Genetic Engineering

Genetic Engineering

1. Genome Modification

   • CRISPR
     • CAS9 cuts dsDNA
     • Uses guide RNA
     • Least specific/ Easiest to program
   • TALEN
     • 34 AAs, 12&13 recognize Nucleotides
     • Recruits FOK1 to cut dsDNA
   • Zinc-finger
     • Most Specific / Hardest to Program
     • Each Module Recognizes 3 bp
     • Recruits FOK1 (Cuts dsDNA)
   • NHEJ/HEJ - Lambda Red Phage
     • FLP Recombinase Recognizes FRT
     • Flank Insert Gene With FRT Sites
     • FLP Expression Plasmid Eliminates Antibiotic Resistance Gene
     • Exo, Gamma, Beta
   • MAGE
     • Alters Many Loci at the Same Time
     • Add Pool of Short Oligos that Create Mismatch/Insertion/Deletion
     • Various Mutants Created
   • CAGE

2. Prokaryotic Gene Regulation

   • RNA Polymerase
     • Holoenzyme = Core + Sigma
       • Core - Transcribes
       • Sigma - Initiates
         • 1st Level of Transcriptional Regulation
         • Regulate Large Numbers of Genes
   • Transcription Factors
     • 2nd Level of Transcriptional Regulation
     • Regulates a Few Genes
   • Lac Operon
     • Lactose Metabolism
       • Lactose - Disaccharide Sugar
       • Lactose Permease
         • Membrane Protein
         • Transport Lactose Into Cell
       • β-Galactosidase
         • Cleaves Lactose
       • Diauxic Shift
         • Small Plateau After Glucose Consumed to Switch to Lactose Usage
         • Carbon Catabolite Repression
           • Presence of Preferred Carbon Source Prevents Use of Secondary Carbon Source
           • CCR in E.coli
             • CAP Protein
       • Lactose Activates Transcription of β-Galactosidase
     • LacZ
       • β-Galactosidase Mutation
     • LacY
       • Permease Mutation
     • LacA
       • Transacetylase Mutation
     • LacI
       • Upstream Mutation
       • Creates a Repressor

3. Eukaryotic Gene Regulation

   • Six Types of Regulation
     • Transcriptional Control
       • Chromatin Mediated
       • Mediators
       • Epigenetic Control

4. Math

   • Gibbs Free Energy
     • When a Reaction will Occur
   • Laws of Thermodynamics
     • 1st Law
     • 2nd Law
   • Boltzmann Distribution

5. Molecular Cloning

   • Traditional Method
     • Digest, Ligate, Transform
     • Uses Restriction Enzymes
   • Gibson Method
     • T5 Exonuclease Chews Back Homologous 5' Ends
     • DNA Fragments Anneal to Each Other
     • Phusion Polymerase Extends 3' Ends
     • Taq Ligase Seals Nicks
     • Doesn't Have to Be Palindromic (Avoids Poisoning)
   • Phage λ Recombination
   • Gateway System
   • In-Fusion Method
   • Golden Gate One Pot Digestion Ligation
   • Modular Cloning

6. Sequencing

   • Sanger
   • Next Generation

7. Nucleic Acids

   • DNA
     • Bases
       • C-G (3 H-bonds)
       • A-T (2 H-bonds)
         • in RNA, T is replaced with U
       • Pyrimidines
         • C, U, T (smaller)
         • Only Anti
       • Purines
         • A & G (bigger)
         • Anti or Syn
     • Replication
       • Semi-Conservative
       • Enzymes
         • RNA Primase
         • DNA Polymerase
         • DNA Helices
         • DNA Ligase
         • Topoisomerase
     • Repair
       • Recognize Errors
         • Exonuclease
         • MutS & MutL
       • Single Strand Repair
         • BER
           • DNA Glycosylase
           • Endonuclease
           • Phosphodiesterase
           • DNA Polymerase
           • DNA Ligase
         • NER
           • Excision Nuclease
           • DNA Helicase
           • DNA Polymerase
           • DNA Ligase
       • Double Strand Repair
         • NHEJ
           • Section of DNA removed
           • Causes Frame Shift
         • HEJ
           • Removes Damaged DNA
           • Uses Sister Chromatid to Repair
     • Types
       • B-DNA
       • B'-DNA
       • A-DNA
       • Z-DNA
   • RNA
     • mRNA
     • tRNA
     • rRNA
   • Glycosidic Bonds
   • Phosphodiester Bonds
   • Sugars
     • C2'-Endo (S)
     • C3'-Endo (N)

8. Proteins