New Video: Recommended Reading

New video - Recommended Reading - this video is part of my "You ask, I answer" series and is my response to a question I was asked the other week.

Are you starting a Bioscience or Biomedical Sciences degree and wondering which textbooks you should buy? You’re not alone! This is one of the most common questions I get asked: “What textbooks do I really need?” In this post, I’ll give some practical advice on buying textbooks and share some recommendations for Biochemistry and Cell Biology textbooks.

Should You Buy Textbooks Right Away?

My short answer is: no, don’t rush into buying textbooks at the start of your course. University libraries will have copies of the textbooks you need. Many students make the mistake of buying books too early, only to find that the ones they purchased aren’t the best fit for their study style.

Libraries often have several types of loans:

• Long-term loans (weeks at a time)
• Short-term loans (48 hours)
• Reference-only (cannot leave the library)

And so are likely to have the books you need. However, there’s a risk that the library copies may all be checked out when you need them—especially during exam season. That’s why it’s important to determine which books work best for you before purchasing.

How to Choose the Right Textbooks

You’ll likely receive a reading list with several textbook options when starting your course. The key is to take some time to evaluate them. Head to the library and look through each recommended book to see which one you like and find easiest to use. When I was a student, I was recommended three different Biochemistry textbooks, and each presented the material differently. 

By reviewing the books in the library first, I could choose the one that made the most sense to me. After I decided on my favourite, it was bought for me as a Christmas present—a great way to save money since textbooks can be expensive; I also picked up a secondhand copy of a second book for a different perspective, which was a cost-effective way to supplement my learning.

The Case for Owning Your Own Textbook

While you don’t need to buy all your textbooks, there are some clear advantages to owning the ones you use most. Having your own copy means you can highlight key information, make notes in the margins, and stick in tabs to mark important pages. You can also use it whenever you need it, without worrying about library loan deadlines.

Some students consider buying electronic versions of textbooks. However, many e-books have restrictions, such as limited access for a set period. If you prefer making digital annotations, this might work for you. However, a physical copy is usually the better investment if you want a resource you can keep and reference throughout your course.

My Textbook Recommendations for Biosciences

For Cell Biology, I would recommend two books:

• Molecular Biology of the Cell" by Alberts – This is a classic in molecular and cell biology and is great for understanding complex concepts - Amazon Affiliate Link
• "Molecular Cell Biology" by Lodish – Another excellent book that offers clear explanations and diagrams - Amazon Affiliate Link

Alberts and Lodish are excellent resources, and I use them in my teaching.

For Biochemistry, there are a few great options:

• "Biochemistry" by Stryer** – This is one of the best textbooks for Biochemistry and has been continually updated over the years. I particularly like the diagrams and how clearly the material is presented - Amazon Affiliate Link
• "Principles of Biochemistry" by Leninger** – While a bit more detailed and dense, it’s another solid choice, especially if you’re looking for a deeper understanding of the material. - Amazon Affiliate Link

Summary

Choosing the right textbook is a personal decision, and finding one that works for you is important. Take advantage of your university library, review your options carefully, and consider waiting before purchasing. When you do decide to buy, consider secondhand editions to save money. And remember, a well-chosen textbook can be a valuable resource throughout your studies—and even beyond.

New Video: What is the most significant change I've seen in the biosciences during my career?

In the video What is the most significant change I've seen in the biosciences during my career?, I look at what I consider to be some of the biggest changes I have seen.

This video is part of my "You Ask, I Answer" series.

Over my career in bioscience, I’ve witnessed some incredible changes that have significantly changed how we do biomedical sciences and how cells work.

Recently, I was asked what I considered the most significant change in the biosciences I had seen. At first, I thought of the rise of omics technologies like genomics and proteomics, but after I thought about it some more, a few key innovations and changes in our thinking truly stood out.

In this blog post, I’ll tell you what I consider to be the major changes I have seen.

The PCR Revolution

When I first encountered PCR in the late 1980s, it seemed almost like a magic trick. The idea that you could heat a solution containing enzymes to 90°C and still get a reaction was mind-blowing. As a biochemist, the method seemed to go against what I had been taught on my degree - enzymes start to denature and stop working once you get above 40 ºC. 

Now, PCR is everywhere in the biosciences. It's used for research, criminal forensics, and disease diagnosis, and the ability to amplify DNA has changed the way we do science.

PCR Videos:

• PCR
• PCR primer design

Mass Spectrometry and Proteomics

Another major technological leap I saw was the rise of easy-to-use mass spectrometers. Early in my career, mass spectrometry was a complex and inaccessible tool. However, the introduction of user-friendly mass spectrometers transformed lab work. Suddenly, we could easily measure the monoisotopic mass of peptides and carry out peptide mass fingerprinting to identify proteins with ease. 

This shift was a game-changer for proteomics and molecular biology, enabling us to quickly identify proteins. The combination of advanced instruments and accessible databases allowed for faster, more detailed analyses that were once considered out of reach for most labs.

Bioinformatics

Bioinformatics has completely altered the way we approach science. Finding a scientific paper or sequence information in the past required extensive library work and manual searches. Now, with databases full of genetic sequences, protein structures, and published studies, the challenge has shifted from finding data to filtering and making sense of it. 

When I started, labs had no computers at all. Now, every researcher has a personal computer linked to a global network of scientific knowledge and a vast array of powerful bioinformatic tools. 

Adipocytes - the cells that changed

Beyond technological advances, our understanding of biology has also changed. 

When I first started studying bioscience, adipocytes (fat cells) were considered passive storage units for energy. Today, they are recognised as dynamic endocrine organs that secrete a range of adipokines, influencing metabolism and overall health.

As we age, we tend to gain more fat cells, effectively adding to the number of these endocrine cells in our bodies. This shift in understanding has implications for obesity research, diabetes treatment, and metabolic health,

Mitochondria - not just a powerhouse

Our understanding of the mitochondrion—the "powerhouse" of the cell—has also changed.

Mitochondria were initially viewed as static structures within cells; however, we now know that mitochondria are dynamic organelles. They fuse, move, and interact with other cellular components, forming complex networks that help regulate cellular energy and even apoptosis (programmed cell death).

Mitochondria Videos

• What is a mitochondria
• Mitochondria function
• Mitochondrial DNA

• Mitochondria fusing

The Golgi

Like the mitochondria, our view of how the Golgi works has also changed. Previously, the Golgi was viewed as a series of membranes that looked like a stack of dinner plates, with the proteins being processed moving through the stack. Now, the machinery processing the proteins comes to the proteins, a radical change in our thinking.

My top picks

So, which do I view as the top change? Well, you will have to watch the video to find out!

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Maths and Chemistry Refresher for Life and Biomedical Scientists - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)
•  📗 - Catchup Maths and Stats - (affiliate link)
•  📗 - Catchup Chemistry - (affiliate link)
•  🌎 - You can practice your lab calculations at: https://maths4biosciences.drnickmorris.com/

New Video: Batch vs Continuous Cultures: Growing Cells in the Lab Explained

In this video, I look at Batch and Continuous Cell Cultures and discuss how they can be used in the lab:

In the lab, we often need to grow bacterial, yeast, or mammalian cells, and we use specific methods to cultivate cells in a controlled environment. 

Two common cell growth approaches are batch cultures and continuous cultures.

Batch Cultures: The Standard Approach

If you’ve ever worked in a biology lab, chances are you’re familiar with batch cultures. 

In a batch culture, cells are grown in a fixed space, such as a flask, dish, or plate. The culture is usually incubated for a set period, often overnight or several days, depending on the type of cells you’re growing.

During this process, the cells go through different stages of growth, represented by the bacterial growth curve:

1. Lag phase: Cells adapt to their environment.
2. Log phase: Cells rapidly divide and grow.
3. Stationary phase: Growth slows as nutrients deplete.
4. Death phase: Cells die as waste products build up and nutrients run out.

One limitation of batch cultures is that you only grow cells for a finite period. When the nutrients in the medium are exhausted, the cells stop growing, and you must start a new batch if you need more cells or products. The number of cells you can produce is limited by space and the number of available nutrients.

Continuous Cultures: Nonstop Cell Growth

In contrast to continuous cultures, cells are grown in a specialised vessel known as a bioreactor, fermentor, or chemostat. 

The continuous culture method continuously adds fresh media to the vessel while an equal amount of media is removed, creating a steady flow. This keeps the cells growing indefinitely, as they are always supplied with fresh nutrients.

One of the primary advantages of continuous cultures is that they maintain cells in the growth phase, meaning they can keep dividing and producing the desired products, whether that’s proteins, enzymes, or other biological compounds. However, setting up a continuous culture can be challenging. If the flow of new media is too slow, cells will run out of nutrients, leading to stagnation and death. But, if the flow is too fast, cells won’t have time to divide, and they could be washed out of the vessel.

The challenge with a continuous culture is finding the right balance in the media flow. The goal is to keep the cells in a state of constant division while also maintaining the product yield. Achieving this balance ensures that the total number of cells in the vessel remains steady over time, which is ideal for industrial applications where continuous production is required.

Choosing the Right Method

When deciding between batch and continuous cultures, the choice depends on the specific requirements of the experiment or production process. Batch cultures are easier to set up and control, making them ideal for smaller-scale studies or experiments with a defined endpoint. Continuous cultures are more complex but offer the advantage of sustained growth and production, which is essential for large-scale manufacturing or long-term studies.

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

New Video: Tagging and Labelling Proteins for Purification and Tracking

In this video - Tagging and Labelling Proteins for Purification and Tracking - I look at why we tag proteins and the methods we use to add a tag to a protein.

The Basics of Protein Tagging and Purification: A Lab Guide

During my career, I have had to produce and purify proteins in the lab, which can be challenging.

In the lab, we tag and purify proteins to understand what a protein does in the cell: how it works, is transported and what it interacts with, and to produce proteins for medical treatments. However, working with proteins in the lab presents some challenges. One of the biggest obstacles is that the cells that produce the protein of interest, they also make their own proteins for survival. So, how do we isolate our desired protein from the rest? The answer lies in tagging and labelling techniques, allowing easier purification and tracking.

Why Tag or Label a Protein?

When we express a protein in cells, whether for research or therapeutic purposes, it’s mixed with the cell’s proteins. Hence, we need to purify our protein of interest from this mix, and that's where tagging comes into play. Adding a specific "tag" to the protein allows us to separate it from other cellular proteins using specialised methods. 

Challenges in Protein Production

Another hurdle is that producing a large amount of protein burdens the cell, slowing its growth and division. To counteract this, we use a controlled system to regulate protein production. A common approach in bacterial systems is to use an expression vector that includes regulatory elements, such as the lac operon. Therefore, by adding a chemical called IPTG, we can switch on protein production at the right time once the cells have grown to the desired number.

Methods for Protein Tagging

When it comes to purification, two main protein tags are commonly used:

1. Histag: This tag consists of a sequence of six or more histidine residues that can be added to either the N- or C-terminal of the protein. After the cells producing the protein are lysed, the tagged proteins can be captured using nickel affinity chromatography. The histidine residues bind to the nickel, making purifying the protein from the cell mixture easy.
2. GST Tag (Glutathione S-Transferase): GST is a small protein that can be fused to the target protein. The fusion protein is purified using glutathione beads. One advantage of this method is that an enzyme can later cleave the GST tag, leaving behind the pure target protein.

Alternative Tagging for Visualisation

While GFP (Green Fluorescent Protein) doesn’t assist in purification, it is often used to label proteins for visualisation. GFP is a fluorescent protein derived from jellyfish, and it allows the movement of proteins to be tracked inside living cells under a microscope. Like Histag and GST, GFP tagging involves cloning the gene for GFP alongside the gene for the protein of interest, so both are expressed as a single molecule.

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

New Video: Three Parent Babies: Mitochondrial Disease Treatment

In the video - Three Parent Babies: Mitochondrial Disease Treatment - I look at how mitochondrial disease can be treated using mitochondrial replacement therapy (MRT) and three-parent babies.

The mitochondria, often called the "powerhouses" of the cell, provide the energy that keeps our bodies functioning. Interestingly, mitochondria carry their own DNA, separate from the DNA in the cell’s nucleus. This mitochondrial DNA (mtDNA) is passed down almost entirely from our mothers, and it plays a key role in producing proteins essential for the mitochondria's function.

However, mutations in mitochondrial DNA can lead to serious and sometimes life-threatening conditions, referred to as mitochondrial diseases. These diseases primarily affect high-energy tissues such as the brain, muscles, and heart, resulting in a range of debilitating symptoms.

What is Mitochondrial Replacement Therapy?

Mitochondrial replacement therapy (MRT) has been developed to combat these inherited mitochondrial conditions. This procedure aims to replace faulty mitochondria and prevent transmitting mitochondrial diseases from mother to child.

Here’s how it works: 

• An egg is taken from a healthy donor, and its nucleus is removed, leaving behind healthy mitochondria.
• Then, the nucleus from the mother’s egg (who has mitochondrial disease) is transferred into the donor egg, essentially creating a new egg with the mother’s genetic material but the donor’s healthy mitochondria.
• This egg is fertilised with the father’s sperm and implanted into the mother’s womb.

The result is a baby who inherits the vast majority of their DNA from their biological parents but receives mitochondria from a third-party donor. This process prevents the faulty mitochondria from being passed on, giving the baby a chance at a healthy life without mitochondrial disease.

Ethical Considerations of MRT

While mitochondrial replacement therapy has successfully prevented mitochondrial diseases, it comes with significant ethical considerations. Since mitochondria contain their own DNA, this procedure changes the genetic makeup of the individual born through MRT and their future offspring. This raises important questions about the long-term impact on the human gene pool and whether we should alter human genetics this way.

Despite these concerns, mitochondrial replacement therapy has already been performed in some countries, offering families the chance to have healthy children free from mitochondrial disease.

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)

New Video: Answering Your Questions on Genetic Engineering: Unintended Consequences, Science Models and Risks

On my video - Genetically Engineering Pigs to Reduce Environmental Pollution - I received a comment which included a series of important questions on genetic engineering. I was going to reply to the comment directly, but instead decided to do a video reply - Answering Your Questions on Genetic Engineering: Unintended Consequences, Science Models and Risks.

The questions raised in the comment can be broken down into four main areas of concern:

1. Understanding Unintended Consequences in Genetic Engineering

One of the central questions was about the potential unintended consequences of genetic engineering. While science has made remarkable progress, particularly with tools like CRISPR, it's essential to acknowledge that we still need to fully understand all biological systems. This incomplete picture means that, despite our best efforts, we can’t always predict every outcome of genetic modification.

For example, altering a gene in an organism may have unexpected downstream effects. In one case, when scientists genetically engineered babies to be resistant to HIV by removing the CCR5 receptor, they found off-target genetic changes that could have unintended consequences. I discuss these issues in my video - CRISPR Case Studies: Ethical Dilemmas and Revolutionary Applications.

Similarly, work in which I was involved on genetically modified potatoes showed that removing a harmful compound caused the plant to produce a different compound with potentially more harmful effects. You can get the full story in my video - Unexpected Challenges in Genetic Engineering: A Case Study on GM Crops.

The lesson? Genetic engineering requires caution. Scientists must always consider not only the intended outcomes but also the potential for unintended consequences.

2. The Incomplete Nature of Scientific Models

In the comment on the pooping pig video, I was asked whether I ever consider the incomplete nature of our understanding—whether scientific models are accurate or could fail in the long term. The simple answer is yes; I am well aware of this.

For example, at the start of my PhD studies, our understanding of insulin signalling was very basic. However, over the three years it took me to complete my PhD, scientists around the world significantly added to our knowledge and understanding. The model went from a simple black box to a complicated network involving many proteins. However, even this model is imperfect, and new discoveries are constantly reshaping our understanding.

This is true for all areas of biology, including genetic engineering. The more we experiment, the more we refine our models. However, the fact that our models are incomplete doesn’t mean we should stop pursuing genetic modification—it simply means we must proceed with caution and a sense of responsibility.

3. Ethical Concerns: Should We Be Playing God?

Another critical question raised was whether scientists are "playing God" by tinkering with nature. The concern here is that humanity might be overstepping its boundaries by altering the genetic makeup of living organisms.

Humans have indeed been manipulating nature for millennia, from selecting plants and animals for desirable traits to breeding dogs with specific characteristics. However, modern genetic engineering tools, such as CRISPR, allow us to make more precise and rapid changes, bypassing the slow process of natural selection and breeding.

While genetic engineering has the potential to solve significant problems—such as developing disease-resistant crops or addressing hereditary health issues—ethical considerations must remain at the forefront. Just because we can modify genes doesn't always mean we should.

4. Safeguards and the Role of Scientific Scrutiny

A significant factor that should be considered is the safeguards scientists put in place to prevent unintended consequences from spiralling out of control. For instance, if we genetically modify an organism, we can sequence its entire genome to ensure no other changes were made. This type of oversight is crucial when dealing with changes that could have long-term effects on ecosystems or human health.

The issue of gene drives—where genetic changes are engineered to spread through a population—is of particular concern as once released, there’s no way to reverse such changes. This makes it all the more important to thoroughly study and assess the implications before moving forward.

A Balanced Approach to Genetic Engineering

Genetic engineering holds immense promise but also carries significant risks. It is the duty of scientists to consider both the benefits and the possible dangers. By continuing to question the work, refine the models, and engaging with ethical concerns, science can harness the potential of genetic engineering in a way that’s safe and beneficial for society.

At the heart of this debate lies a critical question: how do we balance innovation with responsibility? And we should always consider the question — just because we can, should we?

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

New Video: Unexpected Challenges in Genetic Engineering: A Case Study on GM Crops

This video - Unexpected Challenges in Genetic Engineering: A Case Study on GM Crops - is a true story from my research career.

In 2004, I was involved in a €3 million research project that brought together labs from Europe and China. The research team included sociologists, psychologists, biochemists, botanists, and chemists, and we were looking at food safety in genetically modified (GM) crops and people's attitudes to 'functional foods'.  ('Functional foods' are foods that have been produced using genetic modifications or have added vitamins and minerals.)

The project focused on two staple foods—rice and potatoes—and our aim was to reduce harmful compounds in these crops that could pose risks to human health. Specifically, we were trying to develop a strain of rice with low levels of phytic acid and a variety of potatoes with reduced glycoalkaloid content.

Phytic acid, though naturally occurring in many plants, can bind essential nutrients like zinc, calcium, and iron, making them unavailable to the body. This is a particular problem in regions where diets heavily rely on rice, leading to widespread iron deficiency and anaemia, affecting one in four people globally. Rice produces phytic acid to store phosphate in the seed to help it grow.

Some of the different glycoalkaloids found in potatoes have been linked to health risks such as cancer. While potatoes are generally safe to eat, reducing the levels of certain glycoalkaloids could further enhance their safety. The potato produces glycoalkaloids to prevent it from rotting.

Our collaborators in China irradiated rice to introduce mutations, grew the plants and screened them for low phytic acid levels. Meanwhile, our colleagues in Aberdeen developed a potato with a gene knocked out that was responsible for producing a specific glycoalkaloid.

As with many scientific endeavours, our project encountered unexpected results. 

In the case of low phytic acid rice, the mutation that blocked phytic acid production also disrupted a key component of the glycolytic pathway. This meant that the rice could only complete one turn of the TCA cycle per glucose molecule, severely stunting its growth. The rice, though low in phytic acid, grew poorly.

The potato project presented its own surprises. While the gene responsible for producing one of the glycoalkaloids was successfully knocked out, the potato plant compensated by activating another gene that produced a different glycoalkaloid. The total glycoalkaloid content remained unchanged.

For me, this project was a powerful reminder that science, particularly genetic engineering, is often unpredictable. Despite our best efforts and the involvement of many bright minds in the field, the natural complexity of these plants outsmarted us. The results we achieved were not what we had hoped for, but they were incredibly valuable in their own right. They highlighted the intricate balance of biological systems and the challenges of modifying them without unintended consequences.

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

New Video: Genetically Engineering Pigs to Reduce Environmental Pollution

In this very short video - Genetically Engineering Pigs to Reduce Environmental Pollution - I look at how genetically engineering a pig has produced an animal that is less harmful to the environment.

The problem of phosphate pollution in the runoff from farms, particularly in pig farming, is a significant environmental concern. Animals' diets are often rich in grains, which contain phytic acid, a compound that pigs cannot digest due to the absence of a specific enzyme. The undigested phosphate-rich phytic acid is then excreted as waste, contributing to environmental issues like algal blooms and water contamination.

Phytic acid, found abundantly in grains, is a form of phosphorus that pigs—and many other animals—cannot utilise because they lack the phytase enzyme to break it down. Without this enzyme, the phosphorus passes through the pigs' digestive system and is excreted, leading to the concentration of phosphate in the environment.

To combat this issue, pigs have been engineered to contain the E. coli appA gene, which enables them to produce phytase, the enzyme needed to digest phytic acid. This modification allows the pigs to break down the phytic acid in their diet, effectively reducing the amount of phosphate in their waste.

What makes this solution particularly innovative is the way the gene is expressed. The E. coli appA gene is under a promoter from a mouse, which regulates the expression of proteins in the mouse salivary gland. This means that the pig only produces the phytase enzyme in its saliva. This targeted expression ensures that the enzyme is active exactly where it needs to be—in the pig's mouth. As the pig chews and swallows, the phytase is mixed with the grain, breaking down the phytic acid before it can pass through the digestive system.

This genetic modification significantly lowers the levels of phosphate pollution associated with pig farming by reducing the amount of undigested phytic acid excreted by pigs. 

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

New Video: How Plants Are Fighting Climate Change and Pollution?

 In this video - How Plants Are Fighting Climate Change and Pollution? - I look at how plants are being used to help fight climate change and polution.

Genetically engineered plants are being explored to combat climate change and environmental pollution. 

Slowing Climate Change with Carbon-Capturing Plants

One area of research focuses on using plants to slow climate change by enhancing their ability to capture and store carbon. Plants have been engineered to produce suberin, a cork-like substance that resists degradation and effectively traps carbon. These plants are also being developed to grow deeper root systems, ensuring that the suberin (and the carbon it contains) is buried deep within the soil, where it can remain sequestered for long periods.

This carbon sequestration will help reduce the amount of carbon dioxide in the atmosphere and slow global warming. 

Building Resilient Crops for a Changing Climate

As climate change continues to impact agricultural productivity, crops must be developed that can withstand extreme conditions such as heat, drought, disease, and high salinity. Researchers are working to make plants more resilient, ensuring that food supplies remain stable even as the environment becomes increasingly unpredictable.

By enhancing plants' natural defences, it is hoped that crops can be produced that can thrive in adverse conditions, reducing the risk of crop failures and food shortages.

Plants as Pollution Fighters

Plants are also being engineered to tackle pollution. 

A particularly interesting approach involves cloning the rabbit gene P4502E1 into Devil's Ivy, a popular houseplant. This gene encodes an enzyme that breaks down harmful chemicals, including potential carcinogens, into less toxic compounds. By introducing this gene into plants, researchers are creating a natural air purification system that can absorb and detoxify pollutants in urban environments and indoor spaces.

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

New Video: The genetically engineered mouse and COVID-19

 In this video - How Genetically Engineered Mice Help Us Study COVID-19 - I look at how a mouse that was genetically engineered for studying one virus, helped us understand COVID-19.

To develop effective treatments and vaccines for COVID-19, we needed to understand how the virus interacted with living organisms. For this, scientists typically use model organisms like mice. However, using mice for COVID-19 research presented a significant problem.

COVID-19, caused by a coronavirus, gains entry into human cells by binding to a specific receptor on the cell surface known as ACE2 (angiotensin-converting enzyme 2). However, the ACE2 receptor in mice differs from the human version, making it impossible for the virus to bind to mouse cells. This meant that mice could not be used to study the virus in a living organism to better understand how it works and to test potential treatments.

Fortunately, genetic engineering provided a solution. 

Back in 2007, during the outbreak of another coronavirus, SARS, scientists developed a line of genetically engineered mice that express the human version of the ACE2 receptor. These “humanised” mice could be infected with SARS-CoV-2, the virus responsible for COVID-19, making them an invaluable tool in the fight against the pandemic.

Using these genetically modified mice, researchers could study how COVID-19 interacted with cells, how it causes disease, and how different treatments might work in a living organism.

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

New Video: Gene Drives: A Powerful and Controversial Genetic Technology

In this video - Gene Drives: A Powerful and Controversial Genetic Technology - I look at gene drives. A genetic engineering technique that I find equally fascinating and scary.

Gene drives are a revolutionary and controversial advancement in genetic engineering. The process allows scientists to introduce self-propagating changes into an organism's genome, bypassing traditional Mendelian inheritance. By ensuring that specific mutations become homozygous in offspring, gene drives have the potential to rapidly spread these changes throughout populations.

The applications of gene drives are both promising and far-reaching. They could play a crucial role in eradicating mosquito-borne diseases such as malaria and Zika, which continue to claim millions of lives globally. Additionally, gene drives could help tackle drug-resistant fungal pathogens that pose a significant threat to human health and be employed to control or eliminate invasive species that disrupt ecosystems.

But how does this technology work? 

Gene drives use CRISPR, a gene-editing tool. In a gene drive, the CRISPR machinery is introduced along with the desired mutation. This machinery then actively copies itself onto the corresponding chromosome, ensuring the organism becomes homozygous for the mutation. This process allows the mutation to "drive" through the population, even if it does not provide a traditional selective advantage.

However, the power of gene drives comes with significant ethical concerns. The ability to alter ecosystems and species at such a fundamental level raises the question: Just because we can, should we? That is, once you introduce a change in a system using gene drive, how could you change or reverse it, or stop it spreading to other organisms?

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

CRISPR Case Studies: HIV resistant babies, cancer and blindness

In the video - CRISPR Case Studies: Ethical Dilemmas and Revolutionary Applications - I look at the illegal use of CRISPR on babies and its legal use to treat some conditions.

CRISPR has emerged as a powerful genetic engineering tool. However, it does come with the ethical implications.

CRISPR

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a gene-editing tool that allows scientists to alter DNA sequences within organisms. Using this technology, researchers can add, remove, or modify genetic material, leading to potential cures for genetic diseases, improved agricultural practices, and innovative solutions to environmental issues.

However, using CRISPR, especially in humans, raises significant ethical issues.

The CRISPR Babies

One of the most shocking uses of CRISPR on humans occurred when the genomes of human embryos were edited, leading to the birth of two genetically modified babies. The primary goal was to disable the CCR5 gene to produce babies that were HIV-resistant. That is, remove the receptor that HIV uses to enter human cells.

The CCR5 gene was selected because a naturally occurring mutation in this gene provides resistance to HIV and the Black Death in about 1% of Northern Europeans.

Researchers attempted to mimic this natural 32-base pair deletion using CRISPR, disrupting the CCR5 receptor's function and preventing HIV infection.

Outcomes and Concerns

The resulting edits did not replicate the exact natural mutation. One baby had a 15-base pair deletion in one copy of the gene, while the other had different insertions and deletions across both gene copies.

These unintended mutations raise serious health concerns as studies suggest that individuals with CCR5 mutations may have a 20% lower likelihood of reaching age 76 and could be more susceptible to other infections and diseases.

There is also the risk of off-target edits, where CRISPR inadvertently alters other parts of the genome, potentially leading to unforeseen health issues.

The scientific community widely condemned this work for its ethical breaches, lack of transparency, and disregard for established guidelines. The researchers involved faced legal consequences, including fines and imprisonment.

This case highlights the ethical dilemmas associated with germline editing:

• Consent: The edited changes are heritable, affecting future generations who cannot consent.
• Risk vs. Benefit: The potential health risks may outweigh the intended benefits, especially given existing alternatives to prevent HIV transmission.
• Regulatory Oversight: The need for strict guidelines and oversight in genetic editing research is evident to prevent misuse and ensure ethical compliance.

Promising and Legal Applications of CRISPR

CRISPR has the potential for legitimate and beneficial medical applications. Here are two examples:

1. Cancer Treatment

Treatment of testicular cancer resistant to conventional therapies.

Patient-derived T cells are collected and genetically modified using CRISPR to disrupt three specific genes that regulate T cell targeting. 

A lentivirus is then used to introduce a new targeting mechanism, directing the T cells to recognise and attack proteins unique to the patient's cancer cells.

The modified T cells are then reintroduced into the patient, aiming to boost the immune system's ability to fight cancer effectively.

This represents a personalised and targeted approach to cancer therapy and demonstrates CRISPR's potential to improve immunotherapy treatments.

2. Treating Childhood Blindness

CRISPR is also being used to address Leber Congenital Amaurosis 10 (LCA10), a cause of blindness in children.

The procedure targets the CEP290 gene, where specific mutations disrupt normal retinal development.

The CRISPR components are packaged into adeno-associated viruses (AAVs), and the system precisely removes the mutation in the CEP290 gene, aiming to restore proper protein function and improve vision.

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

Exploring Genetic Engineering: How It's Protecting Crops and Enhancing Food

In this video - Exploring Genetic Engineering: Protecting Crops and Enhancing Food - I look at three examples of how genetic engineering has been used to protect crops from insects and enhance the nutritional value of food.

1. BT Cotton: A Solution with Unintended Consequences

BT cotton is a prime example of how genetic engineering can provide a targeted solution to agricultural problems. This cotton variety has been engineered to produce a toxin derived from a specific type of bacteria. The toxin crystallises within the plant, and when an insect eats the leaves, the toxin dissolves the insect’s gut, eliminating the pest without the need for widespread insecticide use. This approach has the added benefit of protecting beneficial insects, as the crop itself becomes a selective defence mechanism.

However, this crop has had some unintended consequences. For example, in India, the high cost of BT cotton seeds has created a debt cycle for many farmers. Additionally, the market has seen a rise in counterfeit seeds, complicating the situation further. 

2. Venomous Cabbage: A Controversial Defense

I do like this one.... what made the researchers think of it?

The venomous cabbage - these cabbages have been modified to express scorpion venom in their leaves. The Diamondback moth larvae, notorious for damaging cabbage crops, eat the leaves of cabbages producing the venom and dying. Luckily, this venom is not toxic to humans, making the cabbage safe to eat.

Would you be comfortable eating such a cabbage?

3. Golden Rice: Tackling Vitamin A Deficiency

I was involved in a similar project - more about that in a later video.

Golden Rice represents a more human-centric approach to genetic engineering. This rice has been enriched with beta-carotene, a compound the human body can convert into vitamin A - making it a potent tool in the fight against vitamin A deficiency, which is prevalent in many parts of the world.

The rice owes its yellow colour to the addition of genes from daffodils and bacteria.

While Golden Rice has the potential to significantly improve public health, it also raises important questions. How do we feel about consuming a staple food that contains genes from other species? This case exemplifies the broader debate surrounding genetically modified organisms (GMOs) and their role in our food supply.

What are your thoughts on these genetically engineered crops? Would you eat them? Let me know in the comments or comment on the video.

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

The Cavendish Banana Crisis: How CRISPR Could Save Our FavoUrite Fruit

I like bananas — there, I have said it. They are one of my go-to fruits. But did you know they are in danger of being wiped out and no longer available?

In this video - How CRISPR Could Save Bananas from Extinction | The Cavendish Crisis Explained -  I look at why bananas are in trouble and how genetic engineering may come to the rescue.

About 99% of bananas we eat come from a single strain known as the Cavendish. Unfortunately, this strain faces a threat that could wipe it out.

The Cavendish Banana: A Monoculture at Risk

As popular as it is, the Cavendish banana has a significant vulnerability. It's sterile, meaning it can’t reproduce through seeds like many other plants. Instead, it’s propagated through cuttings. While this has allowed us to produce vast quantities of genetically identical bananas, it also means that if a disease affects one plant, it can quickly spread to all Cavendish bananas worldwide.

Fusarium wilt tropical race 4, or TR4, is a fungus threatening to wipe out the Cavendish banana. TR4 attacks the plant's roots, eventually killing it. Because the Cavendish is sterile, traditional breeding methods can’t be used to introduce resistance to this fungus, making the banana especially vulnerable.

Worryingly, this isn’t the first time a banana strain has faced extinction due to a fungal disease. Before the Cavendish, the Gros Michel banana was the world's favourite. However, attacked by a different strain of Fusarium wilt, leading to its near-total disappearance from the market. The Cavendish was introduced as a replacement, but now it’s facing a similar fate.

How Can We Save the Cavendish Banana?

Traditional breeding can't be used as the Cavendish is sterile. Hence, scientists are using genetic engineering to save the banana. One of the most promising approaches involves tweaking the banana’s genome to make it resistant to TR4. 

One explored method is inserting a resistance gene from wild bananas into the Cavendish. The wild bananas have naturally evolved to resist the fungus, and by transferring their genes, we could give the Cavendish the same level of resistance.

However, an even more interesting approach would be to use CRISPR to make precise changes to the banana’s DNA. In the case of the Cavendish banana, CRISPR could be used to activate a gene that has been silenced but could provide resistance to TR4. Additionally, CRISPR could deactivate genes that make the Cavendish susceptible to the fungus.

Why CRISPR is a Game-Changer

The advantage of using CRISPR is that it doesn’t involve inserting foreign DNA into the banana. This means the resulting banana wouldn’t be considered transgenic, which could ease regulatory hurdles and public concerns about genetically modified organisms (GMOs). 

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

New Video Posted: Introduction to Genetic Engineering: Ethics, Science, and Innovation

I have posted a video - Introduction to Genetic Engineering: Ethics, Science, and Innovation.

Genetic engineering often evokes strong emotions and heated debates. When people hear the term, they might immediately think of genetically modified organisms (GMOs), "Frankenstein foods," or the ethical dilemmas surrounding altering life at its most fundamental level. But what exactly is genetic engineering, and what are the implications of this technology?

In its simplest form, genetic engineering directly manipulates an organism's DNA to achieve desired traits. Humans have used genetic engineering through selective breeding to cultivate crops and livestock that yield better produce and more reliable outcomes for centuries. But today’s technology allows scientists to bypass traditional breeding processes and make precise changes to DNA in a laboratory setting. This raises a profound question: just because we can modify life in this way, should we?

In the video, I look at the ethical and scientific complexities of genetic engineering and wonder where society should draw the line. Is it acceptable to engineer a potato or a chicken for better production? What about a cow? How about a human? These questions are not just theoretical but have real-world implications as technology continues to advance.

I also discuss how genetic engineering can be further divided into two broad categories of genetic engineering: research and applications. Research involves using genetic engineering to understand biological systems and diseases, while applications focus on improving crops, livestock, and human health. The ethical dilemmas become particularly acute when considering human health. Should genetic modifications be limited to somatic cells (which don’t get passed on to offspring), or is it ethical to alter germ cells, thereby affecting future generations?

Finally, I wrap up the video by explaining the difference between transgenic and non-transgenic organisms. Transgenic organisms have DNA from a different species introduced into their genome, while non-transgenic organisms involve changes made to the organism’s own DNA. But this distinction leads to another intriguing question: Is DNA truly species-specific?

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

New Video Posted: Site-Directed Mutagenesis Explained | Understanding the Basics

In this video - Site-Directed Mutagenesis Explained | Understanding the Basics - I look at how you can mutate DNA in the lab.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Site-directed mutagenesis is a powerful technique in molecular biology that can introduce specific mutations into a DNA sequence. The method has been instrumental in understanding how proteins function and how genes are regulated. 

The Process of Site-Directed Mutagenesis

Site-directed mutagenesis involves several key steps:

1. Preparation of DNA: The DNA sequence of interest is either cloned into a plasmid or selected from a plasmid library.
2. Designing the Primer: A primer is designed to bind specifically to the region of DNA where the mutation is desired. This primer is about 20 bases long, with one crucial difference—it contains the new base that will introduce the mutation. More information on Primer Design.
3. Creating a Single-Stranded DNA Template: The double-stranded plasmid DNA is converted into a single-stranded form. This single strand serves as the template for the primer to bind.
4. DNA Synthesis and Ligation: Once the primer is bound, DNA polymerase synthesises the complementary strand, incorporating the new base. DNA ligase is then added to seal the DNA strand, completing the synthesis.
5. Introduction into Bacteria: The newly mutated plasmid is introduced into bacteria, where the bacteria's natural DNA repair mechanisms take over. These mechanisms either repair the mismatched DNA back to its original form or incorporate the new mutation.

Applications of Site-Directed Mutagenesis

The ability to change a single base in a DNA sequence provides scientists with a powerful tool to explore how specific mutations affect protein function and gene expression. This technique has enabled numerous studies, allowing researchers to pinpoint individual amino acids' roles in proteins and dissect complex genetic regulatory networks.

From Site-Directed Mutagenesis to CRISPR

While site-directed mutagenesis has been a common lab technique for decades, the advent of CRISPR technology has revolutionised the field of genetic engineering. CRISPR offers a more efficient and precise method for making targeted genetic changes, but the foundational principles of mutagenesis laid the groundwork for these modern advances.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)

New Video Posted: DNA Sequencing: Sanger Method and Beyond Explained

In the video DNA Sequencing: Sanger Method and Beyond Explained, I explain the Sanger method and look at some of the new approaches and methods that can speed up the DNA sequencing process.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

DNA sequencing allows scientists to decode the genetic information that dictates everything from the colour of our eyes to how our cells function. One of the most widely used laboratory methods for DNA sequencing is the Sanger method, also known as the chain-terminating dideoxynucleotide method.

The Basics of Sanger Sequencing

The Sanger sequencing process begins like a PCR (Polymerase Chain Reaction), with a template DNA that you want to sequence, a single primer (and not two primers as in PCR), DNA polymerase, and nucleotides (dNTPs). However, the method also includes special nucleotides called dideoxynucleotides (ddNTPs), which play a key role in the sequencing process.

In the sequencing method, the reaction goes through 25-30 cycles of denaturing, annealing and extension, just as you would in PCR. The DNA polymerase builds new DNA strands during the reaction and adds nucleotides to the growing chain. The twist comes with the ddNTPs; when one of these is incorporated, the chain is terminated, and no further nucleotides can be added. Each ddNTP is tagged with a different fluorescent marker corresponding to one of the four DNA bases (A, T, C, or G). This allows the sequence to be read by analysing the fluorescent tags after the reaction.

Once the reaction is completed, the next step is separation. The DNA fragments are separated using gel electrophoresis. As the fragments move through the gel, a laser scans the gel for the fluorescent tags, and the sequence of the DNA is determined by the fluorescence colour at each position.

While the Sanger method has been incredibly valuable, it has limitations. It can only read between 500 to 1000 bases per reaction, making it labour-intensive and unsuitable for large-scale projects.

Moving Beyond Sanger: Advanced Sequencing Methods

As the need for faster and more efficient sequencing has grown, new methods have been developed. One such method is shotgun sequencing, where the genome is broken into random segments. Each segment is sequenced individually using the Sanger method, and then the fragments are pieced together like a puzzle to reconstruct the full genome.

Another significant advancement was Next-Generation Sequencing (NGS), also known as massively parallel sequencing. NGS technologies have revolutionised genomics by allowing the simultaneous sequencing of many DNA molecules. This high-throughput approach can detect the sequence using either fluorescent nucleotides or pH changes.

Finally, we have Third-Generation Sequencing methods, which include techniques like nanopore sequencing. In nanopore sequencing, DNA is passed through a nanopore, and the sequence can be determined by detecting changes in electrical current.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Additional Resources

•  📗 - The Biosciences Glossary - Google Play Book Store
•  📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
•  📗 - Molecular Cell Biology (Lodish) - (affiliate link)
•  📗 - Biochemistry (Stryer) - (affiliate link)
•  📗 - Principles of Biochemistry (Lehninger) - (affiliate link)
•  📗 - Catchup Chemistry - (affiliate link)

New Video Posted: DNA Libraries: Genomic vs. cDNA - Key Differences and Applications

 I have posted a video on the key differences between genomic DNA (gDNA) and complementary DNA (cDNA) libraries - DNA Libraries: Genomic vs. cDNA - Key Differences and Applications.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

DNA libraries are essential tools for researchers to study and manipulate genetic material. If you're working in a lab, you'll likely encounter two main types of DNA libraries: genomic DNA (gDNA) libraries and complementary DNA (cDNA) libraries. Though both serve important roles in research, they are fundamentally different in their composition, creation, and applications. 

What Are DNA Libraries?

A DNA library is a collection of DNA sequences cloned into vectors, small pieces of DNA that can carry foreign DNA into a host cell, such as bacteria. These libraries allow scientists to store, access, and manipulate specific DNA sequences for various research purposes.

Genomic DNA (gDNA) Libraries

A genomic DNA library is created from the complete genomic DNA of an organism. This means it contains all the genetic material necessary to build that organism. In eukaryotes, this includes both the coding regions (exons) and the non-coding regions (introns) of genes. 

How is a gDNA Library Made?

1. DNA Extraction: Genomic DNA is extracted from cells.
2. DNA Fragmentation: The extracted DNA is cut into smaller fragments using restriction enzymes.
3. Cloning: These fragments are then cloned into plasmids, which are circular DNA molecules.
4. Transformation: The plasmids are inserted into bacteria, which replicate the DNA fragments, creating a library.

When to Use a gDNA Library?

gDNA libraries are ideal when studying the full structure of genes, including regulatory elements, or when exploring gene functions across the genome.

However, there are some limitations. Because a gDNA library contains both introns and exons, it can complicate the task of isolating and studying the sequences that actually code for proteins. Additionally, genes in a gDNA library may be fragmented across multiple clones, making it challenging to reconstruct the complete gene sequence through sequence alignment.

Complementary DNA (cDNA) Libraries

cDNA libraries are derived from messenger RNA (mRNA) in a cell. This means that a cDNA library only contains the sequences actively expressed as proteins when the library is made. Therefore, the content of a cDNA library depends on the type of cell, the time of day, and the cell's conditions.

How is a cDNA Library Made?

1. mRNA Isolation: Cells are lysed, and the mRNA is purified from the lysate using affinity chromatography, which often involves oligo-dT beads that bind to the poly-A tails of mRNA molecules.
2. Reverse Transcription: Using reverse transcriptase, the mRNA is used as a template to synthesise complementary DNA (cDNA). This gives a mRNA/DNA molecule.
3. RNA Removal: The original mRNA is removed from the mRNA/DNA molecule using an enzyme (RNase H). The now single-stranded cDNA is converted into a double-stranded DNA molecule using DNA polymerase.
4. Cloning: The cDNA is cloned into plasmids and inserted into bacteria.

Isolating mRNA can be tricky because it is easily degraded by enzymes released during cell lysis or by RNases in the environment. Moreover, because cDNA libraries only reflect the genes being expressed at a specific time, they may not provide a complete picture of an organism's genome.

When to Use a cDNA Library?

cDNA libraries are particularly useful for studying gene expression, identifying specific mRNA sequences, and producing recombinant proteins.

Choosing the Right Library

It is essential to understand the distinctions between gDNA and cDNA libraries. A gDNA library is the right choice if the goal is to study the entire genome or understand gene regulation. However, a cDNA library must be used if you're interested in the proteins a cell produces or need to work with specific mRNA sequences.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Additional Reading

The video was produced with help from the following resources:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)

New Video Posted: PCR Primer Design: Tips for Accurate DNA Amplification

I have posted a video on PCR primer design - PCR Primer Design: Tips for Accurate DNA Amplification.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Polymerase Chain Reaction (PCR) is an important lab technique that allows scientists to amplify specific DNA sequences. However, the success of a PCR largely depends on correctly designing the PCR primers. 

Why Primer Design Matters

The importance of primer design in PCR cannot be overstated. If your primers are poorly designed, the DNA amplified during the reaction might not be the correct region of DNA, or you will get a low product yield. To avoid such issues, you must carefully consider three primary factors when designing primers: 

1. Melting temperature (Tm)
2. Disruptive secondary structures
3. Specificity of the primers

1. Melting and Annealing Temperatures

Two crucial temperatures play a role in PCR primer design:

1. the melting temperature (Tm)
2. the annealing temperature

The Tm is the temperature at which 50% of the primer would dissociate from a double-stranded to a single-stranded form. The primers bind to the denatured DNA template during each PCR cycle at the annealing temperature. Typically, the ideal Tm is 55-65 °C, and the annealing temperature is set 3-5 °C below the Tm to ensure optimal primer binding.

In the lab, you would use a computer program to determine the Tm of a primer. The program usually uses the nearest neighbour method to calculate the Tm accurately. This method considers the specific sequence of bases and the concentrations of components in the reaction.

2. Disruptive secondary structures

It is essential to ensure your primers won’t form problematic secondary structures like hairpin loops or primer-dimer pairs. Hairpin loops occur when a primer folds back on itself, creating a loop that can be difficult to melt during the PCR process, leading to inefficient amplification. Primer-dimer pairs form when two primers bind to each other instead of the target DNA. This can result in an inefficient reaction and the production of the wrong DNA.

In the lab, you would use a computer program to test for hairpin loop formation and primer-dimer pairs.

3. Specificity of the primers

A standard PCR primer is usually 20 to 30 bases long and has an ideal Tm of around 55-65 °C. The Tm and the length of the primer help ensure specificity. 

Finally, it is advisable to perform a BLAST search with your primer sequences, which can help confirm that they do not have unintended matches with other sequences in the species. If the DNA species is not human, a BLAST search against a human database should be performed to check for the possibility of a result if the reaction is contaminated with human DNA.

Advanced Techniques: Overcoming Challenges in PCR

In some cases, even with well-designed primers, you might need help getting the correct DNA to amplify, especially when dealing with low amounts of template DNA or needing to add restriction sites to your product. To address this, you can use a nested primer approach. This involves using two sets of primers, one set falling inside the other, to improve specificity and yield through a two-round PCR process.

For adding restriction sites, primers are designed with the desired restriction site sequences at the 5’ end. However, this introduces a mismatch between the primer and the template DNA. To compensate, the initial PCR cycles are performed at a lower annealing temperature to help the primers bind more effectively. Once the restriction site is incorporated into the amplified DNA, the annealing temperature is raised to the standard level for subsequent cycles. A GC clamp at the 3’ end of the primer, where the last two bases are guanine (G) or cytosine (C), can also help improve binding due to the stronger hydrogen bonds these bases form.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Additional Reading

The video was produced with help from the following resources:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)

New Video Posted: Bacterial Transformation: Natural vs. Artificial Methods Explained

In this video - Bacterial Transformation: Natural vs. Artificial Methods Explained - I look at how bacteria can take up DNA from the environment and how we can get bacteria to take up DNA in the lab.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Bacterial transformation is where bacteria take up external DNA, which can lead to genetic changes within the cell. This process can occur naturally or be induced artificially in a laboratory setting. 

Natural Transformation

In natural transformation, bacteria acquire DNA from their surroundings, typically from nearby bacteria that have lysed (broken apart). The free-floating DNA can then be integrated into the bacterial chromosome or replace an existing gene, leading to potential new traits or functions. This ability to naturally take up DNA is a trait of "competent" cells, which possess specific genes that encode the machinery necessary for DNA uptake.

Artificial Transformation 

In a lab, we can induce transformation using artificial methods. Unlike natural transformation, this process requires deliberate manipulation of the bacteria to make them more likely to accept new DNA. First, the bacteria must be made competent so they can take up the DNA. Techniques such as heat shock, electroporation, or polycations are then used to encourage bacteria to take up the DNA. Each method works differently but ultimately serves the same purpose: introducing new genetic material into bacterial cells, enabling researchers to study gene function, produce recombinant proteins, or create genetically modified organisms.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Additional Reading

The video was produced with help from the following resources:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)

New Video Posted: How Bacteriophages Transfer DNA Between Bacteria

In this video - Understanding Transduction: How Bacteriophages Transfer DNA Between Bacteria - I look at the process of DNA exchange, transdcution, between bacteria by bacteriophages.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Transduction is driven by bacteriophages, which are viruses that specifically target and infect bacterial cells. In the video, I explain the mechanics of transduction and look at how bacteriophages can inadvertently transfer DNA from one bacterium to another.

Bacteriophages operate through two distinct life cycles: the lytic and lysogenic cycles. During the lytic cycle, the bacteriophage attaches to a bacterial cell, injects its DNA, and takes over the bacterium to produce new viruses. This ultimately leads to the bursting (lysis) of the bacterial cell, releasing the newly formed phages to continue the infection cycle. In the lysogenic cycle, the bacteriophage DNA is integrated into the bacterial chromosome, where it can lie dormant until conditions favour a return to the lytic cycle.

Some bacteriophages, such as the P22 phage, can mistakenly package bacterial DNA instead of their own during the assembly of new viruses. When these phages go on to infect other bacteria, they transfer the captured bacterial DNA, effectively driving genetic exchange between bacteria. This mechanism contributes to bacterial evolution in the wild and serves as a valuable tool in the lab for gene mapping and studying bacterial genetics.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Additional Reading

The video was produced with help from the following resources:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)

New Video Posted: Gene Mapping in Bacteria Using Conjugation and Interrupted Mating

Gene mapping in bacteria is a process that has helped scientists understand the order and location of genes on a bacterial chromosome. 

In this post, I will look at a method of gene mapping using bacterial conjugation, specifically focusing on a technique known as interrupted mating. Although this technique is historical and has been largely replaced by genome sequencing, it remains an important method that sheds light on genetic exchange in bacteria.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

 

The Role of Hfr Cells in Gene Mapping

The process of using conjugation to map genes in bacteria was developed using a specific type of bacteria discovered in the 1950s, known as Hfr (High Frequency of Recombination) cells. These cells are particularly efficient at transferring genes during conjugation, a process where DNA is exchanged between two bacterial cells.

In Hfr cells, the F (fertility) factor, which is normally an independent plasmid, gets incorporated into the bacterial chromosome. This integration means that during conjugation, the F factor and parts of the bacterial chromosome can be transferred to a recipient cell.

Two outcomes can occur during this process:

1. F' Factors: If the F factor is excised from the chromosome, it may carry with it some bacterial genes, forming what is known as F' factors.
2. Chromosome Transfer: Alternatively, the entire bacterial chromosome, including the F factor, can be transferred to the recipient cell.

The Concept of Interrupted Mating

Interrupted mating is a technique that was used for gene mapping. To understand how it works, let’s consider an example involving two bacterial strains:

• Donor Bacteria (Hfr positive): This strain has the genes to produce amino acids leucine and threonine but is sensitive to the antibiotic ampicillin.
• Recipient Bacteria (Hfr negative): This strain lacks the genes for leucine and threonine production but is resistant to ampicillin.

In this experiment, the donor and recipient bacteria are mixed and allowed to conjugate. The goal is to map the location of the genes that produce leucine and threonine on the donor’s chromosome. Here’s how the process unfolds:

1. Conjugation Begins: The bacteria are mixed, and DNA transfer begins from the Hfr donor to the recipient.
2. Interrupted Mating: At specific time intervals, the mating process is interrupted, effectively stopping the transfer of DNA.
3. Plating on Selective Media: After interrupting the mating, the bacteria are plated on media that either lacks leucine or threonine and contains ampicillin. The donor bacteria, which are sensitive to ampicillin, will die. The recipient bacteria will only grow if they have received and integrated the necessary genes from the donor to produce the missing amino acids.
4. Mapping Gene Order: By examining which plates show bacterial growth at different time points, scientists can determine the order in which the genes were transferred. For instance, if bacteria start growing on the leucine-lacking plate before the threonine-lacking plate, it indicates that the leucine gene is closer to the origin of transfer than the threonine gene.

Applications and Limitations of the Technique

This method of mapping was useful because it allows scientists to estimate the relative positions of genes on a bacterial chromosome. For example, using this technique, researchers were able to map the E. coli K12 genetic map into a timeline of 100 minutes, representing the time required for the full chromosome to transfer at 37 °C.

However, the technique has limitations:

• It is mostly applicable to E. coli and closely related bacteria.
• Mapping the entire chromosome is rare and works best for genes that are close to each other.
• The F plasmids involved are large, of low copy number, and may not be ideal for genetic manipulation.

Despite these limitations, the interrupted mating technique provides valuable insights into genetic exchange mechanisms in bacteria. While modern approaches like genome sequencing have largely replaced it, understanding this historical method helps us appreciate the exchange of DNA between bacteria.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

 

Additional Reading

The video was produced with help from the following resources:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)

New Video Posted: Genetic Exchange in Bacteria: Conjugation, Transduction, and Transformation

In this video, I look at the three methods bacteria use to share genetic information: Conjugation, Transduction, and Transformation.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

Bacteria typically increase their numbers through a process akin to cloning; that is, they make exact copies of themselves. While this method is efficient, it presents a significant evolutionary limitation: genetic diversity can only arise through random mutations. In the absence of a mechanism for exchanging genes, bacteria would be stuck in a genetic standstill, unable to benefit from the rapid spread of advantageous traits. Bacteria solve this problem by horizontal gene transfer (HGT), which is also called lateral gene transfer (LGT).

To help the spread of advantageous genes in a bacteria population, the bacteria use three methods to exchange DNA:

1. Conjugation: This process involves direct physical contact between two bacteria through a structure called a sex pilus. A donor bacterium with an F plasmid (F+) forms a bridge to a recipient bacterium (F-), transferring a copy of its DNA. This method not only promotes genetic diversity but also allows for the rapid spread of advantageous traits such as antibiotic resistance.
2. Transduction: In this method, bacteriophages (viruses that infect bacteria) play a crucial role. When a virus infects a bacterium, it sometimes incorporates fragments of the host's DNA into its own genetic material. As the virus infects new bacterial cells, it transfers these DNA fragments, facilitating genetic exchange.
3. Transformation: This process occurs when bacteria take up free-floating DNA from their environment, often released by dead bacterial cells. The acquired DNA is then incorporated into the recipient's genome, providing new genetic traits that can be beneficial for survival and adaptation.

In the video, I examine the three methods.

Blog Bonus: Free information sheet summarising the video and defining the key terms - download.

 

Additional Reading

The video was produced with help from the following resources:

• 📗 - The Biosciences Glossary
• 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
• 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
• 📗 - Biochemistry (Stryer) - (affiliate link)