Week 7: Biotechnology and Bioengineering

Genetic Engineering

The modification and manipulation of an organism's genome using technology, began in the early 1970s

Herbert Boyer and Stanley Cohen

Created the first GMO in 1973 by inserting antibiotic resistance genes into E. coli

Genentech

Produced the first commercial GMO product (human insulin) in 1978 using recombinant E. coli

Genetic Vectors

DNA molecules used to carry foreign genetic material into host organisms

Plasmids

Primarily used to transform bacteria, some (shuttle vectors) work in eukaryotes

Bacteriophage

Derived from λ and M13 genomes, used to transduce bacteria, Can integrate (lysogenic) or not integrate (lytic) into the host genome

Cosmids

Hybrid of plasmid and λ phage DNA, used to transform E. coli

Artificial Chromosomes

Circular (BAC) or linear (YAC) vectors for large insert sizes

BAC (Bacterial Artificial Chromosome)

Can carry inserts up to 100–300 kb

YAC (Yeast Artificial Chromosome)

Can carry inserts up to 1–2 Mb

Plasmid Vectors

Small, circular, double-stranded DNA molecules engineered to carry, replicate, and express foreign genetic material

Key Components of Plasmid Vectors

Origin of replication, multiple cloning site, promoter/regulatory elements, selectable marker, and reporter genes

Origin of replication

Allows the plasmid to replicate independently within the host cell

Multiple Cloning Site

Contains unique sequences for inserting foreign DNA

Promoter/Regulatory Elements

Drive the transcription of inserted genes

Selectable Marker

Antibiotic resistance genes to select for positive cells

Reporter Genes

Visual markers like lacZ (β-galactosidase) or GFP (green fluorescent protein)

Restriction Enzyme Cloning

A technique for generating recombinant DNA using restriction enzymes to cut DNA and DNA ligase to join fragments

Sticky Ends

Single-stranded DNA overhangs created by restriction enzymes, used for ligation

Type IIS Restriction Enzyme

Generates user-defined sticky ends for Golden Gate Assembly

Golden Gate Assembly

A restriction enzyme-based technique for assembling multiple DNA fragments into a single construct

DNA Ligase

Joins compatible sticky ends of adjacent DNA fragments

Gibson Assembly

A cloning technique that joins linear vector and DNA inserts into a circular DNA molecule without restriction enzymes

5’ Exonuclease

Chews back the 5’ ends of DNA fragments in Gibson Assembly

DNA Polymerase

Fills gaps by extending the 3’ ends in Gibson Assembly

DNA Delivery Methods

Techniques for introducing DNA into host cells

Transformation

DNA delivery into prokaryotic cells, methods include heat shock and electroporation

Transfection

DNA delivery into eukaryotic cells, methods include lipofection, gene gun, and microinjection

Heat Shock Transformation

A method of transformation using calcium chloride and heat to make cells competent

Electroporation

Uses electric pulses to create temporary membrane pores for DNA entry

Lipofection

DNA encapsulated in cationic lipid vesicles (liposomes) for delivery into cells

Gene Gun

DNA-coated gold particles propelled into cells, common in plants

Microinjection

DNA injection using a fine needle, used for oocytes or embryos

Viral Vectors

Engineered viruses used to deliver genetic material into plant and animal cells

Lentivirus

Integrates into the host genome, effective for dividing and non-dividing cells

Adenovirus

Does not integrate into the genome, leads to transient expression

Adeno-associated Virus

Provides stable but non-integrating gene expression

Retrovirus

Integrates into the host genome of dividing cells

Protein Engineering

The design and modification of proteins to enhance or alter their function, stability, or specificity

Directed Evolution

A technique that mimics natural selection to improve or modify protein function

Random Mutagenesis

Introduces random mutations via error-prone PCR or DNA shuffling

Screening/Selection

Identifies improved protein variants from a library of mutants

Rational Design

An engineering approach involving knowledge-based modifications to improve or alter protein function

Site-directed Mutagenesis

Introduces specific mutations to improve protein function

De Novo Design

The computational and experimental creation of entirely new proteins from scratch

Computational Design

Uses AI and molecular modeling to predict sequences for stable, functional 3D structures

CRISPR/Cas

A gene-editing technology derived from a bacterial immune defense mechanism

Cas9

A protein that forms a complex with guide RNA (sgRNA) to cleave complementary DNA sequences

sgRNA

Small guide RNA that directs Cas9 to a specific DNA sequence for cleavage