4 Genetic Engineering Flashcards

Flashcards covering theoretical foundations of genetic engineering, including basic lab techniques, enzymology, molecular cloning, and vectors.

Basic steps of molecular cloning of genes

Construction of recombinant DNA molecule, Transport into the host cell, Multiplication of recombinant DNA molecule, Division of host cell, Numerous cell divisions resulting in a clone

Step 1 of molecular cloning

A fragment of DNA containing the gene to be cloned is inserted into a vector to produce a recombinant DNA molecule.

Step 2 of molecular cloning

The vector transports the gene into a host cell, which is usually a bacterium.

Step 3 of molecular cloning

Within the host cell, the vector multiplies, producing numerous identical copies of itself and the gene it carries.

Step 4 of molecular cloning

When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place.

Step 5 of molecular cloning

After a large number of cell divisions, a colony, or clone, of identical host cells is produced, each containing one or more copies of the recombinant DNA molecule.

Methods for obtaining fragments to be cloned

PCR, chemical synthesis, cDNA synthesis

Forms in which Plasmids can be found

CCC DNA (Covalently Closed DNA), OC DNA (Open Circular DNA), Linear DNA

Migration of Supercoiled DNA in electrophoresis

Supercoiled DNA migrates faster

Size of Plasmids

Vary in Nucleotide Length and Molecular Mass

Origin of Plasmids

Prokaryotes, except for plasmid 2 mm (yeast)

Non-conjugative Plasmids

Cannot be transferred by conjugation; generally high number of copies

Conjugative Plasmids

tra genes: transfer by conjugation between bacteria; generally few copies

Autonomous or non-integrative Plasmids

Independently replicated

Episomes

Integrated into the chromosome and replicate when it replicates

ori in plasmids

Origin of replication

Main replication proteins encoded in the chromosome near the ori

DNA polymerases

Function of the ori

Confer bacterial host specificity

Relaxed Plasmids

High number of copies

Stringent Plasmids

Low number of copies

Incompatibility in Plasmids

Plasmids with very similar characteristics that are incompatible with each other within the same bacterium due to the same replication control mechanism.

Incompatibility groups

they include plasmids that are incompatible with each other

Basic features for use in cloning

Small size, selectable, unique restriction sites

Advantages of small and low molecular weight plasmids

Easier cloning, more resistant to breakage, largest number of copies

Methods to select colonies with a vector

Antibiotic resistance and reverse auxotrophy

Examples of antibiotic resistance genes

Ampicillin, Kanamycin, Chloramphenicol, Tetracycline, Streptomycin

Reverse auxotrophy

Mutant bacteria that only grow in minimal medium when they have incorporated the plasmid

ccdB gene

Induces the cleavage of bacterial DNA by DNA gyrase

Tracking Gene (screening or reporter genes)

Genes that are inactivated when the insert enters the vector, allowing for visual examination to identify colonies with vector + insert

Examples of Tracking Gene (screening or reporter genes)

lacZ gene encoding β-galactosidase

lacZ SYSTEM

Requires adding Xgal + IPTG to the medium

Genotype of E. coli DH5α strain

fhuA2 lac(del)U169 phoA glnV44 Φ80' Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17

Polylinker or Multiple Cloning Site (MCS)

The area of the vector containing the unique restriction sites

Plasmid to use as the plasmid vector to express gfp protein in bacterial cells

pARA plasmid

Reasons pARA plasmid is the best choice

Once the gfp DNA fragment is inserted, the pARA plasmid could express the gfp protein. The pARA recombinant plasmid would be maintained in bacterial cells as they replicate.

Two basic structural types of phages

Head-and-tail and Filamentous

Lytic phages

Endogenous gene replication of the phage, Ex. Phage T4, T3, T7

Lysogenic phages

Incorporation into the bacterial genome lytic phase destroys host cells E.g. Phage l (lambda)

cos sites of phage lambda

Allows recirculation when injecting the DNA and circle replication rolling

Cloning strategies with phage lambda

Circular and linear strategies

Types of vectors based on phage lambda

INSERTIONAL and REPLACEMENT