Cloning freestyle Vol. 2: DIY biology solutions
I was helping students from my community college think about research projects a few months ago, and when I heard that they were interested in molecular biology, I suggested they start a biofoundry. Here are some of the suggestions I threw their way. Cover art is a laboratory fallacy that happens in science all the time.
Preface
The target audience of this post isn’t well equipped and highly funded academic labs. I wrote this with a community college audience in mind, which IMO would 1) appreciate the exercise in learning the fundamentals of molecular biology and 2) find the experience simply working in a laboratory valuable.
I think that as we see significant advances in molecular biology, the fundamentals should become more accessible. For example, look at DNA sequencing: you can go old-school sanger, you send your samples for next generation sequencing, or you can buy a nanopore sequencer and do it yourself. In this cascade, new tech will lower the cost of sequencing across the board (whole plasmid sequencing for <$50 from plasmidsaurus!!) For wetlab though, new reagents don’t lower the cost of of older reagents; the old reagents are just phased out. So, I figure that, with technological advances in biomedical research in general, its not too much to ask for a community college to produce its own reagents and equipment, with a little help from a scientist.
Personally, I would have loved to try to purify enzymes and do biology experiments in my free time when I was at my community college (shoutout DC3!) and with the citizen science movement picking up, I think community colleges are the perfect place to start.
Disclaimer
Do science at your own risk and follow safety regulations (or at least don’t sue me.) Some states let you work with antibiotic resistant bacteria as long as you bleach them dead; buyer beware. Speaking of, Etsy shutdown a shop selling fluorescent protein (FP) plasmids, which I don’t think is illegal, but you do run that kind of risk.
I 1000% discourage biohacking. IMO, its tacky and underwhelming. Off the top of my head, plasmids/proteins should get degraded if you eat them. if you inject yourself with a plasmid Bioshock style, you will probably not express the protein of interest. Even if it does express, it will be quickly degraded because of metabolism. If it’s not degraded, it probably won’t show efficacy cuz of PK/PD. However, there is a good chance of going septic or suffering from endotoxemia.
I have “good hands”, meaning my technique in the laboratory is good and I can get new experiments to work probably the first time, but that’s not enough to make biohacking safe. What would it take for biohacking to be safe IMO? Many years of in vitro and in vivo (animal) studies, controlled clinical trials, extensive, documentation, cGMP production, and hundreds of highly trained scientists trying to poke holes in your entire project from every possible direction (AKA the FDA approval process!)
Accessing papers and designing projects
It sounds weird, but if a paper you want is behind a paywall, try emailing the authors asking for it. I love sharing my papers and most journals allow for authors to share their work this way. For open access right out of the gate, look at preprint repositories like Biorxiv, a preprint server for scientific papers under review at journals, or in preparation. Articles haven’t necessarily completed the peer review process though, so buyer-beware. Ideally, once articles are revised (and ultimately published), the authors choose to update the figures, which could make it a good place for cutting-edge work. Alternatively, I will also provide this quote from this article from Nature, which is not advice, legal or otherwise.
Sci-Hub, the popular website that offers access to millions of pirated research papers and books
Now, at some point, you WILL need equipment, so why not ask engineering students help with that? Some cool stuff I’ve found doing a quick search on biorxiv for DIY:
Plasmids 101: Addgene
In case you haven’t heard, Addgene is a nonprofit that distributes plasmids to universities for about $75+shipping. You get an agar stab 2-5 weeks after ordering. For example, here is a link to a DNA polymerase plasmid from addgene and here is a link to the paper, for reference. They also have a lot of great, free resources and tutorials!
#OverlyHonestMolecularBiologist
Some questions I can answer based on experience:
How long do bacteria last in the fridge? i.e. How long do they last at 4 degrees? In my experience, bacteria on agar plates “work” for several years. Your antibiotic will probably be gone after about a month tho, so be careful. I found some old (stored for 2-7 years) plates and picked a colony, spread it on a fresh ampicillin plate, and grew the insert no problem (sequence verified!) If its not moldy and obviously contaminated, why not try it? Preparing a glycerol stock is obviously the best practice, but -80s are expensive.
What about glycerol stocks? How long do competent cells last? Probably longer than the manufacturer says, unless your -80 melted down or you freeze-thawed them a bunch. Don’t store them at -20 if you don’t have to. That said, when I used to prepare glycerol stocks and run way too many experiments at the same time, I would sometimes forget the glycerol stocks at -20 over the weekend. I doubt that killed them, and you can try storing them for longer (report the results in the comments please!) but definitely don’t try to store competent at -20. Lowkey, I’m running this experiment at DC3; will update with the results eventually. Also, Pipette Jockey has a super cool post exploring the potential of storing bacteria for long periods of time at room temperature using treehalose. Sounds like a fun project!
When do enzymes expire? How long do restriction enzymes last? When do DNA polymerases expire? How long do RNA polymerases last? That’s not a question I can answer, only someone with end to end experience working with the reagents can answer that question. When I was inexperienced, fresh reagents didn’t work sometimes. Expired reagents also didn’t work. Maybe someone before me left them out or contaminated them? Maybe I contaminated them and didn’t notice? Who knows. What I do know is that I’ve used reagents 1-13 months past expiration dates (cuz I had to) and they produced results at least half the time. I was the only one working with them, and I was confident in my technique, so when I had to, I used them. Freeze-thaw cycles kill stuff, so minimize that to increase your chances of success. Still, if you knowingly use stuff past the expiration date, you shouldn’t be surprised if your experiment fails, even if you have years of experience, so don’t take risks you unnecessarily.
Note, these are proteins you’re working with, so you have to be careful. That said, I’ve heard of people storing antibodies incorrectly (-20 when they should’ve been 4, and vice versa) and them working afterwards. Its bad practice, but they could still work. I’ve also heard of 20 year old antibodies purified by hand still working, although those were stored at -80.
Do I have to work under a flame with bacteria? I didn’t. Its probably a good practice, but I’ve worked in a lab with no gas access, and all I did was always wear gloves, obsess over the sterility of my microtubes/loops/picks/, spray the area and my gloves with 70% ethanol/propanol, and was mindful not to contaminate anything. I’ve heard of people working without gloves, and that’s fucking crazy to me. I wear gloves when doing ephys, making buffers, opening the lab fridge, looking at bottles, etc. Labcoat? Do your thing, but doing experiments without gloves? You will never catch me slipping.
DIY molecular biology reagents
I would start by checking out this link for an open access article on preparing molecular biology reagents. Here is another link to a more recent article describing the traditional laboratory production of a Taq polymerase. Useful IMO if you have lab equipment but not a lot of funding. This article also describes a very nice labmade PCR enhancer solution (spoiler: the 5X solution contains: “2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 lg/ml BSA and was stable at 20 C for at least 3 months.” Their touchdown PCR reaction mixture contained “65 mM Tris–Cl, 16.6 mM (NH4)2SO4, 3.1 mM MgCl2, and 0.01% (v/v) Tween 20 at a pH of 8.8” and they also produced their Taq DNA polymerase in their own lab.
Some links to open source lab instrument projects; variable cost:
A little more advanced: cell free expression systems, which are reported to be a useful and low-cost alternative for molecular biology reagents (see this link to a paper for the specifics.) Note: the plasmids are in addgene already!
I acknowledge that there are gaps I am missing; what about competent cells, cloning and purification protocols, etc? Well, that’s the fun part of science, but I wouldn’t leaving you hanging! Check out openwetware: an open molecular biology protocol wiki. DIY/low-cost molecular biology is not easy, but it is absolutely possible!
Open bioeconomy resources
The Open Bioeconomy lab’s sounds mission pretty dope and might be a good resource for some. From their website:
We focus on developing open source tools for biotechnology. The pace of openly licensed and off-patent biological reagents entering the public domain is growing rapidly. However, open licenses and contracts are insufficient when practical routes for global access to and use of materials are not in place. Currently, access to reagents is a major impediment to scientists in the Global South, Africa and in low resourced settings like DIYBio labs.
Plus, they worked closely with the FreeGenesProject at Stanford to create open source enzymes, plasmids, reporters, etc. It seems like an easy process to place orders.
Other resources/stuff
Here are some lab stuff I found on Amazon:
DNA polymerase kit
“PowerPCR Taq DNA Polymerase Features 5 U/µl (1µl contains 5 units Taq DNA Polymerase) Amplify DNA target up to 6 kb Elongation velocity is 1kb/min Lacks of 3' to 5' exoneclease activity Makes 3'-dA overhangs PCR product, good for TA cloning 10x Reaction Buffer is included”
cheap dNTPs
Really just browse through the Bioland store on Amazon
I’m old school and prefer Ethidium bromide (EtBR) and a UV light source for visualizing bands, but you may choose to go with sybersafe and light source if you can find or make one (i.e. use blue LEDs for absorption, which will emit green; check LED wavelength tho)
Miscellaneous: A github repository for lab hardware (3D printable stuff as well as code)
For educators, the following journal may be of interest: “Biochemistry and Molecular Biology Education is an international journal aimed to enhance teacher preparation and student learning in Biochemistry, Molecular Biology, and related sciences such as Biophysics and Cell Biology, by promoting the world-wide dissemination of educational materials.”
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Additional thoughts
Here are some last minute tips/thoughts for molecular biology
Consider an instapot autoclave
You’ll need 100% alcohol; I prefer ethanol but its hard to obtain without a license so you can usually go with isopropanol
I used a lot of distilled deionized water for many things; worst case scenario, you may have to distill water and autoclave it (probably fine for bacteria) but I’d still prefer molecular grade water for molecular grade work
You will need a -20 C freezer
You’ll need sterile Glycerol to make glycerol stocks
Defintiely consider looking into for methods for storing bacteria at -20*C for long periods of time if you can’t get your hands on a -80 freezer
You’ll need powdered ampicillin (cheaper) or a stock of liquid ampicillin. Do not freeze a large amount, instead aliquot to avoid freeze thaw cycles which destroy it. Also make sure to get the soluble salt form
Make 70% alcohol and spray your working area and gloves anytime you think you might touch a contaminated surface
If you expand and grow your own competent cells from Zymo (they have free samples!), I’m not sure the cells remain “mix and go”-able. You might have to look into heat shock transformation protocols. Maybe not though!
You’ll need sterile picks for picking colonies; consider autoclaving toothpicks
Don’t touch the sides of microtubes; if pipetting use the sides as a guide for the top of your tip
Run negative controls for good measure (e.g. for colony picking, touch autoclaved toothpick to a plate that didn’t have bacteria plated on to it, or just dip it directly into a stock of LB) without touching anything)
I can’t stress enough how good this this free resource is: https://openwetware.org/wiki/Main_Page
The first time you run an experiment: make sure to print out the protocol and do a practice run a few days before you actually do the experiment, so that you have time to order stuff if you realize you don’t have it
Multiple water baths will probably be needed, so find thermometers for monitoring the bath. If you need to make a waterbath cheaply, maybe a sous vide and a container would work? This one on Amazon lowkey looks like a waterbath.
You’ll need a microwave for making agarose gels
DNA ladder for agarose gel (I’ve heard this can be made but can’t remember the source)
You’ll need an L shaped cell spreader. Instead of plastic consumables, you can flame-melt a glass pasteur pipette for resuse, you just gotta dip into 70% alcohol, set on fire, then put it out and let cool before use, all without contaminating. It’s a little tricky and dangerous, but it works.
