02 - Code Breakers: Using CRISPR to Rewrite Genetics
Experiment Summary THE CELL •
LyphoCells™ E. coli Purple: The host bacteria have been genetically engineered to pro- duce a purple pigment protein in the presence of IPTG. The genome has been modified to contain the gene for T7 polymerase under the control of the lac operon. The bacteria have also been transformed with a plasmid containing the Purple Chromoprotein under the control of the T7 promoter. When IPTG is added it de-represses (activates) the lac operon which allows the cell to synthesize T7 polymerase. This polymerase binds to the plasmid and turns on production of the purple protein.
THE PLASMIDS •
pCas9-PurpleGuide: This plasmid contains the Cas9 gene and a gRNA that targets the purple chromo- protein gene. It also contains a chloramphenicol resistance gene. pCas9-ControlGuide: This plasmid contains the Cas9 gene and a non-targeting guide RNA with a random sequence. This sequence was carefully checked against the E. coli purple genome and confirmed to be absent, ensuring that it won’t target any genomic sites during the experiment. The plasmid also contains a chloramphenicol resistance gene.
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THE PLATES This experiment uses three additives to the plates: • IPTG (Isopropyl β-D-1-thiogalactopyranoside):
This compound induces the expression of genes controlled by the lac operon. It mimics the natural inducer of the lac operon, lactose, but unlike lactose, it is not metabolized by the cell, making it a more controlled and reliable inducer for gene expression. • Chloramphenicol: A popular antibiotic and selective agent in bacterial culture media. Effective against a range of bacteria by inhibiting their protein synthesis. • Ampicillin : A broad-spectrum antibiotic belonging to the penicillin group that works by inhibiting the synthesis of bacterial cell walls.
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