Genetic Engineering

Genetic Engineering

Genome Modification
1. CRISPR
  1. CAS9 cuts dsDNA
  2. Uses guide RNA
  3. Least specific/ Easiest to program
2. TALEN
  1. 34 AAs, 12&13 recognize Nucleotides
  2. Recruits FOK1 to cut dsDNA
3. Zinc-finger
  1. Most Specific / Hardest to Program
  2. Each Module Recognizes 3 bp
  3. Recruits FOK1 (Cuts dsDNA)
4. NHEJ/HEJ - Lambda Red Phage
  1. FLP Recombinase Recognizes FRT
  2. Flank Insert Gene With FRT Sites
  3. FLP Expression Plasmid Eliminates Antibiotic Resistance Gene
  4. Exo, Gamma, Beta
5. MAGE
  1. Alters Many Loci at the Same Time
  2. Add Pool of Short Oligos that Create Mismatch/Insertion/Deletion
  3. Various Mutants Created
6. CAGE
Prokaryotic Gene Regulation
1. RNA Polymerase
  1. Holoenzyme = Core + Sigma
    1. Core - Transcribes
    2. Sigma - Initiates
      1. 1st Level of Transcriptional Regulation
      2. Regulate Large Numbers of Genes
2. Transcription Factors
  1. 2nd Level of Transcriptional Regulation
  2. Regulates a Few Genes
3. Lac Operon
  1. Lactose Metabolism
    1. Lactose - Disaccharide Sugar
    2. Lactose Permease
      1. Membrane Protein
      2. Transport Lactose Into Cell
    3. β-Galactosidase
      1. Cleaves Lactose
    4. Diauxic Shift
      1. Small Plateau After Glucose Consumed to Switch to Lactose Usage
      2. Carbon Catabolite Repression
        Presence of Preferred Carbon Source Prevents Use of Secondary Carbon Source
        CCR in E.coli
        CAP Protein
    5. Lactose Activates Transcription of β-Galactosidase
  2. LacZ
    1. β-Galactosidase Mutation
  3. LacY
    1. Permease Mutation
  4. LacA
    1. Transacetylase Mutation
  5. LacI
    1. Upstream Mutation
    2. Creates a Repressor
Eukaryotic Gene Regulation
1. Six Types of Regulation
  1. Transcriptional Control
    1. Chromatin Mediated
    2. Mediators
    3. Epigenetic Control
Math
1. Gibbs Free Energy
  1. When a Reaction will Occur
2. Laws of Thermodynamics
  1. 1st Law
  2. 2nd Law
3. Boltzmann Distribution
Molecular Cloning
1. Traditional Method
  1. Digest, Ligate, Transform
  2. Uses Restriction Enzymes
2. Gibson Method
  1. T5 Exonuclease Chews Back Homologous 5' Ends
  2. DNA Fragments Anneal to Each Other
  3. Phusion Polymerase Extends 3' Ends
  4. Taq Ligase Seals Nicks
  5. Doesn't Have to Be Palindromic (Avoids Poisoning)
3. Phage λ Recombination
4. Gateway System
5. In-Fusion Method
6. Golden Gate One Pot Digestion Ligation
7. Modular Cloning
Sequencing
1. Sanger
2. Next Generation
Nucleic Acids
1. DNA
  1. Bases
    1. C-G (3 H-bonds)
    2. A-T (2 H-bonds)
      1. in RNA, T is replaced with U
    3. Pyrimidines
      1. C, U, T (smaller)
      2. Only Anti
    4. Purines
      1. A & G (bigger)
      2. Anti or Syn
  2. Replication
    1. Semi-Conservative
    2. Enzymes
      1. RNA Primase
      2. DNA Polymerase
      3. DNA Helices
      4. DNA Ligase
      5. Topoisomerase
  3. Repair
    1. Recognize Errors
      1. Exonuclease
      2. MutS & MutL
    2. Single Strand Repair
      1. BER
        DNA Glycosylase
        Endonuclease
        Phosphodiesterase
        DNA Polymerase
        DNA Ligase
      2. NER
        Excision Nuclease
        DNA Helicase
        DNA Polymerase
        DNA Ligase
    3. Double Strand Repair
      1. NHEJ
        Section of DNA removed
        Causes Frame Shift
      2. HEJ
        Removes Damaged DNA
        Uses Sister Chromatid to Repair
  4. Types
    1. B-DNA
    2. B'-DNA
    3. A-DNA
    4. Z-DNA
2. RNA
  1. mRNA
  2. tRNA
  3. rRNA
3. Glycosidic Bonds
4. Phosphodiester Bonds
5. Sugars
  1. C2'-Endo (S)
  2. C3'-Endo (N)
Proteins

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	Criado por Alex Schiffer7426 aproximadamente 9 anos atrás