Restriction Enzymes and Selection
The specific modification on parental DNA adenine bases that DpnI recognizes before digestion
What is methylation?
The specific strain of E. coli used to transform and replicate plasmids in this experiment.
What is NEB-5α E. coli?
The gene targeted for tracking specific structural changes introduced by the barcode fragment.
What is the ADE2 gene?
The error-prone repair pathway that takes over when no repair template is available
What is Non-Homologous End Joining (NHEJ)?
The visual phenotypic shift indicating successful knockout of the ADE2 gene.
What is yeast turning from white to red?
The reason why PCR products do not contain methylation required for DpnI digestion
What is that PCR cannot amplify methylation?
The key advantage of using E. coli over yeast for initial plasmid replication.
What is that E. coli is easier to transform than yeast?
The primary repair pathway that utilizes the repair mutation carried by the editing fragment.
What is Homology-Directed Repair (HDR)?
The specific types of uncontrolled genetic mutations caused by NHEJ repair.
What are random insertions and deletions (indels)?
The 3rd-generation sequencing technology used to read DNA sequences and confirm target mutations.
What is Nanopore sequencing?
The restriction enzyme used to cleave parental plasmids while leaving newly synthesized recombinant plasmids intact
What is DpnI?
The cellular machinery within E. coli used to yield high copy numbers of plasmid DNA.
What is bacterial replication machinery/origin of replication?
The precise genetic outcome made possible when the editing fragment provides a donor repair mutation.
What is precise gene editing?
gRNA and Cas9 activity if the editing fragment is forgotten during transformation.
What is functioning normally (gRNA still takes Cas9 to dsDNA, increases affinity, and cas9 cuts)?
The molecular technique combined with gel electrophoresis to compare product band sizes against a control.
What is PCR (Polymerase Chain Reaction)?
The two plasmid forms (parental and recombinant) that cannot be differentiated without DpnI
What are pCAS and pCAS+gRNA?
The step that must occur with bacterial colonies prior to carrying out co-yeast transformation.
What is plasmid isolation?
The role of the barcode fragment.
What is tracking exactly where the specific change is inserted in the ADE2 gene?
The reason precise gene editing fails when the barcode fragment is omitted.
What is that HDR cannot happen without a repair template/mutation?
The parameters compared during gel electrophoresis to verify gene editing success against wild-type control.
What are DNA bands/fragment size?
The exact mechanism allowing DpnI to selectively cleave parental plasmids instead of recombinant plasmids
What is DpnI recognizes methylation on adenine to digest the parental pCAS plasmid, while unmethylated PCR products (pCAS+gRNA) remain intact?
The purpose of transforming plasmids into E. coli when yeast is the ultimate target organism.
What is using E. coli replication machinery to make multiple copies of plasmids for isolation before co-yeast transformation, because E. coli is easier to transform than yeast?
The functions of the barcode/editing fragment in host yeast cells.
What is carrying the repair mutation for HDR to allow precise gene editing, and acting as a barcode to track where the specific change was inserted into ADE2?
The complete cellular consequence of forgetting to transform the barcode/editing fragment into competent yeast.
What is gRNA and Cas9 still cutting dsDNA normally, but cell repair defaults to error-prone NHEJ (causing random indels) instead of precise HDR editing due to the missing repair template?
Three distinct laboratory techniques that could be used to confirm successful genome editing in this experiment.
What are: 1) Phenotypic change (white to red), 2) 3rd-gen Nanopore sequencing, and 3) PCR + gel electrophoresis comparing band sizes against a control?