – there are no dominant/recessive properties like in diploid cells
- Bacteria allow biochemical studies – enzyme reactions, growth in media with/without
nutrients for production of amino acids
- Mutations allow us to understand what genes do – genetic analysis
Bacterial mutants
- Prototrophs obtain energy from light because they have the enzymes to do this and can
make most of the complex nutrients they need to grow
- Nutritional mutants – some can make amino acids, some cannot. So loss of enzymes
disables bacteria from growing in basic simple medium which means a particular nutrient
must be added. These mutants are also called auxotrophs.
o Carbon-source mutants such as lactose gene mutants cannot use lactose and can
only grow if an alternative carbon source is supplied to grow
- Antibiotics mutants – bacteria can be selected using antibiotics if they have certain genes,
e.g. the genes for streptomycin antibiotic resistance are designated str-r. This allows
selection against mutants without the streptomycin resistance gene and finds bacteria with
this gene active.
Genetic variation - Transformation
- We get genetic variation in bacteria by passing DNA between them.
- Transformation – DNA can be added to bacteria and taken up at low frequency. In nature
bacteria that spontaneously lyse release DNA into the medium and this can be taken up by
other bacteria. This DNA can recombine with existing DNA in the bacteria to produce a new
genotype/phenotype.
- Transformation also allows gene mapping. Transformation usually occurs for 1 in 1000 cells
(10-3). If two genes are to be taken up then frequency is 10 -3 x 10-3 = 10-6. If the two genes are
close together on one piece of DNA then they can transform together therefore 10 -3.
Transformation allows us to find how near genes are to one another → 10 -3 (close), 10-6 (far
apart)
, Genetic variation – Conjugation
- Conjugation is defined as the unidirectional transfer of genetic information between cells by
cell-to-cell contact. This uses genes and sequences present on primitive plasmids called F
plasmids.
- ‘Unidirectional’ means one copy of the DNA is transferred from one cell → from the ‘donor’
to the ‘recipient’.
- The site of initiation of transfer is called oriT (origin of transfer) or mob region (mobility)
referring to a sequence on the bacterial DNA. Proteins produced by bacteria enable this to
happen
- The plasmid codes for the F factor (Fertility factor) containing the tra (transfer) genes. The
genes code for the formation of the pilus which draws the donor and recipient cells together
making a bridge. This also promotes transfer and replication.
- The F factor encodes at least 20 or more genes. It is used to show genes are ‘linked’
o Donors have the F factor termed F+
o Recipients do not have the f factor and are therefore termed F-
- Transfer is always accompanied by replication called the rolling circle
- The F factor can exist in different states:
o ‘F+’ refers to a factor on a plasmid in an extrachromosomal state
o ‘Hfr’ (high frequency recombination) state describes the situation when the factor
has integrated itself into the chromosome
- The rolling circle
o Tra genes code for a specific single-strand nuclease that nicks the DNA at the oriT
site and binds primases to the free 5’ end of the DNA. These proteins lead the
replication of the transferred DNA.
o A single-strand is then transferred from the 5’ end of the donor to the recipient
while DNA replication occurs by “rolling circle” replication in the donor.
o If the DNA being transferred is an F plasmid, it is made double-stranded and
circulated in the recipient
o The F plasmid can mobilise other plasmids that are not non-conjugative as long as
they have an oriT.
o A plasmid lacking both the tra-functions and oriT functions would be classified as
non-conjugative and non-mobilisable.
- In the Hfr state, the F plasmid has inserted into the bacterial genome. During the process of
transfer of a Hfr DNA during conjugation, the bacterial chromosome can break whilst being
transferred the break occurs at the beginning part of the F segment so the rest of the F
sequence is transferred late or not at all, and the recipient cells usually remain F-
- This process allows time-entry mapping to identify the order of genes on the bacterial
genome.
- The frequency by which the genes are apart are turned into minutes. Genes are mapped by
selecting each of the bacteria in medium showing transfer of the gene following growth in
medium where nutrients are not supplied.
- A cross between an Hfr strain and an F- strain will result in transfer of chromosomal makers
at high frequency (10-2 to 10-5). This transfer is orientation and time dependent.
- Since transfer begins at the oriT site in the F factor, a portion of the F factor is transferred
first followed by the remainder of the chromosome. If the entire chromosome is transferred,
then the other portion of the F factor is transferred. The F factor itself does not integrate
into the recipient as there is no homology for such integration by the chromosomal DNA
which has been transferred can recombine in by homologous recombination
- The entire E.coli chromosome takes about 100 minutes but often you get spontaneous
breakage of the mating pair. Such breakage means that markers (genes) transferred late are
often not transferred at al yielding a gradient of transfer
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