Biology 1002B Lecture Notes - Lecture 10: Restriction Enzyme, Channelrhodopsin, Dna Ligase
13 Jun 2018
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Lecture 20 Outcomes:
Signalling Pathways
- EGF (Epidermal Growth Factor): signalling pathway, signal is detected at the surface, then
passed along to through the net to the nucleus
- once the signal reaches the nucleus, the regulatory protein can bind DNA to cause a change
in expression
basic mechanism of propagation of action potentials in neurons
- propagated by movement of ions across membranes (selectively permeable membrane)
causes the neuron to depolarize
- ions flow into the cell causing depolarization
- useful because if you can control the polarization you can control the firing of the neurons,
therefore you can control the brain
how channelrhodopsin can control action potentials in neurons
- channelrhodospin opens when light is shined on it, therefore it allows ions to flow through
- blue light causes neurons to depolarize and fire
- channelrhodopsin was taken from chlamy cells and put into the neurons of animals
possible sources of genes (ie. channelrhodopsin) for use in making transgenic
organisms.
- genomic digest: use restriction enzymes to create recombinant DNA that includes that section
of the chlamy genome
- cDNA: get mRNA from chlamy & use reverse transcription to produce a double stranded
molecule of DNA
- DNA synthesis: Various type of DNA synthesizers or printers can make genes to order
role of restriction endonuclease enzymes in creating recombinant DNA
- restriction enzymes made sequence specific cuts, leaving staggered ends, to make
recombinant DNA using DNA ligase
Basic mechanism for creation of cDNA
- use reverse transcription to produce a double stranded molecule of DNA (cDNA) from a
single-stranded RNA
Ways in which cDNA versions of genes would be different than genomic versions.
- the cDNA wouldn’t have a promotor (not transcribed, not in RNA therefore not going to be in
cDNA)
- cDNA would not have introns
- cDNA would not include a TATA binding site
- cDNA has a poly-A tail and a poly-T tail
-
role of plasmid (expression) vectors in genetic engineering
find more resources at oneclass.com
find more resources at oneclass.com
- transformation: put plasmid into a flask (plasmid vector) and get the cells to take it up from
their environment (by altering the ion concentration, temperature, and permeability of the
membrane, or electroporation: use electric shock to get the cell to take it up)
- expression vector: specialized plasmid that contains all the sequences/components for
recombination (promotor, transcription terminator, places to cut the DNA)
- a DNA molecule used to artificially carry foreign genetic material into another cell, to be
replicated or expressed
role of antibiotic resistance genes on vectors in genetic engineering
- the transformed bacteria will survive on the vector containing the antibiotic, and therefore will
reproduce to grow a population of resistant bacteria
mechanisms for introducing transgenes into microbes, plants and animals
- Bioistic gene guns deliver transgenes to plant nuclei or organelles
- can transform chloroplasts, plants such as tobacco used
- TI plasmid: (Rhizobium radiobacter - prokaryote) natural system that knows how to send
some of its genes to the nucleus of a eukaryote
- put our own DNA into the TI plasmid and it will move the DNA for us
- Viruses are natural genetic engineering machines, they deliver DNA into the nucleus of cells
(animals, and plants)
- adenovirus popular vehicle for delivering DNA
- Microinjections: can inject DNA into the nucleus using a tiny needle to grow a transformed
being
- Transformation: grow cells on a culture containing the gene of choice, cells that take up the
genes (vector) survive, other don’t
- then reintroduce transformed cells into the animal embryo and let it undergo cell division
- will produce mosaic offspring that could produce some transgenic gametes, then the next
generation could have homozygous babies if the mosaics are crossed
role of reporter genes (eg. GFP) in genetic engineering
- confirm uptake
- makes a product that is visible
- ex. GFP green florescent protein, lacZ: Xgall turns blue in the presence of lacZ (luciferase is
the enzyme organisms use to biofluoresce)
strategies for expressing channelrhodopsin in specific neurons.
- if the transferred gene is chlamy, the mouse would die if it was exposed to blue light because
it has channelrhodopsin in all their cells not just neurons
- channelrhodospin should only be present in the neurons of the organisms brain
- can use a virus that only infects neurons (such as adenovirus)
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Egf (epidermal growth factor): signalling pathway, signal is detected at the surface, then passed along to through the net to the nucleus. Once the signal reaches the nucleus, the regulatory protein can bind dna to cause a change in expression basic mechanism of propagation of action potentials in neurons. Propagated by movement of ions across membranes (selectively permeable membrane) causes the neuron to depolarize. Ions flow into the cell causing depolarization. Useful because if you can control the polarization you can control the firing of the neurons, therefore you can control the brain how channelrhodopsin can control action potentials in neurons. Channelrhodospin opens when light is shined on it, therefore it allows ions to flow through. Blue light causes neurons to depolarize and fire. Channelrhodopsin was taken from chlamy cells and put into the neurons of animals possible sources of genes (ie. channelrhodopsin) for use in making transgenic organisms.
