Synthetic biology. A lab manual

Description

Contents About the Authors Foreword Preface Acknowledgements Abbreviations and Acronyms 1. Introduction What is synthetic biology, exactly? The iGEM outbreak A synthetic biology lab manual 2. Genes, Chromoproteins and Antisense RNAs E. coli DNA: Chromosomes, plasmids and copy number Coupling of transcription and translation in bacteria Promoter and terminator for transcription Ribosome binding site (RBS) Codon bias Chromoproteins Small regulatory RNAs (sRNAs) 3. Lab Rooms and Equipment The physical lab spaces Equipment Autoclave Machine Burners Gel Equipment Centrifuges Micropipettes Laboratory Bench 4. Safety is Priority #1 Fires Chemicals Ethidium Bromide Biological safety and disposal Dangerous equipment 5. Lab Course Projects Time and resources Project overview and learning objectives The lab notebook Lab section 1. Preparation of chemical solutions and agar plates Lab section 2. Coloring bacteria by adding a promoter to a chromoprotein gene Colorimetric Assays for Chromoprotein Expression Lab section 3: Rational engineering of chromoprotein expression level Lab section 4. Other experiments BioBuilder 1. Eau that smell 2. iTune device 3. Picture this 4. What a colorful world The “dreaded” exam 6. Protocols Introduction Protocol 1. Preparation of solutions and agar plates Introduction SOB Medium LB Medium LB Agar Plates and Addition of Antibiotics Protocol 2. Overnight cultures with antibiotics, and glycerol stocks Overnight Cultures with Antibiotics Cell Glycerol Stocks Plasmid Preparations Protocol 3. BioBrick™ 3A assembly and gel analysis Introduction Components Procedure Protocol 4. Agarose gel electrophoresis Introduction Components Procedure Protocol 5. Preparation of competent E. coli cells using CaCl2 Introduction Components Procedure Protocol 6. Transformation of CaCl2-competent E. coli cells Introduction Components Procedure Protocol 7. Bacterial re-streaking techniques Introduction Procedure Alternative 1 Procedure Alternative 2 Procedure Alternative 3 Procedure Alternative 4 Protocol 8. Lysis of E. coli cells with lysozyme Introduction Components Procedure Protocol 9. Polymerase chain reaction (PCR) Introduction Components Choice of Thermostable DNA Polymerases Primer Design Dissolving Oligos PCR Reaction Protocol 10. Inverse PCR mutagenesis Introduction Primer Design Phosphorylation of Primers Inverse PCR with Phusion®HF DNA Polymerase Digestion with DpnI Ligation Protocol 11. Colony PCR Introduction Components Procedure Protocol 12. Gibson assembly Introduction Components Procedure Trouble-Shooting by Overlap Extension PCR 7. Advanced Methods Flow cytometry and cell sorting Recombination in plasmids and the chromosome Lambda Red Recombineering Single-Stranded Recombineering and Multiplexed Automated Genome Engineering (MAGE) Site-Specific Recombination Other Recombination Methods Electrocompetent cells 8. The International Genetically Engineered Machine (iGEM) Competition How to start an iGEM team Uppsala iGEM 2011 — Show color with color Uppsala iGEM 2012 — Resistance is futile Uppsala iGEM 2013 — Lactonutritious — it’s delicious 9. Appendices References Text Boxes Index