GENE EDITING
Genetically Modifying E. Coli Bacteria At Home
From failure to success, it was a great experience!
Growing up, LEGO was the absolute greatest. I can always remember the times when I’d buy a new LEGO set and spend hours struggling, learning, and eventually building!
The reason why it was so special to me was because, even though I struggled and failed, it was all for a goal in mind — to let my curiosity shine.
But, let me ask you this question. What would your experience with LEGO be if you could only read the instruction manual?
Over the past few weeks, I’ve grown a great interest in gene editing (GE). However, most of the work I’ve completed relating to it has been theoretical. I’ve researched GE applications for neurological diseases, genetic blindness, and more. Yet, I haven’t applied that learning.
In other words, this entire time, I’ve been reading an instruction manual. And so, I asked, how exactly can I apply the knowledge in a hands-on experience? How can I build that LEGO set?
I searched the internet for an answer and came across this:
A DIY Bacterial Gene Engineering CRISPR Kit — the answer to my question.
I bought the kit right away and received it recently. Over the past few days, I have worked on and successfully completed the experiment!
In this article, I will be going through a step-by-step process on the experiment, explaining the science behind the steps, and more!
Experiment Goals + Importance.
Before diving in, let’s take a minute to understand exactly what gene editing is and how it will apply to this experiment.
Gene-editing is the process of modifying an organism’s genetic material (i.e. DNA) in order to display a certain characteristic.
In many current applications, this technology is being used to fight diseases, improve plant characteristics, and more!
It’s most efficient + effective tool is CRISPR/Cas9 (which is what we’ll be using).
But, for the scale of this at-home experiment, what exactly will we be editing?
We’ll be using the CRISPR/Cas9 system in order to genetically alter the genomic DNA of a strain of E. Coli (bacteria) so that it can grow and survive in conditions it would normally not be able to.
Really cool process, but before we get into the experiment, let’s talk a bit about what exactly CRISPR/Cas9 is doing in this experiment.
The Function of CRISPR/Cas9 in This Experiment.
In order to survive, all organisms need to produce proteins.
Think of yourself as a car, and the proteins as the engine. 🏎
Your proteins (the engine) are what allow you (the car) to function — it keeps you running, and it keeps you healthy.
Our cells produce these proteins using the DNA code — the four bases that make up our DNA:
Adenine — A
Cytosine — C
Thymine — T
Guanine — G
Every three sequential letters of these bases code for a single amino acid. Groups of these amino acids are what we know as proteins.
In a cell, this whole manufacturing process (of proteins) is conducted in ribosomes— the factory of protein synthesis. If our ribosomes are not functioning, our cells aren’t able to make proteins. If this happens, cell growth will cease, and we will die.
The same applies to this experiment dealing with the E. Coli bacteria.
The plate we are trying to grow our bacteria on contains a molecule called streptomycin. Normally, this molecule would bind to the ribosomes of our bacteria, preventing it from functioning (i.e. preventing the growth of proteins → death). However, through this experiment, we will be modifying the rpsL protein of our E. Coli bacteria, which will prevent the streptomycin from binding to its ribosomes. This will allow the bacteria to grow on the streptomycin plate.
The genome of the E. Coli bacteria is over 4 million DNA bases in size, but with the help of CRISPR, we will be finding the single base that needs to be mutated!
Let’s get started!
Steps.
I’ll be breaking down the steps for this experiment into general areas/categories, and explaining the importance + function of each of these areas to the overall experiment.
Creating the Plates. 🧫
There are two types of plates we will be creating in this experiment (LB Agar and LB Streptomycin).
LB Agar Plate.
The LB Agar plate is crucial to this experiment. The purpose of this plate is to grow the E. Coli bacteria in a cell-cultured environment (i.e. a favourable environment). This plate is a nutrient-rich broth that specifically accelerates the growth of the E. Coli bacteria.
In other words, this plate will allow us to quickly grow our bacteria in high yields.
Creating the LB Agar Plate.
- The first step in creating this plate is to mix the powdered LB Agar Media with 150mL of water. The mixture is then poured into a 250mL glass bottle.
We are mixing the powder media with water so that we can liquify the LB Agar.
2. With the LB Agar as a liquid (and in the bottle), we microwave the bottle for a few seconds at a time.
Before microwaving, the mixture in the bottle will be opaque (because the contents of the LB Agar have not yet fully dissolved into the water). By heating up the mixture, the contents will break down and mix. This will turn it from opaque → transparent.
⚠️ Note: Do not screw the bottle’s lid down tight. Instead, just rest it on top. This will prevent pressure from building up in the bottle and potential safety hazards.
⚠️ Note: If the agar starts to boil in the microwave, take it out immediately and cool it down for a few minutes. If the agar is heated too much, it will lose it’s function.
3. Once the mixture is transparent (after microwaving), leave it to cool for 20–30 minutes. Once the time is up, begin pouring the mixture into the empty plates. Let the plates sit for a couple of hours or overnight.
Leaving the plates out will allow the condensation to evaporate, and it will slightly solidify the mixture so that our bacteria can be added on top.
⚠️ Note: Make sure to only fill the bottom half of the plate.
LB Streptomycin (Strep) Plate.
The process for creating the Strep Plate will be the exact same as the LB Agar, except, instead of the powdered LB Agar, we will use the LB Strep Media.
The reason why we created the two plates was so that we could grow the bacteria in a favourable environment (using the LB Agar Plates), which would allow us to scrape off the grown bacteria and add it to the CRISPR mixture (detailed later in the article). The bacteria and CRISPR mixture would then be added to the streptomycin plate to verify for the CRISPR modification (i.e. growth on the strep plate, which would be impossible before the CRISPR transformation).
Creating Competent Bacterial Cells. 🦠
The next step is to create competent cells.
These are cells that are able to intake foreign DNA. In this experiment, we will be making our bacteria competent so that it can intake the CRISPR mixture (i.e. the intended foreign DNA).
The process for doing this is simple.
In order to access our bacteria DNA in the freeze-dried DHA5α centrifuge tube, add 100μL (microlitres) of water (which can be measured using the pipette given) and shake the mixture for one minute to ensure that it is dissolved. This mixture is now our competent cell mixture.
Once dissolved, pour and spread the bacteria onto the LB Agar Plate and let it grow for 1–2 days at 37 °C (or room temperature).
Creating the CRISPR Mixture. 🧬
In order to edit our bacteria, we need to create a CRISPR mixture. Essentially, this mixture is what allows us to modify the rpsL protein of our bacteria, to prevent the streptomycin from binding to it.
To create this mixture, we add 55μL of water to the Bacterial rpsL DNA Template, Bacterial gRNA Plasmid, and the Bacterial Cas9 Plasmid tubes separately (all of our perishable items). Shake until a yellow liquid is visible.
Let’s take a quick pause. ⏸
I’m giving you the names of these tubes, but, what exactly do they do?
The Bacterial rpsL DNA Template contains our target sequence. In order for us to make an edit to the genome of the E. Coli (in our case, to modify the rpsL protein), we need to insert a new sequence to fulfill that. The DNA Template contains the sequence we are adding.
But, if there are millions of bases in the bacteria, how exactly does it edit the single intended base?
The answer is through the gRNA. The target sequence (DNA template) and gRNA plasmid help the CRISPR system by guiding it to a matching template DNA (of the rpsL protein). Using the gRNA, both strands of DNA (from E. Coli and our template DNA) will then bind together, allowing the Cas9 system to do what it does best — cleave the intended sequence, and induce a repair.
But, as of this stage of the experiment, we have not yet added these CRISPR components to the bacteria (that will be conducted in the next stage).
Here’s a quick visual representation of the process outlined above:
Inserting the CRISPR/Cas9 System Into The Cells. 👨🔬
In order to add our CRISPR mixture to the E. Coli, we need to scrape off some bacteria grown on the LB Agar Plate (with an inoculation loop) and add them to the competent cell mixture.
Then, add 10μL of the Bacterial rpsL DNA Template, Bacterial gRNA Plasmid, and the Bacterial Cas9 Plasmid tubes to the competent cell + bacteria mixture (created in the image above). Mix by shaking each tube for ~1 minute.
Once this tube is mixed, store it in the fridge for ~30 minutes. ⏳
You now have the CRISPR system in your competent cell mixture.
When the 30 minutes is up, take the tube (from B, in the image above) and incubate it into very warm water (~42 °C) for 30 seconds. This will apply a “heat shock” to the mixture, which will open up its pores and allow external contents to enter.
To create the external contents we need, add 1.5mL of water into an LB media microcentrifuge tube, and shake to dissolve.
Then, add 250μL of this LB + water tube and add it to your competent cell mixture.
At this point, your competent cell mixture (we’ll be calling it the CRISPR mixture from now on) should include the contents of:
- All perishable items (Bacterial rpsL DNA Template, Bacterial gRNA Plasmid, and the Bacterial Cas9 Plasmid),
- E. Coli from the LB Agar Plate, and the
- LB Media tube
If you have this final mixture, store it at room temperature (or ~30 °C) for 1–4 hours (incubate longer if at room temperature).
This part of the experiment is 🔑 to its success, so if you can, I’d recommend leaving it out overnight.
You have officially edited the E. Coli’s rpsL protein! As you’re waiting, the CRISPR system is being guided to and will be modifying the rpsL protein of the E. Coli bacteria!
Adding the CRISPR Mixture to the Plate. 🧪
Once leaving out the mixture overnight, grab one of the LB Strep Plates you previously created. Using the pipette, add all of the CRISPR transformation (competent cell) mixture onto the plate. Then, spread the mix around the plate and store it at room temperate for ~2 days.
Results.
If, after ~2 days, the bacteria started to grow on the LB Strep Plates (as it previously did with the LB Agar Plate), it means that we were able to make the intended mutation to the E. Coli bacteria (i.e. modify the rpsL protein so that the streptomycin could not bind to it).
And, with that, you’ve successfully conducted the experiment!
Overall, the experiment was filled with new learnings, laughs, and lessons. And, as my first CRISPR kit, it was definitely a wonderful experience!
If it didn’t work out for you, that’s alright, try again. Science is all about learning from your failures and improving to succeed.
And, don’t worry, I made a lot of mistakes too…
Failures and Keys to Improvement
As my first CRISPR experiment, I failed many times. Although I was able to successfully achieve the E. Coli modification, many screw-ups led to that moment. In fact, I failed the experiment two times before succeeding.
Part of the reason why this happened was because of some silly mistakes…
Here’s a photo of me while pipetting a mixture and accidentally ejecting the pipette tip into the tube. Yikes!
When I first started the experiment, I thought that by reading the manual, I’d be alright. I’m here to tell you that that was a huge mistake!
The reason for that is because you don’t learn by reading the manual, you learn by understanding what you are doing to build that LEGO set. For this experiment, there were many times when I didn’t know what I was actually doing — I was simply following the instructions.
That’s why, after my second failed experiment, I knew it was enough — I ended up spending a day searching up some of the terms used for this experiment, learning their function, and more. This gave me the opportunity to not only understand what I was working with, but how I was using these different tools and resources to conduct the experiment.
Once I was able to understand that, it made the process so much easier and more enjoyable.
🔑: If you don’t know, learn — don’t just assume you know. Understanding the process and failing > being clueless and succeeding.
The Uncertain Future of GE.
Although our simple experiment didn’t change the world, many GE applications are doing just that.
In the near future, GE can (and will) be used in endless ways to cure genetic alterations in many people who are immutable. However, with increasing controversy, applications for this technology are being slowed down dramatically.
The founder of The ODIN, Josiah Zayner, has been banned from several media platforms (e.g. YouTube, Amazon, LinkedIn, etc.) because of what he calls “The Crime of Curiosity”.
Although GE has many benefitial applications, could it also be used for harm? Over the past few years, Josiah and his company have been under direct scrutiny because of their mission to make GE applications accessible to the public.
If more people are able to access these kits, would it increase the odds of benefitial or harmful applications?
Here’s what I believe:
Innovation in science doesn’t happen with restrictions, it happens with limitless curiosity. It happens when innovators are able to explore and dive into their interests to make them a reality for the world. That’s how innovation happens.
Publicly available kits as used in this article, is helping innovators feed on their curiosity — the very essence of innovation.
But, with governments and organizations restricting this growth, is the future of GE really a reality?
“Democratizing genetic engineering won’t suddenly unleash bioterrorism upon the world. Instead, what would be unleashed is a wave of innovation similar to what we see in software. Imagine only having the ability to program while in a highly-structured environment, like at a school computer lab. This doesn’t catalyze innovation, and, more importantly, it doesn’t inspire people.” — Josiah Zayner
We can’t use training wheels on this single path anymore, we need to take them off — take that risk, and find new paths. The world’s biggest problems need new solutions.
Solutions that will come through innovation…and innovation that will emerge from curiosity. 💡
