genetic engeering

8/8/2019 Genetic Engeering

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Summary of the fundamental processes underlying genetic engineering


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Finding the right clone

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

specific antibody. (b) Method for detection of recombinantclones by colony hybridization with a radioactive nucleicacid probe. Although both parts of the figure showdetection involving radioactivity, many other types ofnonradioactive detection systems are now being employed.


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Synthesis of complementary DNA (cDNA) from an isolated mRNAusing the retroviral enzyme reverse transcriptase


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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 (andthe cloned gene) would be transcribed by the trcpromoter, which is immediately upstream of the lacoperator (lacO).Immediately upstream of the polylinker is a sequence that encodes a ShineDalgarno site on the resulting mRNA. Downstream ofthe polylinker are two transcription terminators (T1 and T2). The plasmid also contains the lacIgene, which encodes the lacrepressor, and a gene conferring resistance to the antibiotic ampicillin. These two genes are under the control of their ownpromoters, which are not shown.


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Medical applications

treatment of disease

pharmaceuticals

gene therapy

vaccine development

diagnosis of disease

research on the molecular basis of disease


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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 orderto make insulin. (b) Chemical synthesis of the insulin gene and suitable linkers, permitting cloning and expression. Thesynthesized fragments were linked via restriction sites EcoRI and BamHI in a plasmid vector in such a way that the insulinchains 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 thefused protein made in the bacteria because the reagent cyanogen bromide specifically cleaves at methionine residues andinsulin does not contain methionine. Two stop codons were incorporated at the downstream end of the coding sequence.


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Agricultural Applications

Plant genetic engineering: insect,disease,

and herbicide resistance

Animal genetic engineering (hormones):

produce more milk, leaner meat

GMO - genetically modified organism


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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

Plant Genetic Engineering:

Agrobacteriumtumefaciens


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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. coliand A.tumefaciens, and spectinomycin and kanamycin resistance markers. The

kanamycin resistance marker can be selected for in plants. (b) The vectorcan be put into cells of E. colifor cloning purposes and then transferred toA. tumefaciensby conjugation. (c) The resident Ti plasmid used fortransferring the vector to the plant (D-Ti) is itself genetically engineered toremove key pathogenesis genes. (d) However, D-Ti can mobilize the T-DNAregion of the vector for transfer to plant cells grown in tissue culture. Fromthe recombinant cell, whole plants can be regenerated.

.Ti is a natural plant transformation system!

Soybeans treated withRoundup,

manufactured by Monsanto

GMO(Glyphosate resistance)


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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 activeunder unusual conditions

waste management: biodegradation of a numberof waste products (e.g., sewage and petroleum

products)


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Social Impact of Recombinant

DNA Technology

many benefits, but also many risks careful analysis of risks and benefits is

imperative to avoid problems


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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


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Ethical and moral concerns

genetic engineering of humans

unethical use of genetic information

obtained from an individual

creation of biological weapons


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Environmental concerns

ecosystem disruption

spread of cloned genes to weeds or other

organisms in environment

genetic engeering

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