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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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New Video Posted: Understanding Mendelian Genetics: Dominance, Codominance, and Incomplete Dominance

In this video, I explain some of the key terms in Mendelian genetics:

• dominant
• recessive
• codominance
• incomplete (partial) dominance

 

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Introduction to Mendelian Genetics

Mendelian genetics is named after Gregor Mendel, who studied inheritance patterns in pea plants and published his findings in 1866. His experiments with pea plants, including traits like seed shape and flower colour, laid the foundation for understanding how traits are inherited. By cross-breeding plants with different traits, Mendel observed how traits reappear in subsequent generations, leading to the formulation of key genetic principles.

Dominant and Recessive Traits

Mendel discovered that certain traits, like round seeds, are dominant, while others, like wrinkled seeds, are recessive. Through cross-breeding experiments, he noted that in the first generation (F1) of true-bred plants that produced round or wrinkled seeds, the offspring all exhibited the dominant trait. However, when these F1 plants were self-pollinated, the second generation (F2) showed a 3:1 ratio of dominant to recessive traits (see Figure 1).

Using modern terminology, this can be explained with uppercase and lowercase letters representing dominant and recessive alleles, respectively. For instance, the round seed trait is represented by "R" and the wrinkled seed by "r." The F1 generation consists of heterozygous plants (Rr), which exhibit the dominant trait. The F2 generation reveals a combination of homozygous dominant (RR), heterozygous (Rr), and homozygous recessive (rr) plants, demonstrating Mendel's observed ratios (see Figure 1).

Figure 1: Crossing round and wrinkled pea plants

Codominance and Incomplete Dominance

Moving beyond simple dominance, we come to codominance and incomplete (partial) dominance.

In codominance, neither allele masks the other; both traits are fully expressed (see Figure 2). For example, in a hypothetical scenario with cows, crossing a blue cow (BB) and a yellow cow (bb) would result in offspring with both blue and yellow spots (Bb).

Figure 2: Codominance

In incomplete dominance (sometimes called partial dominance), the traits blend rather than mask one another (see Figure 3). Using the same example of cows, crossing a blue cow and a yellow cow would produce green offspring (Bb), illustrating a blend of the two parent traits.

Figure 3: Incomplete Dominance - also called partial dominance

Key Takeaways

1. Dominant and Recessive Traits: Dominant alleles mask recessive ones in heterozygous pairings.
2. Codominance: Both alleles are fully expressed in the phenotype.
3. Incomplete (Partial) Dominance: The traits blend, creating an intermediate phenotype.

Additional Resources

For further assistance, a Bioscience Glossary with over 2000 terms, chemical structures, and supporting videos is available. This glossary can help clarify additional terms and concepts in biosciences.

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New video posted: Genetics and Mendel — DNA, genes, dominant and recessive traits

In this video, I examine Genetics and Mendel and discuss DNA, genes, and dominant and recessive traits.

The video introduces Gregor Mendel, an Austrian scientist and Augustinian friar known as the "father of modern genetics". I discuss his classic rounded and wrinkly pea experiments and describe how he came up with the idea of dominant and recessive traits.

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