Module 8: Molecular Biology Revolution

• sanger sequencing is used for small amounts of DNA
• illumina and oxford nanopore sequencing are used for large amounts of DNA
• illumina sequencing: short sequences, finds base pairs
• oxford nanopore sequencing: long sequences, finds overall structure
• kBase RASTtk: finds genes in DNA sequence, predicts open reading frame and protein sequence
• how can you predict protein function?

  GenBank database and BLAST program
• kbase metabolic model puts protein functions into pathways and predicts amylases
• mobile genetic elements are sequences of DNA capable of hopping between chromosomes
• what are the 2 types of mobile genetic elements?

  insertion sequences and transposons
• insertion sequences code for functions that move DNA
• transposons are useful genes for hosts that cause mutations (ex. antibiotic resistance)
• plr27 transposon is outside the insertion sequence of tn5
• prl27 has hypertransposase: high frequency of transposition
• what is unique about the prl27 origin of replication?

  e. coli specific, suicide plasmid if moved to different organism
• prl27 origin of transfer allows conjugation when tra genes are present
• crispr is bacteria immune system
• steps of crispr:

  1. restriction enzymes attack viral DNA
  2. crispr adds viral DNA to repeat spacer array
  3. DNA is transcribed into guide RNA and association with cas9 protein
  4. cas9 digests reintroduced viral DNA
• how is crisp used in genome editing?

  repairs doubled stranded DNA break by homology-directed repair (eukaryotes) and recombination (bacteria)
• cpec clonin uses pcr with overlapping ends of target plasmid
• cpec cloning steps:

  1. amplified target sequence is incorporated into vector
  2. second PCR reaction transfers plasmid to bacteria
• what are the 4 major protein expression systems?

  1. bacteria
  2. yeast
  3. insect cells
  4. mammalian cells