Genetic Engineering

•The simple addition, deletion, or manipulation of a

single trait in an organism or a molecule to create a
desired change
•Major tool: recombinant DNA technology

•Genetic material can be shared across different organisms

•Bacteria can be engineered (e.g., to express eukaryotic proteins)
•Potential is virtually endless
Summary of the fundamental processes underlying genetic engineering
Finding the right clone

(a) Method for detecting production of protein by use of

specific antibody. (b) Method for detection of recombinant
clones by colony hybridization with a radioactive nucleic
acid probe. Although both parts of the figure show
detection involving radioactivity, many other types of
nonradioactive detection systems are now being employed.
Synthesis of complementary DNA (cDNA) from an isolated mRNA
using the retroviral enzyme reverse transcriptase
 Reverse translation

Deducing the best sequence of an oligonucleotide probe from the amino acid sequence of the protein. Because of
degeneracy, many probes are possible. If codon usage by the same organism is known, then a preferred sequence can be
selected. It is not essential that complete accuracy be achieved because a small amount of mismatch can be tolerated.
 A partial genetic map of the expression vector pSE420

The polylinker is a site containing many different restriction enzyme recognition sequences to facilitate cloning. This region (and
the cloned gene) would be transcribed by the trc promoter, which is immediately upstream of the lac operator (lacO).
Immediately upstream of the polylinker is a sequence that encodes a Shine–Dalgarno site on the resulting mRNA. Downstream of
the polylinker are two transcription terminators (T1 and T2). The plasmid also contains the lacI gene, which encodes the lac
repressor, and a gene conferring resistance to the antibiotic ampicillin. These two genes are under the control of their own
promoters, which are not shown.
 Applications of Genetic
Engineering
• medical applications
• industrial applications
• agricultural applications
Medical applications…
• treatment of disease
– pharmaceuticals
– gene therapy
• vaccine development
• diagnosis of disease
• research on the molecular basis of disease
 Genetic engineering for the production of human insulin in bacteria

(a) Structure of human proinsulin. The peptide shown in yellow must be removed from between the A and B chains in order
to make insulin. (b) Chemical synthesis of the insulin gene and suitable linkers, permitting cloning and expression. The
synthesized fragments were linked via restriction sites EcoRI and BamHI in a plasmid vector in such a way that the insulin
chains are formed as a fusion protein with a portion of a gene found on the vector (note that the EcoRI site is part of this
coding region). The methionine coding sequence was inserted to permit chemical cleavage of the A and B chains from the
fused protein made in the bacteria because the reagent cyanogen bromide specifically cleaves at methionine residues and
insulin does not contain methionine. Two stop codons were incorporated at the downstream end of the coding sequence.
 Agricultural Applications

•Plant genetic engineering: insect,disease,

and herbicide resistance
•Animal genetic engineering (hormones):
produce more milk, leaner meat
•GMO - genetically modified organism
 Plant Genetic Engineering:
Agrobacterium tumefaciens

• Agrobacterium tumefaciens is a bacterium that causes a

disease known as crown gall in plants.
• Infects plants by transferring its genetic material into plant
cell.
• Agrobacterium transformation is the most common
technique for genetically engineered plants
Production of transgenic plants using a binary vector system in Agrobacterium tumefaciens

(a) Generalized plant cloning vector containing ends of T-DNA (in red),
foreign DNA (in yellow), origin of replication elements for both E. coli and A.
tumefaciens, and spectinomycin and kanamycin resistance markers. The
kanamycin resistance marker can be selected for in plants. (b) The vector
can be put into cells of E. coli for cloning purposes and then transferred to
A. tumefaciens by conjugation. (c) The resident Ti plasmid used for
transferring the vector to the plant (D-Ti) is itself genetically engineered to
remove key pathogenesis genes. (d) However, D-Ti can mobilize the T-DNA
region of the vector for transfer to plant cells grown in tissue culture. From
the recombinant cell, whole plants can be regenerated.

….Ti is a natural plant transformation system!

GMO
(Glyphosate resistance)

Soybeans treated with Roundup,

manufactured by Monsanto
Industrial Applications
• manufacturing proteins using bacterial, fungal,
and mammalian cells as factories
• strain improvement for existing bioprocesses
• development of new strains for new bioprocesses
• more efficient catalysts: e.g., enzymes active
under unusual conditions
• waste management: biodegradation of a number
of waste products (e.g., sewage and petroleum
products)
 Social Impact of Recombinant
DNA Technology
• many benefits, but also many risks
• careful analysis of risks and benefits is
imperative to avoid problems
Safety concerns
• widespread infections caused by genetically
modified organisms (GMOs)
• spread of genes from GMOs to other
microorganisms in environment
• release of GMOs currently regulated by
several federal agencies
Ethical and moral concerns
• genetic engineering of humans
• unethical use of genetic information
obtained from an individual
• creation of biological weapons
Environmental concerns
• ecosystem disruption
• spread of cloned genes to weeds or other
organisms in environment