Genetic Engineering

Alex Schiffer7426
Mind Map by Alex Schiffer7426, updated more than 1 year ago
Alex Schiffer7426
Created by Alex Schiffer7426 over 5 years ago


Basics of Genetic Engineering

Resource summary

Genetic Engineering
1 Genome Modification
1.1.1 CAS9 cuts dsDNA
1.1.2 Uses guide RNA
1.1.3 Least specific/ Easiest to program
1.2.1 34 AAs, 12&13 recognize Nucleotides
1.2.2 Recruits FOK1 to cut dsDNA
1.3 Zinc-finger
1.3.1 Most Specific / Hardest to Program
1.3.2 Each Module Recognizes 3 bp
1.3.3 Recruits FOK1 (Cuts dsDNA)
1.4 NHEJ/HEJ - Lambda Red Phage
1.4.1 FLP Recombinase Recognizes FRT
1.4.2 Flank Insert Gene With FRT Sites
1.4.3 FLP Expression Plasmid Eliminates Antibiotic Resistance Gene
1.4.4 Exo, Gamma, Beta
1.5 MAGE
1.5.1 Alters Many Loci at the Same Time
1.5.2 Add Pool of Short Oligos that Create Mismatch/Insertion/Deletion
1.5.3 Various Mutants Created
1.6 CAGE
2 Prokaryotic Gene Regulation
2.1 RNA Polymerase
2.1.1 Holoenzyme = Core + Sigma Core - Transcribes Sigma - Initiates 1st Level of Transcriptional Regulation Regulate Large Numbers of Genes
2.2 Transcription Factors
2.2.1 2nd Level of Transcriptional Regulation
2.2.2 Regulates a Few Genes
2.3 Lac Operon
2.3.1 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
2.3.2 LacZ β-Galactosidase Mutation
2.3.3 LacY Permease Mutation
2.3.4 LacA Transacetylase Mutation
2.3.5 LacI Upstream Mutation Creates a Repressor
3 Eukaryotic Gene Regulation
3.1 Six Types of Regulation
3.1.1 Transcriptional Control Chromatin Mediated Mediators Epigenetic Control
4 Math
4.1 Gibbs Free Energy
4.1.1 When a Reaction will Occur
4.2 Laws of Thermodynamics
4.2.1 1st Law
4.2.2 2nd Law
4.3 Boltzmann Distribution
5 Molecular Cloning
5.1 Traditional Method
5.1.1 Digest, Ligate, Transform
5.1.2 Uses Restriction Enzymes
5.2 Gibson Method
5.2.1 T5 Exonuclease Chews Back Homologous 5' Ends
5.2.2 DNA Fragments Anneal to Each Other
5.2.3 Phusion Polymerase Extends 3' Ends
5.2.4 Taq Ligase Seals Nicks
5.2.5 Doesn't Have to Be Palindromic (Avoids Poisoning)
5.3 Phage λ Recombination
5.4 Gateway System
5.5 In-Fusion Method
5.6 Golden Gate One Pot Digestion Ligation
5.7 Modular Cloning
6 Sequencing
6.1 Sanger
6.2 Next Generation
7 Nucleic Acids
7.1 DNA
7.1.1 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
7.1.2 Replication Semi-Conservative Enzymes RNA Primase DNA Polymerase DNA Helices DNA Ligase Topoisomerase
7.1.3 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
7.1.4 Types B-DNA B'-DNA A-DNA Z-DNA
7.2 RNA
7.2.1 mRNA
7.2.2 tRNA
7.2.3 rRNA
7.3 Glycosidic Bonds
7.4 Phosphodiester Bonds
7.5 Sugars
7.5.1 C2'-Endo (S)
7.5.2 C3'-Endo (N)
8 Proteins
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