Chapter 20: Molecular Technologies

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Imagine holding a universal toolkit in your hands.

One that lets you read, copy, cut, paste, and even rewrite the very blueprint of life itself, DNA.

Wow.

What incredible discoveries would you unlock?

What profound mysteries could you finally solve?

It's truly remarkable to consider the sheer power of these tools.

I mean, they don't just let us observe life's most fundamental processes.

They allow us to actively engage with them.

Absolutely.

So today we're embarking on a deep dive into the fascinating world of molecular technologies.

These aren't just, you know, abstract ideas from textbooks.

They are the groundbreaking methods that have revolutionized genetics, enabling scientists to manipulate and analyze DNA and its products with astonishing precision.

It's like understanding the operating system of biology, really.

Yeah, exactly.

Our guide for this journey is Chapter 20 from Genetics, Analysis and Principles, Seventh Edition by Robert J.

Brooker.

A solid source.

Our mission is to unpack these core technologies, understand their mechanisms, and reveal how they've transformed our understanding of life.

From the initial breakthroughs in gene cloning to the, well, cutting edge of gene editing.

And you're about to get a shortcut to understanding the foundational tools underpinning so much of modern biology and medicine.

We'll reveal some truly surprising facts about how scientists work with the invisible world of genes.

So let's unpack this, starting with the bedrock, gene cleaning.

Okay, gene cloning.

At its heart, it's basically the process of making many, many identical copies of a specific gene.

Before this technology emerged, study a single gene among the thousands present in an organism was a true needle in a haystack problem.

I mean, almost impossible.

Just solve that.

Okay.

So when did scientists first crack this?

When did they start actually manipulating DNA like this?

Well, the initial breakthrough arrived in the early 1970s.

There were independent groups at Stanford, one led by David Jackson, Robert Simons and Paul Berg, and also Peter Lobin and Adele Kaiser.

They achieved something pivotal.

They successfully created the first recombinant DNA molecules.

Recombinant DNA, meaning?

Meaning they literally linked DNA from two different sources together.

That paved the way for putting these new molecules into living cells so they could be replicated.

Got it.

And why does gene cloning matter to you, the listener?

What's the real world impact beyond the lab?

Oh, the impact is immense.

I mean, fundamentally, it lets researchers investigate specific gene sequences, understand their structure, see how they contribute to traits or diseases.

Okay, the basic science stuff.

Right.

But practically, it's the foundation for so much more.

I think gene therapy,

screening for genetic conditions and the commercial production of vital proteins like human insulin for diabetics.

That was a game changer.

Huge.

And it's also used in agriculture to create transgenic plants, you know, with better traits.

Okay.

So to do this kind of molecular cut and paste, you need incredibly precise tools.

What are the key players involved?

Yeah, that's the core of it.

First, you need your source material, chromosomal DNA, which holds the gene you're interested in.

Right.

Then you need a carrier, a vector DNA.

These are typically small circular DNA molecules called plasmids, often found naturally in bacteria or sometimes modified viruses are used.

Plasmids, like tiny biological couriers.

Right.

What makes them so good for this?

Exactly couriers.

They have two critical features.

One is an origin of replication.

This lets them replicate independently inside a host cell, and it kind of determines how many copies get made.

Okay.

And the second is selectable markers.

These are genes often for antibiotic resistance, like the AMP -R gene gives resistance to ampicillin.

Ah, and why is that marker so important?

It's crucial because it lets scientists easily figure out which host cells, like bacteria, have actually taken up the plasmid.

Only the ones with the plasmid survive when you add the antibiotic.

Clever.

Okay, now for the actual cutting.

This really does sound like molecular surgery.

It kind of is.

This is where restriction enzymes or restriction endonucleases come in.

They are the molecular scissors.

Scissors, got it.

These enzymes recognize and bind to very specific DNA sequences.

Often they're palindromic, meaning the sequence reads the same forwards and backwards on opposite strands,

like E.

cari recognizes 5 -eur GATC3 -euri.

Okay.

And they cut the DNA backbone at precise spots, creating what we call sticky ends.

Sticky ends.

That sounds useful.

Exactly.

They're short, single -stranded overhangs that can hydrogen bond, temporarily, with complementary sticky ends from other DNA fragments.

So they sort of find each other.

Precisely.

But those bonds are temporary.

So once they line up, you need the molecular glue.

This enzyme forms strong, covalent bonds, permanently linking the DNA fragments together.

Okay, so you've got your source DNA, your vector plasmid, the restriction enzyme scissors, and the DNA lig is glue.

How do scientists actually do the cloning?

Walk us through it.

Right.

It's basically a step -by -step process, maybe six key steps.

Okay.

First, cut.

You take your chromosomal DNA and your plasmid vector, and you cut both with the same restriction enzyme.

That ensures they have matching sticky ends.

Makes sense.

Second, lag it.

You mix the cut DNA fragments with the cut vector.

The sticky ends pair up, and DNA ligus seals them, forming recombinant vectors that your gene popped into the plasmid.

Sometimes the vector just seals back up on itself, too.

Okay, so you get a mix.

Yeah, a mix.

Third, transform.

You introduce this DNA mixture into host cells, usually bacteria like E.

coli, that have been treated to make them permeable to DNA.

It's called making them competent.

Only a tiny fraction actually take up a plasmid.

Very inefficient, then.

Can be.

That's why step four, select, is key.

You grow these bacteria on a plate with that antibiotic, say ampicillin.

Only the cells that took up a plasmid because it has the amperexistence gene will survive and form colonies.

Ah, so that filters out all the bacteria that didn't get anything.

Exactly.

But you still don't know if they got the recombinant plasmid or just an empty one.

So step five is identify, often using a trick like blue -white screening.

Blue -white.

How does that work?

Well, if inserting your gene disrupts another gene on the plasmid, say the lacZ gene, which makes colonies blue on certain media, then the colonies with your recombinant plasmid will be white because lacZ is broken.

Colonies with the empty recircularized plasmid will be blue.

Brilliant.

So you just pick the white colonies.

You got it.

And finally, step six, amplify.

Once you find a bacterial cell with your recombinant vector, you grow it up.

The plasmid replicates many times inside each cell, and the bacteria divide rapidly.

Overnight, you get millions of cells, each containing many copies of your cloned gene.

Wow.

From one needle to a whole haystack of needles.

Pretty much.

Now what about cDNA or complementary DNA?

We hear that term a lot.

How's it different?

Ah, cDNA.

It's distinct because it's synthesized from an RNA template, not directly from DNA.

From RNA, okay.

Yeah, specifically messenger RNA or mRNA.

The crucial advantage is that cDNA lacks introns.

Introns.

The non -coding bits in eukaryotic genes.

Exactly.

Bacteria can't splice out introns like eukaryotic cells can.

So if you want to express a eukaryotic protein in bacteria, you need to give them the cDNA version, which only has the coding sequence.

Or sometimes you just want to focus on the protein coding part.

So it's like a streamlined, ready -to -go version of the gene for bacteria.

Precisely.

It's made using an enzyme called reverse transcriptase.

This enzyme is actually found naturally in retroviruses like HIV.

Interesting.

You purify mRNA from cells, and then reverse transcriptase uses that mRNA as a template to build a complementary DNA strand.

Got it.

And this ties into DNA libraries, right?

What are those exactly?

Yeah.

When you do a cloning experiment like we described, you often generate a whole collection of different DNA fragments inserted into vectors.

A DNA library is just that collection of recombinant vectors stored, usually in bacteria.

If you started with an organism's entire chromosomal DNA, chopping it up and cloning all the pieces, that's a genomic library.

It represents, theoretically, the whole genome.

The whole blueprint.

Right.

But if you started with mRNA from a specific cell type or condition and made cDNA, then cloned that.

It's a cDNA library.

This library only represents the genes that were actually being expressed as mRNA in those cells at that time.

Ah.

So one is the complete instruction manual, the other is like a list of instructions being used right now in a specific room.

That's a great analogy.

Exactly.

A snapshot of gene expression versus the whole genome.

Perfect.

Now, let's pivot.

We've cloned genes.

Let's talk about a technology that completely changed the game for amplifying DNA without cloning, super fast, polymerase chain reaction,

or PCR.

PCR.

Yeah.

Developed by Carey Mullis back in 1985.

Truly revolutionary.

It's an in vitro method, meaning it happens entirely in a test tube.

No living cells needed.

Right.

It lets you rapidly make millions, even billions of copies of a specific DNA region.

Think about forensics, how it changed almost overnight.

PCR meant you could potentially get a DNA profile from a single hair follicle or a tiny speck of blood.

No bacteria, no vectors.

Wow.

What's the recipe for this powerful reaction?

What do you need?

It's surprisingly simple, actually.

You need four key things in the tube.

OK.

One,

template DNA.

The DNA sample containing the sequence you want to copy could be complex, like the whole human genome.

Two.

Primers.

Two short, synthetic DNA sequences.

You design these to be complementary to the very ends of your target region flank it.

So they define what gets copied.

Exactly.

Three, deoxyribonucleoside triphosphates, or DNTPs.

These are the A's, T's, C's, and G's, the building blocks for the new DNA strand.

And the fourth?

The magic ingredient.

A thermostable DNA polymerase.

Most famously, TAC polymerase, isolated from bacteria living in hot springs, Thermus aquaticus.

It needs to withstand really high temperatures.

Why the high heat?

How does the chain reaction actually work?

It's all about temperature cycling, done automatically in a machine called a thermocycler.

Three repeating steps.

Deep one.

Denaturation.

You heat the reaction tube to about 95 degrees C.

This high heat breaks the hydrogen bonds holding the double -stranded template DNA together, separating it into single strands.

Okay, strands separated.

Step two, primer annealing.

You cool the tube down, maybe to 55, 65 degrees C.

This allows those short primers to find and bind, or anneal, to their complementary sequences on the single -stranded templates.

Primers attached.

Step three, primer extension.

You raise the temperature slightly, usually to around 72 degrees C, which is the optimal temperature for TAC polymerase.

It latches onto the primer and starts synthesizing a new complementary DNA strand, using the template strand as a guide and the DNTPs as building blocks.

And that's one cycle.

That's one cycle.

But the key is, each new DNA strand made in one cycle becomes a template for the next cycle.

Ah, the chain reaction part.

Exactly.

So the amount of target DNA doubles with each cycle, roughly.

It's exponential growth.

After just 20 or 30 cycles, you can get millions or billions of copies from potentially just one starting molecule.

It's incredibly powerful amplification.

Amazing.

So PCR amplifies DNA.

What about reverse transcriptase PCR, or TPCR, and real -time PCR, QPCR?

They sound related.

They are.

And they build on the power of PCR.

RTPCR is used when you want to detect or amplify specific RNA molecules, not DNA.

Like mRNA for gene expression studies?

Precisely.

The key difference is the first step.

You isolate RNA,

then use that enzyme reverse transcriptase again to make a single -stranded cDNA copy of your RNA target.

Okay.

Then, that cDNA becomes the template for a standard PCR reaction.

It's incredibly sensitive.

You can detect really small amounts of a specific RNA, sometimes even from just one cell.

Wow.

And for quantifying, figuring out how much DNA or RNA was there to begin with.

That's where real -time PCR, or QPCR, comes in.

The Q is for quantitative.

It allows you to measure the initial amount of a specific DNA or cDNA derived from RNA in your sample.

How does it do that in real -time?

It uses a special thermocycler that can measure fluorescence during the PCR cycles.

You add fluorescent probes or dyes to the reaction.

For example, a TAC man probe has a fluorescent reporter molecule and a quencher molecule kept close together.

Quencher stops the fluorescence.

Right.

But as TAC polymerase extends the new DNA strand, its natural exonuclease activity chews up the probe, separating the reporter from the quencher.

The reporter is now free to fluoresce.

Ah, so fluorescence increases as more product is made.

Exactly.

The machine measures this fluorescence each cycle.

The cycle threshold, or seat value, is the cycle number at which the fluorescence signal crosses a certain threshold, rising significantly above background noise.

And what does the seat value tell you?

A lower seat value means you hit the threshold earlier, which indicates there was a higher initial concentration of your target DNA or RNA in the sample.

You can compare seat values of your unknown samples to standards with known concentrations to get absolute quantities, or compare them to a reference gene to get relative quantities.

It's hugely important for measuring gene expression levels.

Incredible precision.

Okay, so we've amplified DNA, we've quantified it.

How do we actually read the code?

The sequence of As, Ts, Cs, and Gs.

Let's talk DNA sequencing.

Right, DNA sequencing.

This is fundamental.

It allows researchers to determine the exact base sequence of a DNA strand.

Knowing the sequence reveals promoters, regulatory elements, the coding sequence itself, basically everything needed to understand gene function and heredity.

And the classic method, the one that really got things started, is the de -deoxy method, or Sanger sequencing.

What's the clever trick behind it?

The cleverness is all about using modified DNA building blocks called de -deoxyribonucleotides, or DD -NTPs.

DD -Oxy?

What's missing?

They're missing a specific oxygen atom on the sugar part, the 3 -hydroxyl group, 3 -DOH.

Normal DNTPs have this OH group, and it's essential for adding the next nucleotide in the chain.

So when DNA polymerase happens to incorporate a DD -NTP instead of a normal DNTP into a growing DNA strand, the chain can't be extended any further.

It terminates the synthesis at that specific base.

A deliberate stop signal based on the base type.

How does that give you the sequence?

In the modern automated version, each of the four DD -NTPs, DDA, DDT, DDG, DDC, is labeled with a different colored fluorescent dye.

Let's say green for A, red for T, yellow for G, blue for C.

Okay, four colors.

You set up a reaction with your target DNA,

usually many copies of a cloned fragment you want to sequence, a primer to get synthesis started, lots of normal DNTPs, a small amount of all four of these colored DD -NTPs, and DNA polymerase.

DNA synthesis starts from the primer.

Most of the time, a normal DNTP is added.

But every so often, randomly,

a colored DD -NTP gets incorporated.

When that happens, synthesis stops for that particular strand.

So you end up with a big collection of strands.

Exactly, a collection of DNA fragments of all different possible lengths, each one ending with a specific fluorescently labeled DD -NTP that corresponds to the base at that position.

And then you sort them out.

Precisely.

You separate these fragments by size, usually using a technique called capillary electrophoresis, which is super high resolution.

The shorter fragments move faster through the capillary gel than the longer ones.

Near the end of the capillary, there's a laser and a detector.

As each fragment, ordered by size, from shortest to longest, passes the laser, its fluorescent dye lights up with its specific color.

The detector reads the sequence of colors.

Like reading a barcode.

Kind of.

The sequence of colors directly tells you the sequence of bases added, corresponding to the complementary sequence of your original target DNA.

You get a printout, often called a chromatogram, showing peaks of different colors.

A typical run can read maybe 700 to 900 bases accurately.

Truly ingenious.

Okay, we've read and copied DNA.

Now the really exciting part for many people, rewriting it, that brings us to gene editing.

Yes, gene editing.

This refers to methods that let researchers deliberately change the DNA sequence within genes, often directly inside cells.

It's absolutely essential for figuring out how specific DNA changes affect gene expression, protein function, and ultimately, traits and diseases.

One earlier method is site -directed mutagenesis.

How does that allow for precise changes?

Right, site -directed mutagenesis is designed to alter the sequence at a very specific, pre -chosen spot, but typically within a cloned piece of DNA first, like in a plasmid.

Okay, so not usually directly in a living cell.

Not usually the first step.

The way it works is you design a short, synthetic DNA primer that's mostly complementary to your target sequence in the plasmid, but you build in a specific mismatch.

That mismatch represents the mutation you want to introduce, like changing one base to another.

A deliberate error in the primer.

Exactly.

You anneal this primer to a single -stranded version of your plasmid DNA,

and then use DNA polymerase to synthesize the rest of the strand.

Now you have a double -stranded plasmid with a mismatch at your target site.

And then?

You introduce this plasmid into a host cell, like bacteria.

The cell's own DNA repair mechanisms will recognize the mismatch and fix it.

Sometimes it fixes it back to the original sequence, but other times it uses the mutant strand as the template, incorporating your desired mutation into both strands.

Then you isolate the mutated plasmid.

So it lets you make very specific changes to study function, like altering one amino acid in a protein.

Precisely.

It's great for that kind of detailed functional analysis.

But the technology that's really taken center stage is CRISPR -Cas technology.

This is the one making headlines.

Oh, absolutely.

CRISPR -Cas is a game changer, because it allows for targeted gene editing directly in living cells, and even whole organisms, much more efficiently than previous methods.

And it comes from bacteria.

Yeah.

It's adapted from a natural defense system bacteria use to fight off invading viruses.

Specifically, the Type II CRISPR -Cas system involves a guide RNA and an enzyme called Cas9.

Cas9 is the scissors part.

Cas9 is the DNA cutting enzyme.

The molecular scissors, again, yes.

Researchers engineer a single guide RNA, sgRNA.

A key part of this sgRNA of the spacer region is designed to be complementary to the specific DNA sequence in the gene they want to target.

So the RNA guides the scissors to the right spot.

Perfectly put.

The sgRNA binds to the target DNA sequence, and it brings the Cas9 enzyme with it.

Cas9 then makes a precise double -strand break in the DNA at that location.

It cuts both strands.

And what happens then?

That sounds dangerous for the cell.

It is dangerous.

And the cell immediately tries to repair that break.

How it repairs it is key to how CRISPR editing works.

And researchers can influence the outcome.

There are two main repair pathways.

OK, what are they?

First, non -homologous end joining, or NHEJ.

This is the cell's sort of quick and dirty default repair pathway when there's no template available.

It just sticks the broken ends back together.

But it's often sloppy and introduces small errors, insertions, or deletions of a few bases called indels.

And those small errors can mess up the gene.

Yes, frequently.

If an indel occurs within the coding sequence, it often causes a frameshift mutation, which scrambles the protein sequence downstream and usually results in a non -functional protein.

So NHEJ is often used to knock out a gene to disable it and study what happens.

OK, so that's one outcome.

Breaking the gene.

What's the other pathway?

The other is homologous recombination repair, or HRR.

This pathway is much more precise.

It uses an undamaged homologous DNA sequence as a template to accurately repair the break.

And researchers can hijack this.

If, along with the CRISPR -Cas components, the researcher also provides a donor DNA molecule, a piece of DNA that matches the sequence around the break, but contains a specific desired change, like correcting a disease mutation or inserting a new sequence, the cell's HRR machinery can use this donor DNA as the template.

So it repairs the break and incorporates the desired edit at the same time.

Precisely.

HRR allows for very precise gene correction or modification.

That's incredible.

The ability to either knock out or precisely rewrite genes directly in living cells.

It's transformative.

It works in human cell lines, mouse embryos, adult animals, various plants.

The potential for basic research and developing therapies is just enormous.

OK, shifting gears slightly.

We've talked about manipulating DNA.

What about understanding what the genes are actually doing in a cell?

How do scientists detect the RNA transcripts and the protein products that genes encode?

Right, that gets into analyzing gene expression.

And for that, we often turn to blotting methods.

Blotting, like ink blotting.

Sort of analogous.

The general idea is you separate molecules, like RNA or protein, by size using gel electrophoresis.

Then you transfer or blot them onto a solid membrane.

And then you use a specific probe to detect your molecule of interest on that membrane.

OK, let's start with northern blotting.

What does that detect?

Northern blotting is specifically for detecting RNA molecules.

It lets you see if a particular gene is being transcribed into RNA in a certain cell type, say comparing muscle cells versus nerve cells.

Or maybe at different times.

Exactly, like during development.

It can also show you if a gene's primary transcript, the pre -mRNA, is being alternatively spliced into different sized messenger RNAs in different tissues.

How's it done?

You extract RNA from your cells,

separate the RNA molecules by size on an agarose gel,

transfer those separated RNAs onto a nylon membrane, and then incubate the membrane with a labeled probe.

Usually a piece of DNA or RNA that is complementary to the RNA sequence you're looking for.

And it's labeled, often radioactively or fluorescently.

This probe will stick, or hybridize, only to your target RNA on the membrane.

Then you visualize where the probe bound, revealing bands corresponding to your RNA.

So you see a band if your RNA is present, and its position tells you the size.

You got it.

Okay, so northern is for RNA.

What about proteins?

For proteins, we use western blotting.

Same basic principle, but different molecules and probes.

It's used to identify a specific protein within a complex mixture extracted from cells.

Again, you could ask, is this protein made in this cell type?

Or at this stage?

How does the protein separation and detection work?

First, you extract proteins, then you typically treat them with a detergent called SDS, which denatures them and gives them a uniform negative charge.

You separate these denatured proteins by size, molecular weight, using SDS -PAGE polyacrylamide gel electrophoresis.

Okay, proteins separated by size on a gel, then blot.

Yep, blot them onto a membrane.

Now the key difference, the probe for westerns are antibodies.

Antibodies, like from the immune system.

Exactly, you use a primary antibody that is highly specific.

It recognizes and binds to a unique feature called an epitope on your protein of interest.

So it only sticks to your protein?

Ideally, yes.

After washing away any unbound primary antibody, you add a secondary antibody.

The secondary antibody is designed to bind to the primary antibody, and it's usually linked to an enzyme, like alkaline phosphatase.

An enzyme?

What does that do?

You then add a colorless chemical substrate.

The enzyme attached to the secondary antibody converts the substrate into a colored product, often a dark purple precipitate, right at the location of your protein band.

So a dark band appears where your protein is?

Precisely.

It tells you the protein is present, and its position on the gel tells you its approximate molecular weight.

Northern for RNA, western for protein.

Got it.

Finally, let's touch on how scientists analyze the interactions between proteins and nucleic acids.

How do they figure out which proteins bind to specific DNA or RNA sequences?

Right, these interactions are fundamental for things like gene regulation.

Two common techniques are EMSA and DNA's footprinting.

Okay, what's EMSA?

EMSA stands for electrophoretic mobility shift assay, sometimes called a gel retardation assay.

It's used to determine if a protein binds to a specific DNA fragment or RNA molecule.

How does it show binding?

The principle is pretty straightforward.

You run your labeled DNA or RNA fragment on a non -denaturing gel.

If a protein binds to that nucleic acid, the resulting complex is now bigger and heavier than the nucleic acid alone.

So it moves slower.

Exactly.

It moves slower through the gel matrix.

So on the gel, you'll see a band corresponding to the free unbound nucleic acid moving faster, and if the protein binds, you'll see another band that's shifted upwards or retarded, representing the protein nucleic acid complex.

Simple but effective.

Very effective for detecting and interaction.

Connelly used to study transcription factors binding to DNA regulatory elements, or RNA binding proteins interacting with mRNA.

And the other method, DNA's eye footprinting, sounds more detailed.

It is.

DNA's eye footprinting tells you the precise region on the DNA where a protein binds its footprint.

How does it map the footprint?

It uses an enzyme called DNA's eye, which randomly cuts the DNA backbone.

But if a protein is sitting on a specific DNA sequence, it physically protects that sequence from being cut by DNA's eye.

Like shielding it.

Exactly.

So the procedure involves taking many copies of a DNA fragment, labeled at just one end.

You divide the sample in two.

One tube gets the DNA binding protein added.

The other doesn't.

Okay.

Protein versus no protein.

Then you treat both samples briefly with DNA's eye, aiming for about one cut per DNA molecule on average.

Then you separate the resulting fragments by size on a high -resolution gel, detecting only the labeled fragments.

What do you see?

In the sample without the protein, DNA's eye cuts randomly all along the DNA, so you see a continuous ladder of bands representing fragments of every possible length.

But in the sample with the protein, there will be a gap in that ladder.

A gap?

Yes.

A region where there are no bands, or significantly fewer bands.

This gap, or footprint, corresponds exactly to the DNA sequence where the protein was bound, protecting it from the DNA's eye cuts.

The missing bands map the precise binding site.

Precisely.

It gives you much more detailed information about where the protein sits compared to MSA.

Wow.

Okay.

We have covered a lot of ground.

We've journeyed through this remarkable landscape of molecular technologies.

We really have.

From the ingenious cutting and pasting of gene cloning and the exponential amplification of PCR,

to the precise reading of DNA sequences with Sanger, the targeted rewriting with CRISPR, and then detecting the gene products with northern and western blotting and analyzing their interactions with MSA and footprinting, these tools are truly the workhorses of modern genetics.

Absolutely.

And you, listening, now have, hopefully, a solid grasp of how scientists manipulate and analyze the very essence of life.

This knowledge isn't just academic.

It really does underpin breakthroughs in medicine, agriculture, and our fundamental understanding of who we are.

It really makes you think.

Consider how these technologies, developed largely since the 1970s, have utterly transformed biology.

Given that rapid progress and the incredible creativity of scientists,

what currently unimaginable scientific questions do you think these tools, or maybe their future descendants, might actually let us answer in the next 50 years?

Something to ponder.

Definitely.

We hope this deep dive has sparked your curiosity and maybe given you some valuable insights into this amazing field.

Thank you so much for being part of the Deep Dive family.

Keep exploring.