Lecture 18: Genetic Engineering Flashcards Preview

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What is genetic engineering?

In vitro isolation, alteration+expression of DNA/RNA


What is recombinant DNA technology used for?

Combine DNA from multiple organisms into one molecule


What is a transgenic organism?

Genes from 2 organisms placed into 1 organism


What is the asilomar conference?

1975 meeting of recombinant DNA scientists
-self regulation and informing the public


The GMO debate focuses on higher organisms usually, what issues does it look at?

1. Safety - ok to eat
2. Ethics - moral responsibilities
3. Economics - trademarking life


GMO's are more accepted amongst prokaryotes, what are some examples?

1st genetic engineering product was Human insulin


When manipulating DNA, what techniques are used and why?

1. Restriction enzymes - cutting DNA
2. Gel Electrophoresis - separating DNA Pieces
3. Nucleic Acid Hybridization - Finding right pies
4. Molecular Cloning - Copying DNA pieces
5. Site-Directed Mutagenesis - Changing DNA pieces
6. Reporter and Fusion Genes - Tracking DNA pieces


How do restriction enzymes work to cut DNA into pieces?

Recognition sequence determines where each RE cuts
-RE is named for source bacterium

Sticky Ends are created due to staggered cuts on different strands
-single stranded overhangs used for cloning


How is gel electrophoresis used to separate DNA pieces?

DNA molecules (-) charged
-electrical field used to migrate DNA


How is Nucleic acid hybridization used to find the right DNA piece?

Denatures DNA
-dsDNA to ssDNA

Add Specific nucleic acid probe which binds to specific complementary DNA that you want


How is molecular cloning used to copy DNA pieces?

After using restriction enzymes to cut pieces, you add a vector that has also been cut with RE, add DNA ligase to insert DNA into vector and form recombinant molecule and insert into host


How is site-directed mutagenesis used to change DNA pieces?

Targeted mutation of gene sequences through base pair changes
-Gene knockouts remove entire gene from genome
-allows study of gene function/effect on organism


How are reporter and fusion genes used to track DNA pieces?

Reporter genes
-code proteins that make it obvious this is the altered gene (fluoresence proteins)

Fusion Genes
-join two separate genes (study gene+reporter gene)


When looking for ideal vectors for gene cloning and expression what are you looking for?

Ligation - accepts new genes

Gene transfer - transport genes into host cells


When looking for ideal hosts for gene cloning and expression what are you looking for?

Gene transfer - accepts vectors with new genes

Gene replication and expression - copy new genes


What are the types of vectors used for gene cloning?

Plasmid Vecotrs
-carry small DNA pieces
-inserted by transformation
-short term storage

Bacteriophage vectors
-carry larger DNA pieces
-inserted by transduction
-long term storage


Ideal vectors are able to detect what?

Successful ligation
-presence of DNA piece in vector

Successful Gene transfer
-presence of DNA + vector in host


What are the important features of plasmid vectors?

(1) Reporter Gene (lacZ) - blue=gene ok, white=disrupt
(2) Antibiotic Resistance (ampicillin)
-gene transfer success = growth
-gene transfer failure = no growth
(3) Multiple Cloning Site (MCS)
-restriction enzyme sites located within reporter gene
(4) Origin of Replication (ori)
-initiates copying of plasmid


What are some advanced plasmid vectors?

Expression Vectors
-Vectors with regulatory gene sequences
-For controlled expression of inserted gene

Shuttle Vectors
-Allows for vector replication of two unrelated hosts
-for vectors w/ multiple ori genes


How do expression vectors work?

lacO Gene is operator and lacI Gene is repressor

No expression of gene until inducer (lactose) is present
-Grow host cells to high numbers
-Activate expression by adding lactose
-harvest newly synthesized proteins


What is the bacteriophage T7 system?

An expression vector that encodes own polymerase and recognizes own promoters
-lactose induces polymerase and only transcribes cloned genes


How does bacteriophage lambda work?

1. Insert DNA piece
2. Repackage into head
3. Assemble virions
4. Infect host cells


What are artificial chromosome vectors?

Synthetic cloning vectors
-designed to carry huge DNA pieces
-Used to clone large genomes

Bacterial Articial Chromosomes (BAC)
-Require modified E. coli host cells
-no RE system and no recomination


What are the general characteristics of ideal hosts?

Fast growth rate

Non pathogenic

Competent (easily transformed w/ foreign DNA)

Stable in culture


What are the types of hosts used for gene cloning?

-most common
-disadvantage: no expression of eukaryotic proteins

-accommodates eukaryotic protein expression
-disadvantage: unstable plasmids


What are some examples of prokaryotic hosts?

Esherichia coli
-potentially pathogenic

Bacillus sutilis


What are some examples of eukaryotic hosts?

Saccharomyces cerevisiae

Cell cultures
-animal and plant cell lines
-delicate and expensive
-limited large scale production


How do we transfer vectors into hosts?

-create small pores in host cell membrane w/ electrical current or heat shock
-plasmids enter cells through pores

-virus mediated transfer



How is transformation accomplished in animal and plant cells?

Animal Cells
-lipid vessicles uptaked by phagocytosis
-microinjections directly into nucleus

Plant Cells
-High velocity microprojectile gun
-agrobacterium tumifaciens (plant pathogen)