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BIO 425: Microbiology > Lecture 18: Genetic Engineering > Flashcards

Lecture 18: Genetic Engineering Flashcards

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1
Q

What is genetic engineering?

A

In vitro isolation, alteration+expression of DNA/RNA

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2
Q

What is recombinant DNA technology used for?

A

Combine DNA from multiple organisms into one molecule

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3
Q

What is a transgenic organism?

A

Genes from 2 organisms placed into 1 organism

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4
Q

What is the asilomar conference?

A

1975 meeting of recombinant DNA scientists

-self regulation and informing the public

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5
Q

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

A
  1. Safety - ok to eat
  2. Ethics - moral responsibilities
  3. Economics - trademarking life
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6
Q

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

A

1st genetic engineering product was Human insulin

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7
Q

When manipulating DNA, what techniques are used and why?

A
  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
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8
Q

How do restriction enzymes work to cut DNA into pieces?

A

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

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9
Q

How is gel electrophoresis used to separate DNA pieces?

A 
DNA molecules (-) charged
-electrical field used to migrate DNA
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10
Q

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

A

Denatures DNA
-dsDNA to ssDNA

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

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11
Q

How is molecular cloning used to copy DNA pieces?

A

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

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12
Q

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

A

Targeted mutation of gene sequences through base pair changes

  • Gene knockouts remove entire gene from genome
  • allows study of gene function/effect on organism
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13
Q

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

A

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)

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14
Q

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

A

Ligation - accepts new genes

Gene transfer - transport genes into host cells

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15
Q

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

A

Gene transfer - accepts vectors with new genes

Gene replication and expression - copy new genes

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16
Q

What are the types of vectors used for gene cloning?

A

Plasmid Vecotrs

  • carry small DNA pieces
  • inserted by transformation
  • short term storage

Bacteriophage vectors

  • carry larger DNA pieces
  • inserted by transduction
  • long term storage
17
Q

Ideal vectors are able to detect what?

A

Successful ligation
-presence of DNA piece in vector

Successful Gene transfer
-presence of DNA + vector in host

18
Q

What are the important features of plasmid vectors?

A

(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

19
Q

What are some advanced plasmid vectors?

A

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
20
Q

How do expression vectors work?

A

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
21
Q

What is the bacteriophage T7 system?

A

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

22
Q

How does bacteriophage lambda work?

A
  1. Insert DNA piece
  2. Repackage into head
  3. Assemble virions
  4. Infect host cells
23
Q

What are artificial chromosome vectors?

A

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
24
Q

What are the general characteristics of ideal hosts?

A

Fast growth rate

Non pathogenic

Competent (easily transformed w/ foreign DNA)

Stable in culture

25
Q

What are the types of hosts used for gene cloning?

A

Prokaryotic

  • most common
  • disadvantage: no expression of eukaryotic proteins

Eukaryotic

  • accommodates eukaryotic protein expression
  • disadvantage: unstable plasmids
26
Q

What are some examples of prokaryotic hosts?

A

Esherichia coli
-potentially pathogenic

Bacillus sutilis
-nonpathogenic

27
Q

What are some examples of eukaryotic hosts?

A

Saccharomyces cerevisiae
-yeast

Cell cultures

  • animal and plant cell lines
  • delicate and expensive
  • limited large scale production
28
Q

How do we transfer vectors into hosts?

A

Transformation

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

Transduction
-virus mediated transfer

Conjugation

29
Q

How is transformation accomplished in animal and plant cells?

A

Animal Cells

  • lipid vessicles uptaked by phagocytosis
  • microinjections directly into nucleus

Plant Cells

  • High velocity microprojectile gun
  • agrobacterium tumifaciens (plant pathogen)

BIO 425: Microbiology (22 decks)

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