Exploring Gene Editing: A Hands-On Journey with E. Coli
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Chapter 1: The Connection Between LEGO and Gene Editing
Growing up, I found LEGO to be the ultimate source of joy. I fondly recall the countless hours spent assembling new sets, grappling with challenges, and ultimately succeeding in my builds! Many of you likely share similar memories.
One of my cherished LEGO sets was particularly meaningful; despite numerous struggles and setbacks, I was driven by a singular ambition — to satisfy my curiosity.
If you're reading this, you might be curious about the link between LEGO and altering E. Coli. Let me pose a question: how enjoyable would your LEGO experience be if you could only refer to the instruction manual? Frustrating, right?
Recently, I have developed a keen interest in gene editing (GE). While I’ve engaged in extensive theoretical research about its applications in neurological disorders and genetic blindness, I yearned for a practical application. I was essentially reading a manual without building anything.
In my quest for hands-on experience, I stumbled upon a DIY Bacterial Gene Engineering CRISPR Kit from The ODIN, which seemed to be the solution I needed. Without hesitation, I purchased the kit and have spent the last few days conducting experiments. Now, I'm excited to share my journey with you!
In this article, I'll walk you through a step-by-step guide of my experiment, explain the scientific principles involved, and much more! So, let’s jump right in! 🎢
Experiment Objectives and Significance
Before we delve into the experiment, it's essential to grasp what gene editing entails and its relevance here.
Gene editing involves altering the genetic makeup (DNA) of an organism to produce specific traits. This technology is currently employed in various fields, such as combating diseases and enhancing plant characteristics. The most effective tool in this arena is CRISPR/Cas9, which we will use in our experiment.
In this home-based experiment, our focus will be on editing a strain of E. Coli, allowing it to thrive in environments it typically could not endure.
The Role of CRISPR/Cas9 in Our Experiment
To survive, organisms must synthesize proteins. Imagine yourself as a car, with proteins serving as the engine. 🏎
Proteins are essential for your function and health, and our cells utilize DNA to manufacture these proteins, which are composed of four bases:
- Adenine (A)
- Cytosine (C)
- Thymine (T)
- Guanine (G)
Every trio of these bases corresponds to a single amino acid, and groups of amino acids form proteins.
This protein synthesis occurs in ribosomes, which act as the factories for protein creation. If ribosomes malfunction, cells cannot produce proteins, halting growth and leading to death.
The same principle applies to our E. Coli experiment. The plates we will use contain streptomycin, a molecule that typically hinders bacterial growth by binding to ribosomes. However, we will modify the rpsL protein in our E. Coli, preventing this binding and allowing the bacteria to flourish on streptomycin plates.
The E. Coli genome comprises over 4 million DNA bases, but with CRISPR's assistance, we will pinpoint the exact base that requires mutation!
Let's get started! 🤩
Steps to Success
I will categorize the experiment into general steps, detailing the significance and function of each in relation to the overall project.
Creating the Plates 🧫
We will prepare two types of plates: LB Agar and LB Streptomycin.
LB Agar Plate
The LB Agar plate is crucial for cultivating E. Coli in a nutrient-rich environment conducive to growth.
Procedure for Creating the LB Agar Plate:
Combine powdered LB Agar Media with 150 mL of water in a 250 mL glass container.
Microwave the bottle in short intervals until the mixture turns transparent, indicating that the contents have dissolved.
⚠️ Ensure the bottle lid is not tightly secured to prevent pressure buildup.
Once transparent, let the mixture cool for 20–30 minutes, then pour it into petri dishes and allow it to solidify overnight.
LB Streptomycin Plate
The procedure for creating the LB Streptomycin plate mirrors that of the LB Agar plate, except for the use of LB Strep Media instead of LB Agar.
The purpose of these two plates is to cultivate E. Coli in a favorable environment before transferring it to the CRISPR mixture for transformation.
Creating Competent Bacterial Cells 🦠
Next, we will produce competent cells that can absorb foreign DNA, in this case, the CRISPR mixture.
Steps to Create Competent Cells:
- Add 100 μL of water to the freeze-dried DHA5α centrifuge tube and shake for one minute to dissolve it.
- Spread the resulting competent cell mixture onto the LB Agar plate and incubate it for 1–2 days at 37 °C.
Creating the CRISPR Mixture 🧬
To edit our bacteria, we must prepare a CRISPR mixture. This mixture facilitates the modification of the rpsL protein, preventing streptomycin from binding.
Steps to Create the CRISPR Mixture:
- Add 55 μL of water to the Bacterial rpsL DNA Template, Bacterial gRNA Plasmid, and Bacterial Cas9 Plasmid tubes separately, mixing until a yellow liquid forms.
Let’s pause briefly. ⏸
The Bacterial rpsL DNA Template holds our target sequence for editing. The gRNA guides the CRISPR system to the corresponding DNA sequence, enabling the Cas9 enzyme to cleave it and initiate repair.
Inserting the CRISPR/Cas9 System Into the Cells 👨🔬
To incorporate our CRISPR mixture into E. Coli, we will scrape bacteria from the LB Agar plate and mix it with our competent cell preparation.
- Use an inoculation loop to transfer bacteria from the LB Agar plate to the competent cell mixture.
- Add 10 μL each of the Bacterial rpsL DNA Template, Bacterial gRNA Plasmid, and Bacterial Cas9 Plasmid to this mixture and shake for about a minute.
- Refrigerate for approximately 30 minutes.
Now, we will apply a "heat shock" by incubating the mixture in warm water (~42 °C) for 30 seconds to facilitate DNA uptake.
Once you've completed these steps, store the competent cell mixture at room temperature for 1–4 hours for optimal results.
Adding the CRISPR Mixture to the Plate 🧪
After letting the mixture sit overnight, take one of the LB Strep plates and add the entire CRISPR transformation mixture to it, spreading it evenly. Store the plate at room temperature for about two days.
Results
If bacterial growth appears on the LB Strep Plates after approximately two days, it indicates that we have successfully modified the E. Coli (i.e., the rpsL protein no longer binds to streptomycin)!
| width: | 800 :alt: E. Coli growth on LB Strep Plate |
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Congratulations on completing the experiment! It was a journey filled with learning and laughter.
If your attempt didn't yield the desired results, don’t be discouraged — science thrives on trial and error. I too faced numerous setbacks before achieving success.
Failures and Lessons Learned 🔑
As a novice in CRISPR experimentation, I encountered multiple failures. It took me two tries before I successfully modified the E. Coli. Many errors stemmed from simple mistakes or a lack of understanding.
Initially, I believed that merely following the manual would suffice, but I soon realized that true comprehension of the process is crucial for success.
After my second attempt, I dedicated time to researching the terminology and functions involved. This understanding made the subsequent attempts easier and more enjoyable.
🔑 Takeaway: If you’re unsure, seek knowledge — understanding the process is more valuable than blindly following instructions.
The Uncertain Future of Gene Editing 🔮
While our modest experiment may not reshape the world, numerous GE applications are making significant strides. In the foreseeable future, GE could revolutionize treatments for genetic disorders. However, growing controversies are slowing progress in this field.
Josiah Zayner, the founder of The ODIN, has faced bans on several platforms due to his quest for accessible GE technology, dubbed "The Crime of Curiosity."
Although GE holds immense potential, could it also pose risks? The debate surrounding public accessibility to GE kits raises questions about the likelihood of beneficial versus harmful applications.
In my view, scientific innovation flourishes in an environment of unrestricted curiosity, allowing individuals to explore their interests. The democratization of genetic engineering fosters innovation rather than hindering it.
As Zayner states, "Democratizing genetic engineering won't suddenly unleash bioterrorism upon the world. Instead, it will spark a wave of innovation akin to what we see in software development."
We must remove the training wheels and embrace the risks to discover new solutions for the world's pressing challenges — solutions that arise from curiosity and innovation. 💡