Using the lens of genomics to create synthetic cells and CRISPER-CAS9 to edit a bacterial genome.
Vocabulary:
Genome: the complete set of genes or genetic material present in a cell or organism.
Transposon: "jumping gene" A peice of DNA that can integrate randomly, into a peice of DNA.
Mirror life: is a hypothetical form of life with mirror-reflected molecular building blocks.
Cytoskeleton: a microscopic network of protein filaments and tubules in the cytoplasm of many living cells, giving them shape and coherence.
Wet science: biologist's slang
Living cell: many deffinitions, but one is "somethign that can be infected by a virus"
Spheroplast: a bacterium or plant cell bound by its plasma membrane, the cell wall being deficient or lacking and the whole having a spherical form.
CRISPR: CRISPRs are specialized stretches of DNA. The protein Cas9 (or "CRISPR-associated") is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA. CRISPR is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments from viruses that have previously infected the prokaryote and are used to detect and destroy DNA from similar viruses during subsequent infections.
PAM sequence: Protospacer adjacent motif (PAM) is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.
TALEN (Transcription activator-like effector nucleases): restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands).
Zinc fingers: Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations.
reference
Molecular scissors: Resriction endonucleases that cleave DNA as a specific site.
Lab notes:
This weeks lab was designed by Pranam, who lead us through testing a few experimental CAS9 alternatives he designed. We mixed the CAS proteins and a guide RNA into cells that already had 3 plasmids for antibiotic resistance. We electroporated the cells so they would intake the new material.
Circuit plasmid and guide plasmid were already in the cell. We inserted a dCAS9 plasmid and if successful the cells would turn green.
The number of cells that worked were measured with a flow cytometer.
Q. What tasks must a cell perform to live?
A. 1. Transporting Molecules 2. Metablolism 3. Reproduction. Reference: Cell Life Functions
Another definition: In a membrain vesicle, producing macromolecules (transport/export), doing everything but reproducing is it alive? What about a sterile human?
When thinking about the definition as "able to be infected by a virus"
Deal with the effects of sloppy enzimes and repair itself.
Q. What bacterial innate immune mechanisms must be overcome to perform genome transplantation?
A. Innate immune mechanisms exist in bateria to fight against viral pathogens. These can also target the mechanisms bringing in the new gene.
If a phage enters a bacterial cell restriction enzimes attack it.
Q. What mammalian innate immune mechanisms must be overcome to efficiently install large DNA molecules in mammalian cells?
Mammalian cells don't like DNA in the cytoplasm. If DNA is in the cytoplasm odds are it came from a virus. These cells do exocytosis, RNAi, all the mechanisms that recognize and tag intruders. Most viruses have preventetive measures to avoid these immune responses.
