How I Genetically Modified Bacteria with Jellyfish GFP

A couple of days ago, I genetically modified bacteria, at my house.

This article gives a brief overview of what Gene Editing is and how it works, how I genetically modified bacteria at my own home, and the future of this technology itself.

There is definitely a lot to cover in this article because of how impactful this topic is but, in order to truly understand it, we need to understand the basics first.

The Basics

When it comes to advanced topics like this, they’re certain terms/techniques you need to know so first let's dive deeper into those:

Gene Editing: This whole article is about Gene Editing, so why not start with it first! Gene Editing is essentially a type of technology that gives people/scientists the ability to manipulate or change a host’s genes, and this is done though a process with CRISPR and CRISPR-Associated protiens (CAS). Sounds pretty cool right? If you are new to this concept, check out my article for more information!

Green Florescent Protein: Green Florescent Protien (GFP) is a type of protien in jellyfish that emits green florescence when exposed to a certain type of light. In my experiment, I genetically modify E. Coli bacteria with GFP, so that the bacteria glows green when exposed to light.

Competent Bacterial Cells: In this case, the term ‘Competent” means the bacteria cells are able to intake foreign DNA. You may know that cell walls, in fact do prevent things from entering but mixing certain chemicals and bacteria changes this. This is done through a process called DNA Transformation!

DNA Transformation: DNA Transformation is where we put all the materials like GFP into synthetic DNA, and then the interesting part is where we trick the bacteria into thinking that our DNA is its own DNA and in doing so, they make the proteins coded into the genes you put into the DNA, which is exactly how I genetically modified bacteria at my house.

Source: The ODIN- Competent cells and DNA Transformation

How I Gene-Edited Bacteria at my House

Now we covered all the basics, time for the fun stuff! First, I will go through what my experiment consists of and how it worked, and then I will go through the steps I took to see super cool results!

In reality, Bacteria and all organisms need to make proteins to survive. Even we do! Think of proteins as tiny macromolecules in an organism that does everything from controlling our metabolism to even keeping our heart beating. I would say that proteins are the most important macromolecule in your body.

Without proteins, you wouldn’t have:

  • Hair
  • Nails/Bones
  • Muscles
  • Energy

In short, proteins are essentially the building blocks that make you into who you are.

Every 3 letters of DNA codes for single amino acid and proteins are just chains of amino acids. The experiment I conducted contains DNA with a gene that codes for the Jellyfish GFP! Like I said before, this type of protein glows green when exposed to blue light. In my experiment, I genetically modify E. Coli (non-hazardous) bacteria that I grew so that the bacteria can fluoresce when I shine blue light on them.

My Objective:

The main objective of this experiment is to grow bacteria in LB Agar plates, and genetically engineer them with jellyfish green fluorescent protein so that the bacteria I grew will eventually glow green when exposed to blue light.

Materials:

  • LB Agar
  • LB Kan Agar
  • 7 Petri Plates
  • Inoculation Loops
  • Gloves
  • Blue Light
  • Bacterial Transformation Mix
  • Jellyfish GFP plasmid
  • Centrifuge Tubes
  • Orange UV Filter Sheet (Optional, but recommended)

Method:

  1. Making Plates: Our first step in this experiment is to make LB and LB Kan Agar plates. The reason we do this is to grow bacteria to genetically modify. Without this, what’s left to gene edit? In order to actually make these plates, we need to mix water, with the LB Agar powder till dissolved. We would usually heat it to dissolve then once it is cooled, we would pour them onto Petri plates. We would not only make LB Agar plates but LB Kan Agar plates as well. This will be used for DNA Transformation.
  2. Making Competent Bacterial Cells: Once we are done making plates, we then need to make Competent Bacterial Cells for DNA transformation. To prepare bacteria, I need to add 100uL of water (to get an accurate amount, we would use a scientific pipette) into the DH5a centrifuge and mix it till all the bacteria is dissolved. We would then put this onto a new fresh LB Agar plate while spreading it with an inoculation loop.
  3. Making our Competent Cell Mixture: White-ish bacteria would eventually grow on the plates we made, so the next step is to scrape that bacteria and mix it into the “Bacterial Transformation Mix”. What we just created is knows as our “competent cell mixture”. This can be stored in the fridge for 1–2 days.
  4. DNA Transformation: Now it's time for the coolest part of the experiment, DNA Transformation. First, we need to take the GFP Plasmid, and using my scientific pipette, I need to add 50uL of water, till the plasmid dissolves. Once it’s dissolved, we need to add that to our competent cell mixture from the previous step.
  5. Incubation: Our next step is to incubate the centrifuge tube so that we are sure the DNA transformation will work. We would start by incubating it in the fridge for 30 minutes, and then a heat shock by incubating the tube for 30 seconds at 42°C water. After incubation, using the same pipette, we need to add LB media into our competent cell mixture containing our GFP and DNA plasmid. Now we need to incubate the tube one last time at 37°C for around 2 hours. In this experiment, I wasn’t actually able to incubate it for as long as 2 hours at 37 degrees, but I did incubate it at room temperature for 4 hours. What this step is doing is it allows the bacteria to recover and replicate the inserted DNA.
  6. Results: The results for this experiment are super cool so let's see how I got these results in the first place. Using a disposable pipette, I added around 4 to 5 drops of our transformation mixture on top of an LB Kan Agar plate. Using an inoculation loop (a spreader works too!), gently spread the bacteria and dry the plate for at least 10 minutes. There will still be condensation, so I flipped the Petri plate upside down so that no condensation is dropping onto our fresh bacteria. Finally, once you see white bacteria grow again, shine blue light onto the plate while holding a UV filter sheet (the UV filter sheet just blocks the blue light so we can see the glowing more clearly) and you should see amazing results.

The Future of This Technology

Right now, Gene editing is only at its first stages and I am already genetic engineering at my house. We can easily see that In the future, this technology will be ready to solve some of the world's hardest, and the biggest problems. Gene editing has the ability to even cure deadly diseases like:

  • Genetic defects
  • Blood disorders
  • Cystic fibrosis
  • Cancer
  • HIV

Not only can it cure diseases but it can also create superfoods, allergy-free foods for those who have allergies. It can even get rid of lactose intolerance in people and the best part is, it's happening right now!

CRISPR gene editing is growing exponentially in the global market and it will continue to rise, which is why I think that this technology will be the next big thing.

Hey guys! My name is Sambhav, and I am an aspiring genetic engineer who is super interested in Machine Learning, Web Development, and Genetic Engineering. Claps would always be appreciated! 🙂

13 year old technology enthusiast, researcher and web developer.