The system or by micro/macro-injection or micro-encapsulation.

The bacteria
is the first organism to be genetically modified. Bacterial engineering is the
science that deals with genetic alteration of bacterial genome for medical, and
agricultural purposes. The use of bacterial systems for genetic modification is
favored due to their rapid reproduction rates, and the ease to produce a
genetically identical population following modification. A gene of interest is usually
required. Therefore the cells can be lysed and the DNA can be isolated. Several
nonbacterial proteins can be produced from bacteria. Famous examples include:
vaccines and insulin synthesis. In our project we will be dealing in details
with several medical achievements using bacterial engineering. We will discuss
the process of vaccines development, drug improvements, cancer therapies and
several types of hormonal synthesis out of bacteria.

Plasmids are
circular extrachromosomal self -replicating pieces of bacterial DNA. They
encode for several proteins that serve as an advantage to the host. Plasmids
can be easily isolated and modified in vitro through deleting, or inserting
specific DNA sequences. Plasmids are also called cloning vectors.

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After
isolating the DNA, it can be introduced directly into a specific host organism,
or into a cell that is fused or hybridized with the host. The main principle of
this process is using specific recombinant nucleic acid techniques to generate
new combinations of transmittable genetic material ( DNA) and then incorporate
this material either through a vector system or by micro/macro-injection or
micro-encapsulation.  If the host belongs
to another type of species, the resulting organism is called transgenic. If the
host belongs to the same type of species, the resulting organism is called
cisgenic.

Getting
the Plasmid In

A
bacteria has several enzymes to cut up the DNA of other enemies bacteria, such
as bacteriophages. Those restriction enzymes, can be used to cut DNA for
engineering bacteria purposes. Researchers have been able to isolate hundreds
of restriction enzymes, and each of these enzymes is able to splice specific
nucleotides sequences of the DNA. Some restriction enzymes produce blund ends,
since they cut across both DNA strands. Others, are staggered generating sticky
ends.  These ends are brought by hydrogen
bonding to similar ends on another DNA segment cut with the same restriction
enzyme.
To clone a gene, we  first identify the
gene of interest in the bacteria .The aim of the final resultant organism is
the main drive for identification. Screens can be performed
to identify potential genes and multiple tests can be carried out to determine
the best candidates. Microarrays, transriptomes and genome sequencing eased the
way to find the suitable genes. The next step is to isolate the DNA from the
bacteria  through specific
restriction enzymes or polymerase chain reaction (PCR) to amplify the gene
segment.  The gene of interest is sniped
from the DNA strand. The gene containing segment is spliced into a plasmid cut
by the same restriction enzyme. Once the bacteria take up the plasmid, they are
allowed to replicate. Prior to gene insertion into the host, the gene must be
combined with other genetic materials. These include a promoter and a
terminator sequence region, which initiate and terminates transcriptions.   The
gene can also be modified at this stage for better expression or effectiveness.
These manipulations are carried out using recombinant DNA techniques, such as restriction digests, ligations and molecular cloning.

 

Usually bacterial cells
are not able to take up plasmids. However scientists are able to use various
tricks to enable the bacteria to do so. This
ability can be induced in other bacteria via stress (e.g. thermal or electric shock), which increases the cell membrane’s
permeability to DNA. Genes can be injected through the cell’s nuclear envelope directly into the nucleus, or through the use
of viral vectors. One of the methods to
insert the plasmid is to put the cells in a solution of calcium chloride. The
bacteria are heat shocked briefly so the plasmid can cross through the plasma
membrane. Another method is electroporation. It is the use of a short
electrical pulse to create pores in the membrane, and thus enabling the plasmid
to pass through.  In plants the DNA is
generally incorporated by the use of Agrobacterium-mediated recombination,53 taking advantage
of the Agrobacteriums T-DNA sequence that allows
natural insertion of genetic material into plant cells.  An alternative method is biolistics, where particles of
gold or tungsten are coated with DNA and then shot into young plant cells or
plant embryos.

Marker genes, like
genes that encode antibiotic resistance, are often engineered into plasmids.
These marker genes allow scientists to identify which type of bacteria have the
plasmids. The antibiotic is added then to the media that is used to grow the
bacteria. Cells that lack the plasmid will not be able to grow and reproduce.

However, the goal of bacterial modification is the synthesis of proteins
encoded in fungi, plants or animals, which contain eukaryotic cells. Eukaryotic
cells contain both exons ( cnding sequences) 
and introns.(non coding sequences ). In those cells, the DNA serves as a
template for the synthesis of mRNA. Following 
mRNA splicing, introns are removed and exons are joined to form the
final  mRNA. The latter  goes to the ribosomes for the production of
proteins. However, bacteria do not have the enzymes responsible to cut up the
mRNA. So inserting an eukaryotic gene inside the bacteria requires special
procedure. First, DNA must be synthesized, t complementary to the already
spliced mRNA. The reverse transcriptase enzyme is used to produce the
double-stranded DNA molecule, using the mRNA as a template. Finally, this
double stranded DNA is introduced in the cloning vector. .

Several tests are conducted confirm the presence of
the gene in the organism. These tests include PCR, southern hybridization and
DNA sequencing. It can also determine the chromosomal location and the number
of copies of the inserted gene. However, the presence of the gene does not
assume it will be expressed at the needed level in the target tissue. Several
methods allow us to look for and assess how much gene products ( RNA and
proteins) are generated. These methods include: quantitative RT-PCR, western
blot, ELISA, northern hybridization and phenolytic analysis. Offspring are
studied to ensure homozygous genotype transfer through mendelian inheritance. We
can insert randomly the new genetic material in the host genome, or we can
target it to a specific location. Homologous recombination is used as a
technique for gene targeting, to make desired alterations to a specific
endogenous gene. The frequency of gene targeting can be greatly enhanced using
genome editing: through artificially engineered nucleases, specific
double-stranded breaks are created at specific desired locations in the genome,
and then use the bacteria’s endogenous repair mechanism to fix the induced
break by the natural processes of homologous recombination and non-homologous
end-joining. Currently there are four categories of engineered nucleases: zinc
finger nucleases, transcription
activator-like effector nucleases (TALENs), meganucleases
and the Cas9-guideRNA system (adapted from CRISPR). These nucleases can also be
used to induce desired mutations at endogenous genes that will generate gene
knockout.

 

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