The Role of Microorganisms in Genetic Engineering
'Genetic engineering' or genetic manipulation as it should properly be called,
relies essentially on the ability to manipulate molecules in vitro. Most biomolecules
exist in low concentrations & as complex, mixed populations which it is not
possible to work with effectively. This problem was solved in 1970 using the molecular
biologist's favourite bug, Escherichia coli , a normally innocuous commensal
occupant of the human gut. By inserting a piece of DNA of interest into a vector
molecule, i.e. a molecule with a bacterial origin of replication, when the whole
recombinant construction is introduced into a bacterial host cell, a large number
of identical copies is produced. Together with the rapid growth of bacterial colonies
all derived from a single original cell bearing the recombinant vector, in a short
time (e.g. a few hours) a large amount of the DNA of interest is produced. This
can be purified from contaminating bacterial DNA easily & the resulting product
is said to have been 'cloned'.
Most vector molecules were originally derived from one of two sources:
Rapidly, the original vector molecules were greatly modified to improve their
usefulness as vectors, e.g:
- Plasmids - small, autonomously replicating circular pieces
of bacterial DNA, which often carry antibiotic-resistance genes.
- Bacteriophages (phages) - viruses which infect bacteria.
- Insertion of selectable marker genes to pick out recombinant
molecules containing foreign inserts (antibiotic resistance genes & genes
which produce a coloured product & therefore a coloured bacterial colony).
- Removal or creation of useful sites for cloning
- Insertion of sequences which not only allow but greatly increase the expression
of cloned genes in bacterial, animal & plant cells.
Vector molecules & cloning are not the only contribution which microorganisms
have made to genetic manipulation. The actual task of altering the DNA at a molecular
level is carried out by the use of naturally-occurring enzymes - most of which
are derived from bacteria or viruses:
- Restriction endonucleases: bacteria in natural environments
are continually exposed to a dilute 'soup' of foreign DNA released from other
organisms which have died & lysed. They are also exposed to bacteriophage
DNA. If unable to protect themselves, they would rapidly become infected &
killed. The answer to this problem are the many different restriction-modification
systems possessed by different groups of bacteria. The enzymes are named after
the organisms from which they were originally isolated, e.g:
EcoRI from Escherichia coli
BamHI from Bacillus amyloliquefaciens
These systems operate by enzymes which recognise specific short regions
of DNA sequence, which are usually palindromic ('Able was I ere I saw Elba'),
The modification part of the system is responsible for covalently modifying
specific nucleotides in this sequence, either by glycosylation or more commonly
by methylation of the bases. Modification prevents the corresponding restriction
endonuclease (internal cleavage, c.f. exonuclease - degradation form the end
of the strand) binding to & cutting the sequence, thus the cells DNA is
protected. Foreign DNA (e.g. a bacteriophage genome) is not modified & is
thus digested by the restriction enzymes & destroyed. These systems are
an effective although not infallible means of protecting bacteria from infection.
Molecular biologists use highly purified preparations of R.E.s to cut DNA into
5' GGATCC 3'
3' CCTAGG 5'
- DNA ligase: provides the ability to rejoin cut fragments
of DNA & form artificial recombinant molecules. The enzyme utilizes ATP
to create new phosphodiester bonds, covalently linking together the ends of
DNA strands. The ligase encoded by bacteriophage T4 is particularly valuable
since it has very good properties in vitro, such as the ability to join together
'blunt-ended' DNA fragments which allows the joining of otherwise non-compatible
fragments, e.g. different restriction enzymes.
- Other modifying enzymes (phosphatases, kinases, single-strand
specific nucleases, etc): allow precise modifications to pieces of DNA to be
made in vitro in order to add, remove or alter the structure of DNA.
- RNA modifying enzymes - e.g. exonucleases, RNA ligase, reverse transcriptase.
RNA is much more difficult to work with in vitro because the enzymes available
are generally not as sophisticated as the set available which modify DNA.
- DNA Polymerases: Allow the synthesis of DNA from a pre-existing
template in vitro, which can then be used for a variety of purposes, including
sequence analysis by chain-termination methods.
Recently, thermostable polymerases have become important,
e.g. Taq DNA polymerase from Thermus aquaticus. This
bacterium has evolved to grow in hot springs at temperatures which kill most
other species. These enzymes allow the amplification of as little as one molecule
of DNA into a large amount by means of repeated cycles of melting, primer annealing
& extension by the enzyme which is not destroyed by the high temperatures
used in this process. This is known as the polymerase chain reaction:
The utility of cloning is partly analytical, i.e it provides
the ability to determine the genetic organization of particular regions or whole
genomes (the human genome will soon be underway). However, it also facilitates
the production of naturally-occurring & artificially-modifed biological
products by the expression of cloned genes.
The ability to take a gene from one organism (e.g. man or a tree), clone it
in E. coli & express it in another (e.g. a yeast) is dependent
on the universality of the genetic code, i.e. the triplets of bases which encode
amino acids in proteins:
In fact, the genetic code is not completely universal - there
are minor differences in the codons which are recognised & used by different
groups of organisms, e.g. animals, plants & bacteria. However, this rarely
presents a difficulty expressing foreign genes, & if it does, the means
exist to modify particular codons to alternatives coding for the same amino
acids which will be recognised by the host organism.
A list of some of the products produced by genetic engineering is given elsewhere,
but this is expanding very rapidly & new products of great medical, agricultural,
environmental & industrial importance are constantly being produced by this
The practical application of molecular biology is
founded on our understanding of microorganisms. Microbiology is thus a central
discipline in modern biology.
L.M.Prescott et al.
A balanced, comprehensive introduction to all major areas of microbiology. The
sixth edition has been updated extensively to reflect the latest discoveries
in the field.
© MicrobiologyBytes 2007.