3Zero to Genetic Engineering Hero
Contents
Authors ................................................................................................................................................................. 6
Junior Editors ...................................................................................................................................................... 7
Preface ................................................................................................................................................................. 8
Who this book is for: .............................................................................................................................................................................. 9
This book is not a typical textbook! ..................................................................................................................................................... 9
Acknowledgements ................................................................................................................................................................................ 9
The Genetic Engineers’ Pledge ........................................................................................................................ 11
Isolating DNA, the Blueprints of Life ............................................................................................................... 12
Equipment and Materials ........................................................................................................................................................ 13
Learning Hands-On: Breaking Cells Open & Extracting DNA ........................................................................................................ 15
Step 1. Create salt water inside the resealable bag ....................................................................................................................... 15
Step 2. Mashing the strawberry into individual cells ................................................................................................................... 16
Step 3. Breaking open the cells with soap ...................................................................................................................................... 17
Step 4. Filtering the cell debris ......................................................................................................................................................... 19
Step 5. Precipitating the DNA ........................................................................................................................................................... 19
Fundamentals: DNA ................................................................................................................................................................ 23
Evolution: It’s natural for DNA to change ........................................................................................................................................... 23
Modern Synthesis .................................................................................................................................................................................. 24
Genetic engineering: The road to precise editing of DNA ............................................................................................................... 26
Atoms, molecules, and macromolecules of the cell .......................................................................................................................... 27
Understanding the nomenclature of DNA ......................................................................................................................................... 32
DNA extractions in the real-world ...................................................................................................................................................... 34
Summary and What’s Next? ................................................................................................................................................... 36
Setting Up Your Genetic Engineering Hero Space ......................................................................................... 39
Getting Started ......................................................................................................................................................................... 40
Do I need government approval? ........................................................................................................................................................ 40
What type of room should I set up my genetic engineering space in? ........................................................................................... 41
Equipment and materials for your Genetic Engineering Hero space ............................................................................................ 42
Materials and Supplies ......................................................................................................................................................................... 44
Cleaning and Other Supplies ............................................................................................................................................................... 45
Experiment kits/wetware ..................................................................................................................................................................... 46
Safety and Best Practices ..................................................................................................................................................................... 46
Just because I Can, does it mean I should? ........................................................................................................................................ 47
Growing E. coli Cells ......................................................................................................................................... 51
Getting Started ......................................................................................................................................................................... 53
Equipment and Materials ..................................................................................................................................................................... 53
Virtual Bioengineer
TM
simulation Breakout Session 2 ..................................................................................................................... 54
Learning Hands-On: Growing K12 E. coli cells .................................................................................................................................. 55
Step 1. Download the instruction manual for the Canvas Kit ....................................................................................................... 55
Step 2. Put on your gloves and lab coat ............................................................................................................................................ 55
Step 3. Create molten LB agar powder ............................................................................................................................................. 56
Step 4. Adding antibiotic ................................................................................................................................................................... 57
Step 5. Pour LB agar plates ................................................................................................................................................................ 58
Step 6. Use & storage of LB agar plates ........................................................................................................................................... 60
Step 7. Streaking E. coli bacteria ....................................................................................................................................................... 61
Step 8. Incubating E. coli cells ............................................................................................................................................................ 61
Step 9. Viewing plates of grown bacteria ........................................................................................................................................ 62
Step 10. Painting living art (bioart) with E. coli bacteria ................................................................................................................ 63
Step 11. Incubating E. coli cells ......................................................................................................................................................... 64
Step 12. Viewing & Preserving your bioart with a Keep-it Kit ...................................................................................................... 64
Step 13. Clean-up and inactivation .................................................................................................................................................. 65
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4 Zero to Genetic Engineering Hero
Fundamentals: E. coli Cells ......................................................................................................................................................67
Introduction to “Lab” E. coli ........................................................................................................................................................... 67
A Tour of the E. coli Microfactory ......................................................................................................................................................... 70
The Fence (A) .......................................................................................................................................................................................... 70
The Outer Wall (B) .................................................................................................................................................................................. 72
The Lobby (C) ......................................................................................................................................................................................... 74
The Inner Walls (D) ................................................................................................................................................................................ 75
The Factory Floor (E)............................................................................................................................................................................. 75
Summary and What’s Next? ................................................................................................................................................... 83
Genetic Engineering Your E. coli Cells ........................................................................................................... 86
Getting Started ..........................................................................................................................................................................87
Equipment and Materials ..................................................................................................................................................................... 87
Learning Hands-On: Transforming K12 E. coli cells with a DNA Plasmid .................................................................................... 89
Step 1. Download the instruction manual for the Engineer-it Kit ................................................................................................ 89
Step 2. Put on your gloves and lab coat ............................................................................................................................................ 89
Step 3. Label your plates .................................................................................................................................................................... 90
Step 4. Make non-selective and selective LB agar plates .............................................................................................................. 90
Step 5. Streaking E. coli and the negative control plates ................................................................................................................ 92
Check Point! ....................................................................................................................................................................................... 94
Step 6. Making chemically competent cells ................................................................................................................................... 95
Step 7. Add DNA plasmids and Heat Shock ..................................................................................................................................... 96
Step 8. Recovery step .......................................................................................................................................................................... 98
Step 9. Plating and incubating your cells ........................................................................................................................................ 99
Step 10. What to expect & inactivation ............................................................................................................................................ 100
Fundamentals: How a cell reads a DNA plasmid .................................................................................................................101
The basic operating environment of a cell: The Four B’s ................................................................................................................ 101
(Bump, Bind, Burst, Bump) ................................................................................................................................................................. 101
The Three Steps to Microfacturing .......................................................................................................................................................... 102
Deoxyribonucleic acid (DNA) vs. Ribonucleic acid (RNA)............................................................................................................... 103
RNA polymerase: The cell machine that transcribes ...................................................................................................................... 105
What is a gene? ..................................................................................................................................................................................... 106
Starting Transcription ......................................................................................................................................................................... 106
During transcription: Direction ......................................................................................................................................................... 109
During transcription: Which DNA strand does RNA polymerase read? ....................................................................................... 110
During transcription: A secret cipher for transcribing DNA to RNA ............................................................................................ 112
Stopping transcription ........................................................................................................................................................................ 113
What can you do with RNA? ................................................................................................................................................................ 114
Summary and What’s Next? .................................................................................................................................................. 116
Extracting your engineered proteins ............................................................................................................. 119
Getting Started ........................................................................................................................................................................ 120
Equipment and Materials ....................................................................................................................................................................120
Learning Hands-On: Culture and lyse engineered E. coli to obtain a protein product extract .................................................. 121
Step 1. Download the instruction manual for the Plate Extract-it Kit ........................................................................................ 121
Step 2. Put on your gloves and lab coat ........................................................................................................................................... 121
Step 3. Transform cells to get fresh colonies (Optional) ............................................................................................................... 121
Step 4. Make selective LB agar plates for amplification ............................................................................................................... 122
Step 5. Culturing: Spread out your freshly engineered cells ....................................................................................................... 122
Step 6. Culturing: Incubate at 37 ˚C for 24-48 hours ................................................................................................................... 123
Step 7. Extraction: Collect cells and start the lysis ........................................................................................................................125
Step 8. Extraction: Lyse the cells ..................................................................................................................................................... 126
Step 9.Extraction: Pellet the cell debris .......................................................................................................................................... 127
Step 10. Extraction: Filter sterilize your proteins ......................................................................................................................... 128
Step 11. Using your proteins ............................................................................................................................................................129
Fundamentals: How cells translate proteins from RNA ...................................................................................................... 131
Step two of the Three Steps to Microfacturing: Translating proteins from RNA .............................................................................. 131
Starting Translation ............................................................................................................................................................................. 132
During Translation: The RNA to protein cipher ............................................................................................................................... 133
During Translation: Locating the starting point for translation .................................................................................................... 137
Stopping Translation ...........................................................................................................................................................................139
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5Zero to Genetic Engineering Hero
Summary and What’s Next? ................................................................................................................................................... 142
Check Point! .......................................................................................................................................................................... 145
Processing Enzymes ....................................................................................................................................... 147
Getting Started ........................................................................................................................................................................ 149
Equipment and Materials ....................................................................................................................................................................149
Learning Hands-On: Process one molecule into another using enzymes .................................................................................. 150
Exercise 1: Enzymatic processing to generate smells .................................................................................................................... 150
Step 1. Download the instruction manual for the Smell-it Kit .................................................................................................... 150
Step 2. Complete the engineering part of the Smell-it Kit ........................................................................................................... 150
Step 3. Culture cells with the substrate ...........................................................................................................................................150
Step 4. Labeling and creating your LB agar plates for culturing ................................................................................................. 151
Step 5. Culture engineered cells on your selective LB agar plate ................................................................................................ 152
Exercise 2: Enzymatic processing to generate color ...................................................................................................................... 153
Step 1. Download the instruction manual for the Blue-it Kit ....................................................................................................... 154
Step 2. Complete genetic engineering and extraction procedures ............................................................................................. 154
Step 3. Dissolve your substrates ...................................................................................................................................................... 154
Step 4. Add cell extract beta-galactosidase to the substrate ........................................................................................................155
Fundamentals: Diving into enzymatic processing ............................................................................................................. 159
The basics of enzymatic chemical reactions .................................................................................................................................... 159
The Four B’s and enzyme function ..................................................................................................................................................... 159
Atoms ..................................................................................................................................................................................................... 161
Bonds ..................................................................................................................................................................................................... 163
Protein enzyme catalysis in cells ....................................................................................................................................................... 168
Summary and What’s Next? .................................................................................................................................................. 174
Manually turning on genes in situ ................................................................................................................... 177
Getting Started ........................................................................................................................................................................ 178
Equipment and Materials ....................................................................................................................................................................178
Learning Hands-On: Manually turning on genes in situ ............................................................................................................... 179
Exercise 1: Inducing a gene using a chemical .................................................................................................................................179
Step 1. Complete the Induce-it Kit engineering exercise. ............................................................................................................ 179
Step 2. Culture your cells ..................................................................................................................................................................179
Step 3. Add your inducer ...................................................................................................................................................................179
Exercise 2: Inducing a gene using temperature .............................................................................................................................. 180
Step 1. Complete the Heat-it Kit engineering ................................................................................................................................180
Step 2. Increase the temperature .................................................................................................................................................... 181
Exercise 3: Inducing a gene using light. ........................................................................................................................................... 181
Step 1. Streak cells ............................................................................................................................................................................ 181
Step 2. Turn on the light! .................................................................................................................................................................. 181
Fundamentals: Diving deeper into genetic ‘switches’ ....................................................................................................... 183
Turn on genes with chemicals ............................................................................................................................................................183
Turn on genes with temperature .......................................................................................................................................................185
Turn on genes with light ......................................................................................................................................................................186
Summary ................................................................................................................................................................................. 188
Periodic table .................................................................................................................................................. 192
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