Lecture 10 - Genetic Engineering

Genetic Engineering

the direct manipulation of DNA to alter an organism's characteristics in a particular way.

How to clone a human gene (insulin) into a bacterium:

1. Purify plasmid

2. Make many copies of target gene DNA (insert)

3. Use restriction enzymes* to cut insert and plasmid

4. Ligate cut insert into cut plasmid with DNA ligase*

5. Transform cells with recombinant plasmid

6. Induce expression of gene

7. Purify products

1. Purifying Plasmid

Plasmid

A small ring of DNA that carries accessory genes separate from those of the bacterial chromosome

Vectors

small pieces of DNA that can be stably maintained by the recipient

can introduce foreign fragments to encode new properties

they have direction; trasmit genes from one organism to another

Cloning Vector

a DNA molecule used to carry and deliver foreign genetic material into a host cell, enabling the replication and amplification of that DNA.

Plasmid Properties

contains a origin of replication

selectable marker (AbxR)

restriction enzyme cut sites

high copy number

small size

2. Make many copies of target gene DNA (insert)

Polymerase Chain Reaction (PCR)

an in-vitro method of amplifying DNA replication of an insert to produce a bunch of copies

PCR X Insulin

insulin gene contains introns so it must be converted to cDNA

Complementary DNA (cDNA)

reverse transciptase performs reverse transcription on mRNA (introns are spliced out) to make DNA without any introns

Distinguish the process to "clone" a prokaryotic gene and a eukaryoticgene into a prokaryote.

Prokaryotic DNA: already intron free

Eukaryotic DNA: has introns, must do reverse transcription (must DNA → mRNA → cDNA)

PCR: Denaturation

Heat (95C) briefly to separate DNA strands

PCR: Annealing

DNA sample is cooled (60C) allowing primers to attach to opposite ends of the target sequence

PCR: Extension

increase temperature - add DNA polymerase and nucleotides to produce two complete strands

3. Use restriction enzymes to cut insert and plasmid

Restriction Enzymes (RE)

endonucleases that cut (hydrolyze the phosphodieester bond of) the phosphate-sugar backbone of dsDNA at specific sequences

Endonuclease

cut inside a DNA strand (e.g., restriction enzymes in defense, or in repair).

Exonucleases

chew DNA from the ends (important in repair and DNA processing)

Restriction-Modification (RM) System

to protect aaginst phase infection

to protect host DNA with recognition sequence

RM System: Enzymes

Restriction Enzymes (RE)

Methylase

To protect against phage infection

a RE cuts dsDNA (e.,g viral genomes) that carry the RE's recognition sequence, which prvents replication of the viral genome

To protect host DNA with the recognition sequence

A methylase that recognizes the same DNA sequence as its RE partner modifies the DNA by adding a methyl group; methylation at its recognition site inhibits the partner RE from cutting the DNA

Gel Purification

isolates the DNA segment that was cut ("insert")

Electrophoresis

A process where DNA fragments are separated according to size using electrical charges, separating the insert

4. Ligate cut insert into cut plasmid with DNA ligase*

DNA ligase joins together stickey ends of the DNA insert and the plasmid = recombinant plasmid

5. Transform cells with recombinant plasmid

perform artificial transformation with recombinant plasmid. then, plate treated cells onto media with selection (e.g., antibiotic) to select for transformants carrying the selectable marker (e.g.,abxR)

Methylation

chemical tag that marks host DNA as "self," and also signals during replication/repair

CRISPR

clustered, regularly interspaced short palindromic repeats

CRISPR-Cas

a system of bacterial immune defense

CRISPR-Cas Mechanism

1. Spacers

2. crRNA

3. Cas9

Spacers

CRISPR array is assembled on bacterial chromosome by the insertion of phage sequences (spacers) and a CRISPR-specific repeated sequence

crRNA

CRISPR array is transcribed to produce precursor RNA (pre-crRNA)

3. Cas9

crRNA that match viral sequence recruits Cas9 which cuts the viral genome, preventing phage replication

CRISPR/Cas9 X Genome Editing

Use Cas9 to cut a specific section of DNA to precisely delete, insert, or modify a gene