The Graduates Guide to Synthetic Biology

“The trouble with having an open mind, of course, is that people will insist on coming along and trying to put things in it.” – Sir Terry Pratchett

Congratulations! You’ve done some, or all, of your graduate degree in Biology, which means you’re probably brimming with knowledge of peptide and nucleotide mechanics.

If you’re really lucky, you’ve also had extensive practical experience in the laboratory. If so, perhaps you should find your way across to the Experts page.

However, if you’re like me, and a shocking number of graduates out there, you’ve ended up with a shiny degree and disappointing lack of PRACTICAL EXPERIENCE.. You did the pracs, you poured the gels and then you promptly forgot the ratio of agarose to TAE. Your restriction digest went perfectly in class… but already you wonder what the name of the buffer that went with EcoRI was.

Sound familiar? Well you’re in the right place comrade. Whether you’re just starting your honours project or striking out as an independent researcher – this resource will be expressly designed to help YOU apply the knowledge of your degree where it matters most. In the lab!

If something seems super obvious and boring, skip it! This resource is intended to be comprehensive enough to fill any gaps in your knowledge, not a prescriptive step-by-step guide. YOU are the greatest expert in what YOU know, so you have my permission to skip sections at will. Now… on to the guide!

Table of Contents: Guide #3
3.0 The Sterile Workspace
3.1 Choosing Your Organism
3.2 Knowing Your Organism
3.3 Mixing Media and Pouring Plates
3.4 Mixing Antibiotic Media
3.5 Sterile Technique
3.6 Mixing Buffers
3.7 DNA101
3.8 Purification of Genomic DNA
3.9 Plasmids101
3.10 Purification of Plasmid DNA (Miniprep aka. Ethanol Lysis)
3.11 Plasmid Insertion
3.12 Agarose Electrophoresis
3.13 Restriction Enzyme Digest
3.14 Phosphorylation
3.15 Ligation
3.16 Polymerase Chain Reaction (PCR)
3.17 Primer Design
3.18 Gradient PCR
3.19 Flanking Restriction Site addition
3.20 Site Directed Mutagenesis
3.21 Gibson Cloning Protocol
3.22 Golden Gate Cloning Protocol
3.23 Confirmation of Successful Edits
3.24 RNA101
3.25 RNA Purification
3.26 RT-PCR
3.27 RT-qPCR
3.28 GBlock Design
3.29 Backbone Selection
3.30 CRISPR/CAS9
3.31 Proteins101
3.32 Induction of Protein Expression
3.33 Cell Lysis Protocols
3.34 His-Tag Protein Purification
3.35 SDS-PAGE
3.36 ELISA
3.37 Western Blot
3.38 Southern Blot?
3.39 Mass Spec Prep
3.40 Cell Assays
3.41 Antibody Production
3.42 In Silica

Tips & Tricks

3.0 The Sterile Workspace

The first thing you’ll need in order to do synthetic biology is a sterile workspace. To create this, you’re going to need a spray bottle containing 70% ethanol and a flame. Gloves are great to have, but are actually an unnecessary precaution while you’re getting started. Spray down your workspace and wipe it dry with a paper towel. Next, spray your hands and wrists; it will dry out your skin, but not do any damage. Gloves are useful if you do this regularly, but also pose the risk of melting on to your hands if they get to close to the Bunsen. Spraying your bare hands with ethanol will be enough to prevent contamination, but not disable RNAses – which will only become a problem further down the road.

PICTURE

Next, light your lighter or match and then turn on the gas of your Bunsen. Hopefully your high school chemistry teacher lectured you on this, but this order of operations is important – don’t let out a big cloud of flammable gas before sparking your lighter! While you can use improvised sources of flame such as a camping stove or a blow torch, a portable Bunsen burner is best. If you’re lucky enough to work in a space that has ready to use gas outlets, you won’t even need to pay for a portable Bunsen!

If your workspace looks something like this… you did it!

3x photos of lab, with each different flame, sad face next to Bunsen with nowhere to be plugged in

You’ve now created a sterile workspace, but how does it work? Ethanol kills the bacteria on the bench and on your hands – while the Bunsen flame creates an updraft in the air around your workspace. This is your CONE OF PROTECTION – and the size of it is dictated by how hot the flame is. I generally consider anything within two feet of a roaring blue Bunsen flame to be safe from contamination… but you’ll learn all about this in the section on Aseptic Technique (3.3).

Picture of Bunsen Flame on within sterile environment, Goku Flames

But before we get there, it’s time to figure out a strategy for sterilising your media. There are three main strategies for sterilisation;

1. Autoclave – These high pressure steam sterilisers are the gold standard for sterilisation of media. They can cost more than AU$2,000 second hand, so they’re unlikely to be useful for solo projects. If you’re at a community lab and can share the cost, this should be one of your first investments. It can be quite astounding just how much stuff you’ll need to sterilise just to keep everyone in a community lab supplied with fresh reagents. 
2. Pressure Cooker – Many solo synthetic biologists do all of their work using a simple pressure cooker on a gas stove. I personally find these spooky and generally fear that they’re prone to blowing up in my face; but fellow lab manager Meow swears by his pressure cooker. Try get one large enough to hold several bottles at once, the volume of the vessel will dictate how much of the day you spend just sterilising stuff. 
3. Microwave – If the two above options do not appeal to you, there is the option of sterilising in a microwave. Any old microwave will do, and it’s generally held that 900 W for 10 min will be enough to sterilise your media. Some bacterial or fungal spores might survive this treatment however, so you should incubate a couple of plates (Room Temperature [RT] and 37°C) after using this method to see if anything grows on them. If they last a week without any apparent growth, you can assume the microwave sterilisation protocol is sufficiently robust for your purposes. 

3 X Photos of Sterilisation equipment

Once you’re able to create a sterile workspace and sterilise media; you’re well on your way to becoming a completely independent scientist! To that end, it’s time to learn how to mix our own media and pour our own plates. But what kind of media…? To figure that out, you’re going to have to make your first important decision:

Protocol: The Sterile Workspace

Tips & Tricks

3.1 Choosing Your Organism

You can skip this section if you’ve already chosen a target or plan to work in E. coli (good choice!)

Want to know something extremely unsexy about synthetic biology? For most of the time, the sum total of your life’s work will look to the layman like this: 

Or this:

If you imagined that this guide was going to let you give yourself super strength, or create a glowing rabbit – TURN BACK NOW. Soon, maybe, later… we will have the power to manipulate genetics with a flick of the wrist, but we aren’t there yet. Legal and ethical reasons aside, our understanding of the sheer complexity of life is still nascent. You’re going to want a simple, single-celled organism to work with. 

I personally recommend anything that can hold and utilise a plasmid, such as Bacteria or Yeast. It is important to choose a non-pathogenic strain, the last thing you want is become infected by your own work. Try to source a well characterised strain from a respected laboratory or supplier.  

Choosing a single-celled target allows you far more power of manipulation over DNA at this stage of technological development. This guide will not involve many ‘In vivo’ experiments, instead we’ll be slaughtering our bacteria in order to extract the sweet, sweet plasmid DNA within. The power of ‘In vitro’ synthetic biology is that we can separate DNA and RNA for manipulation without worrying about killing the already deceased bacteria.

Now read the above paragraph, but replace the word “bacteria” with the word “bunny rabbit”, and this should help you understand why you won’t be working with higher-order mammals. If not, you’re a monster but I respect your honesty. 

A very important step while choosing your target organism is legality. I can’t answer this for every country, so spend some time googling your government’s stance on genetic manipulation. The language of these laws can be quite dated – such as Australia’s Gene Technology Act (2001):  so if you’re worried, try calling your country’s regulator, such as Australia’s OGTR. The well trod path will often be the easiest answer (K12 derived E. coli strains) but worry not; we will teach you to do miraculous things in this admittedly dull-to-the-naked-eye organism.

Protocol: Synthetic Biology Host Selection Criteria

Tips & Tricks

3.2 Knowing Your Organism

Again, skip this section if you plan to work in E. coli.

All protocols henceforth will deal primarily with Escherichia coli (E. coli). If you’ve chosen to work with something else (such as Yeast or Bacillus strains), the methods contained within will still be useful. However, you may need to innovate on the fly.

The secret to doing this is to KNOW YOUR ORGANISM. Spend some time googling culture conditions, ideal media, growth curves, morphology – anything that might be useful for improving your success rate. Then, if my protocols tell you to incubate at 37°C, but you know your critter grows best at 30°C, you’ll already be able to make the expert’s decision to optimise your method!

I’ve already stated the implication of this, but here we go again: The well trod path will often be the easiest. An abundance of literature on culturing conditions will help you breeze through these early chapters and on to more advanced techniques. A novel bacteria that is both difficult to culture and poorly understood will be an uphill battle.

But a word of hope to the innovators out there: nature holds many juicy mysteries to uncover. Perhaps the platonic ideal of a synthetic biology host already exists somewhere in nature, and we fools who toil with E. coli work in the ignorant bliss of a so called superior understanding.

Protocol: Culturing Conditions

Tips & Tricks

3.3 Mixing Media and Pouring Plates

I’m unsure how, but I finished my degree having never poured an agar dish. Most new lab members arrive with a vague memory of doing it in the first year, but from pre-prepared media. Without further ado, here is a simple protocol for LB (Luria-Bertani) media and agar:

PROTOCOL: LB Liquid Media

PROTOCOL: LB-Agar Mixing and Plate Pouring

This will be your stock standard E. coli media, but don’t be afraid to hunt down other recipes. Open Agar is likely still in development, so google “Best media (your organism)” and hunt down a recipe! 

Tips & Tricks

3.4 Mixing Antibiotic Media

Antibiotic media is biotech on easy mode. Just a few drops of this secret sauce is enough to purge your media of anything that doesn’t have your chosen plasmid. Well almost anything… The one caveat to this blissful state is that the mixing of antibiotic media/agar requires the one thing biologists fear most of all – maths. Worry not however! We’ve thought ahead to ensure that once you mix the tube of antibiotic stock, all the maths can be left at the door. 

Protocol: Preparing Antibiotic Stock Solutions

Protocol: Addition of Antibiotic Stock Solutions to Media

Tips & Tricks

3.5 Sterile Technique

Okay you’ve just made some really tasty microorganism food for your chosen critter, now you must learn to defend it from the wider world. For some microbiologists; Sterile Technique is more than just jargon, it’s a way of life. It’s also the first thing a nosy onlooker will comment on – so knowing WHY we use this technique is just as important as learning HOW to do it. It can feel superfluous to aim for perfect technique when working with antibiotics (because it’s easy mode) but I recommend you go through the motions anyway. But it’s also worth remembering that failure at sterile technique can yield fascinating and bewildering results; I’m sure that Alexander Fleming and Bob Ross would agree that “we don’t make mistakes… just happy little accidents”

Sterile Technique, aka. Aseptic Technique is the core discipline of microbiology. It is one of those easy to learn, hard to master skills that you could refine for your entire life without perfecting. I draw criticism from time to time for a misplaced hand, a handle touching glass, or for not wearing gloves at every single moment. I don’t take this criticism to heart, and neither should you if your mentor ever scolds you for technical errors. You know it is coming from a good place – they’re just trying to help you minimise the chance of a non-experimental variable affecting your results. 

Technical errors only matter if they bring non-experimental variables into the mix. 

Success in synthetic biology is measured by the quality of your results, measurable through a number of robust protocols that you will soon learn. If you’re getting contamination on your plates, unexpected morphologies or weird growths – that is a clear sign that your sterile technique needs work.

However, if you’re able to reliably grow plates without contamination and verify the results of experiments through PCR and restriction digest; the original set up and quality of your space does not matter. The nuances of your technique do not matter. If it works for you and you can show results – don’t let anyone get you down.

One of Bio foundry’s PhD mentors has recently had her GARAGE in Australia PC1 approved, allowing her to do various gene editing experiments… in a GARAGE. Her technique is obviously outstanding, and she regularly produces reliable and interesting results.

Another hero of the DIY biology world; David Ishee, has been working to genetically modify dogs to fix a number of negative mutations brought about by generations of selective breeding. He revels in the joy of success using a rudimentary setup and I can’t deny that watching genetic modification occur in a converted tool shed is a compelling sight.

The point is, government willing – you can do biotech anywhere. How important technique is to your result is actually a factor of experimental design. I’m going to be starting us off with basic ‘plasmid’ work using antibiotic media. This really is biotech on easy mode. In a lot of cases, you could probably cough on your antibiotic-plate and it would have zero impact on the colonies that are growing. 

So if you want to become a really bad-ass microbiologist, practise on media without antibiotics and using just a Bunsen burner. Become the master of your organism, ensure it grows only when and where you want it to. Become the master of your media, protecting your organism from all outside contaminants.

If however this is your first time, don’t feel intimidated by these techniques honed by wise and ancient scientists over the last century. Working with antibiotic media and plasmid DNA is akin to bowling with the bumpers up. Your mistakes are rarely costly and more often the fault of the antibiotic rather than yourself as a scientist.

I’d like to take this chance to give you the first of many reminders to come; that Positive and Negative controls will be your second set of bumpers in this extended bowling analogy. When things go tits up, you’re going to be glad you included a positive and negative control plate to identify the problem. In this case, your negative control would involve the addition of bacteria without a plasmid onto an antibiotic-plate. If the plate gets growth, you’ll know the antibiotic is borked and your experimental plate is no longer trustworthy. More on that later, for now – head over to the Sterile Technique page to learn more about this core discipline. 

Protocol: Sterile Technique

Back so soon? Or did you read ahead before checking out the sterile technique information? Well I’ve got some good news for you – nested within Sterile Technique was some other core tools of microbiology – and these are literal tools, not just an abstract set of skills. 

So, just in case you missed it in the shuffle, lets learn how to use…

THE Micropipette! (Hyperlink) (Picture)
The Inoculating loop (Hyperlink) (Picture)
The Inanimate glass rod (Hyperlink) (Picture)

Holy moly, you’re now self-sufficient! Just a bit of protein, yeast extract, salt, water and agar – that’s all it takes to get E. coli to grow. With merely a flame and some ethanol, you’re able to create your own controlled environment. Add in 3 tools and you can now do all the poking, prodding and squirting you want. It’s magnificently simple, and cheap as chips – so watch the guides and don’t fall into the trap of buying pre-poured plates!

Tips & Tricks

3.6 Mixing Buffers

Well you’re pretty self-sufficient, but there’s still a lot of resources you’re going to need to buy if you wish to work with plasmids.
Buffers are mixtures of chemicals that contain competing molecules which allows the liquid to resist changes in pH. We use them for nearly everything, but the prices for even the simplest buffers can be outrageous from commercial suppliers, not to mention the lead time on shipping. As such, learning to mix your own buffers can be both a time and money saver. But.

DIY buffer mixing is a different beast entirely from pouring your own plates, carrying significantly more dangers as you handle deadly compounds in their pure form. That said, it’s going to save you A LOT OF MONEY. If you’re a solo hobbyist who will struggle to source their own pure Hydrochloric acid or Guanidine Thiocyanate, I recommend you buy your buffers pre-mixed. It will cost you more, but save on headaches caused by supply chains and chemical fumes. However, if you’re in a small community lab such as Biofoundry; then mixing your own buffers can cut thousands from your budget – just be sure to suit up in your PPE!

Protocols: DIY Buffer Mixing

3.7 DNA101

This guide will need to be built after the protocols are done. Make a list of first principles…
DNA101 If you’re reading this guide before it is complete, this may be a good time to learn more about DNA. If you have a good working knowledge, feel free to skip ahead.

3.8 Purification of Genomic DNA

Genomic DNA is the core code of any organism and depending on the species it can come in a variety of forms and be located in a variety of places within the cell. As humans, our genomic DNA is encoded as 46 chromosomes located within the nucleus. We pass this genomic DNA to our children via 23-chromosome gametes (sperm or eggs). We share this trait with all other Eukaryotic organisms, from Bakers Yeast to Blue Whales.

The other two Domains of life are Prokaryotes, consisting of Bacteria and Archaea. These domains differ in many ways, but as Prokaryotes, they both lack a nuclear membrane and simply leave their genomic information floating around within the cell. This “bacterial chromosome” is most often a giant circle of DNA consisting of several hundred-thousand to several million nucleic acids. There are plenty of exceptions to this rule, such as multi-chromosome bacteria, something to look into if you’re using a unique target. Bacteria pass their genome on to their offspring through the process of binary fission, using instructions contained within the chromosome to order self-replication by dividing in two.

Viruses and bacteriophages don’t get included in the above discussion because they lack the replication machinery necessary to be classified as “life”. They do however have genomes, and they come in the widest variety yet. Single-stranded DNA, Double-stranded DNA, RNA-only, some bacteriophages even have genomes larger than some bacteria. The purification methods required will vary based upon the genome type. If you’re trying The following link will help you decide on a genomic DNA extraction protocol for the needs of your experiment.

Protocols: Genomic DNA Extraction

If you’re using E. coli, lucky you – the genome is a nice and simple 4,583,637 base pair (bp) single chromosome that replicates using a process similar to the diagram above. There is an easy ‘boil purification’ method that requires minimum reagents which will allow you to extract the bacterial chromosome.

Protocol: Boil Purification of E. coli Genomic DNA

BUT! Before you jump right in to slaughtering your bacteria in a heat block, ask yourself – what do I want to do with this chromosome afterwards?

Chromosomes are really hard to get back into a cell once they’ve been extracted. Furthermore, most cells already have a chromosome! There are some fascinating projects that focus on generating a synthetic cellular membrane around a chromosome, but that is perhaps a decade away from being useful and far too expensive for the likes of us.

Don’t lose heart, nature has handed us the most incredible tool for ‘gently’ adjusting the genetic expression of bacteria, yeast and potentially higher order organisms; THE PLASMID.

So with that teaser of what you’re about to learn, I offer you these protocols for extracting Genomic DNA. Put them in your back pocket for when you’ve learnt about Plasmids, PCR and Agarose Electrophoresis. “Genomic DNA Extraction” is the sort of protocol you rarely need until you absolutely do. Until then, it’s time to become a master of plasmid manipulation.

3.9 Plasmids101

If you’ve ever written computer code, you could imagine plasmids as “add-ons” or “plugins” – files that you attach to a larger program to change its function in some way. But as a warning, don’t lean too deep into that allegory, as my professor Dom told me;

“Writing computer code is easy, everything is laid out nice and simply on a silicon computer chip. Synthetic biology is a different beast entirely, it’s like trying to write code inside of a burrito”

Inside this “burrito” are thousands of intersecting cellular messages that slip and slide over each other in their haste to perform their multitude of functions. Simply the presence of a surplus of one molecule may turn on several enzymatic pathways, leading to a cascade of unpredictable results.

You’re about to barge into this complex system with new code, rewiring systems on the fly to suit your desires. If you aren’t careful, you’ll do this with little care for the ability of your organism to survive. In synthetic biology, it is very possible to be catastrophically successful within an experiment e.g. you produced a huge amount of green-glowing protein, but all the cells died during the first generation.

Fortunately for you; the manipulation of bacteria using plasmids is now a 30-year-old discipline and you’ll have plenty of excellent examples of success and failure to learn from. Thanks to technological developments such as CRISPR/CAS9, you also will be able to use plasmids to make extremely selective modifications to the aforementioned genomic chromosome without ripping it out of the cell first. Nice!

It’s time to synthesise your growing knowledge of your target organism with an understanding of plasmids and their interaction with your target.

Plasmids101: If you’re reading this guide before it is complete, this may be a good time to learn more about plasmids. If you have a good working knowledge, feel free to skip ahead.

3.10 Purification of Plasmid DNA (Miniprep aka. Ethanol Lysis)

3.11 Plasmid Insertion Protocols

3.12 Agarose Electrophoresis

Gels by Benj

3.13 Restriction Enzyme Digest

3.14 Phosphorylation

3.15 Ligation

3.16 Polymerase Chain Reaction (PCR)

3.17 Primer Design

3.18 Gradient PCR

3.19 Flanking Restriction Site addition

3.20 Site Directed Mutagenesis

3.21 Gibson Cloning Protocol

3.22 Golden Gate Cloning Protocol

3.23 Confirmation of Successful Edits

3.24 RNA101

3.25 RNA Purification

3.26 RT-PCR

3.27 RT-qPCR

3.28 GBlock Design

3.29 Backbone Selection

3.30 CRISPR/CAS9

3.31 Proteins101

Zeeshan’s Zone

3.32 Induction of Protein Expression

3.33 Cell Lysis Protocols

3.34 His-Tag Protein Purification

3.35 SDS-PAGE

3.36 ELISA

3.37 Western Blot

3.38 Southern Blot?

3.39 Mass Spec Prep

3.40 Cell Assays

3.41 Antibody Production

Gonna learn this from Torsten one day

3.42 In Silica

I reckon by the time we get to this part of the guide, some nifty hacker will have protocols for us.

Tips & Tricks