Flashcards in Lecture 18: Genetic Engineering Deck (29)
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?
-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?
-code proteins that make it obvious this is the altered gene (fluoresence proteins)
-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?
-carry small DNA pieces
-inserted by transformation
-short term storage
-carry larger DNA pieces
-inserted by transduction
-long term storage
Ideal vectors are able to detect what?
-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?
-Vectors with regulatory gene sequences
-For controlled expression of inserted gene
-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
Competent (easily transformed w/ foreign DNA)
Stable in culture
What are the types of hosts used for gene cloning?
-disadvantage: no expression of eukaryotic proteins
-accommodates eukaryotic protein expression
-disadvantage: unstable plasmids
What are some examples of prokaryotic hosts?
What are some examples of eukaryotic hosts?
-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