Lecture 16. Plant Cell Engineering 1 Flashcards
What does the top down approach to synthetic biology in plants result in?
Excising plants can be tweaked for higher efficiency
What does the bottom up approach to synthetic biology in plants result in?
A new plant can be built from scratch using individual parts
Why are plants more complicated than existing synthetic models?
- Have interconnected complex signalling pathways 2. More organs (chloroplasts) and cell walls
- Very large genomes
- Multicellular Organism
- Ethical issues of synthetic biology and the Genetically Modified Organism (GMO) phobia
What is golden gate cloning/molecular cloning?
Molecular cloning simply means copying a DNA sequence (usually genes, and usually for the purpose of expression)
What two things are required for molecular cloning?
DNA to be amplified and cloned
Vector (or: plasmid) to carry and replicate the DNA in bacteria (insert plasmid into amplified or cloned DNA)
What is autosequence?
Put in known sequence, and get double stranded DNA
What is the role of regulatory elements of plants in synthetic biology?
Plant promoters are often very long and include series of responsive elements or enhancer sequences that allow activation by different transcription factors Synthetic promoters can be designed to include the required elements for desired localisation or expression profile in a short sequence
How do bacteria use restriction enzymes normally?
These enzymes cut up “foreign” DNA that invades the cell, recognise DNA chains at specific sites, bind and cleave the DNA
What is the difference between type II and IIS restriction enzymes?
Type IIS cuts several base pairs after recognised site
What happens in golden gate cloning?
Digestion – Type IIS enzymes cut the DNA, creating unique overhangs.
Ligation – A DNA ligase joins the fragments in the correct order.
One-pot reaction – The simultaneous digestion and ligation increase efficiency
What happens in genome editing?
Genetic engineering at specific locations in genome via engineered nucleases (resulting in insertions, deletions or replacements in DNA)
Nucleases recognise specific sequences in DNA and create double strand breaks
What are TALENs?
Transcription Activator-Like Effector Nucleases
Artificial restriction enzymes
Generated by fusion of TAL effector DNA-binding domain with a endonuclease DNA cleaving domain
Efficient, programmable, and can be used for DNA cleavage at specific target sites
Powerful tools for genome editing in cells
Frequency of TALEN hits: 1 in 30-50 nucleotides
Can TALENs be sent everywhere?
Most places yes, some no
How has TALEN been used?
Acute lymphoblastic leukemia treated by allowing patient to receive modified immune cells from healthy donor
TALEN to tweak donor T cells to target leukemia but not harm host patient (by disabling CD52 in donor T cells to mask from patients immune system)
What can modified TALENs be used for?
Can be modified to make synthetic transcription factors by fusing TALE DNA-binding domains to endogenous activation domains
Huge but outshone by CRISPR/Cas9
