Activity 2-0 - Introduction to Plasmid and Enzyme p450

Nucleotides – The Building Blocks of DNA

DNA, or Deoxyribonucleic Acid, is the molecular blueprint of life, storing genetic information that dictates everything from eye color to disease susceptibility. At its core, DNA is made up of nucleotides, which are the basic building blocks. Each nucleotide consists of three components: a deoxyribose sugar, a phosphate group, and a nitrogenous base. The sugar is a five-carbon molecule, and the carbons are numbered from 1' (one prime) to 5' (five prime). This numbering becomes crucial in understanding DNA's directionality and how the molecule is assembled. The four nitrogenous bases are Adenine (A), Thymine (T), Guanine (G), and Cytosine (C)—and they are what make up the unique code of genetic information. For example, a gene that codes for the insulin protein will have a specific sequence of these bases that is different from the gene that codes for hemoglobin.

Phosphodiester Bonds and Directionality (5' to 3')

Nucleotides connect to each other via phosphodiester bonds, which form between the 3' hydroxyl (–OH) group of one sugar and the 5' phosphate group of the next nucleotide. This linkage forms a sugar-phosphate backbone that is both strong and flexible, providing structural support to the DNA molecule. Because of this bonding, each DNA strand has directionality—one end has a free 5' phosphate group, and the other end has a free 3' hydroxyl group. This is critical during DNA replication and transcription, as enzymes like DNA polymerase and RNA polymerase can only add new nucleotides to the 3' end, so DNA is always synthesized in the 5' to 3' direction. Think of it like writing a sentence in English: you must start from the left (5') and move to the right (3')—writing in the reverse would result in gibberish or unusable code.

Although a single DNA strand can carry information, functional DNA in living cells exists as a double-stranded helix. Two DNA strands align in an antiparallel fashion—one runs in a 5' to 3' direction, and the other runs in the 3' to 5' direction. This orientation is necessary for the nitrogenous bases to hydrogen bond correctly in the center of the helix. If both strands ran in the same direction, the base pairs would not align properly for hydrogen bonding, and the helix would not form. Imagine a zipper: both sides must align oppositely for the teeth (bases) to interlock. The classic image of DNA’s double helix, first revealed by Rosalind Franklin’s X-ray crystallography and modeled by Watson and Crick, is built upon this principle of antiparallel strand alignment.

Complementary Base Pairing and Stability

The two DNA strands are held together through specific base pairing—Adenine (A) always pairs with Thymine (T), and Guanine (G) always pairs with Cytosine (C). These pairings are stabilized by hydrogen bonds: A-T pairs form two hydrogen bonds, while G-C pairs form three. The extra bond in G-C pairs makes them more stable and harder to break. This difference has practical implications. For instance, genes or DNA regions rich in G-C content are more stable and harder to denature, which is an important consideration in PCR (polymerase chain reaction) when designing primers or setting annealing temperatures. Similarly, promoter regions in some species tend to have more A-T content, making them easier for the transcription machinery to unwind and access.

One fascinating aspect of DNA is that any two single strands with complementary sequences can find each other and anneal—or stick together—via base pairing. This principle is widely used in biotechnology and molecular biology. For example, during PCR, synthetic DNA primers bind (anneal) to specific complementary sequences on target DNA to initiate replication. In gene cloning, scientists design complementary overhangs to ligate DNA fragments. In Southern blotting, labeled DNA probes bind to complementary DNA in a sample, allowing for gene detection. This ability of DNA to self-recognize and pair with its complement underpins much of modern genetic technology.

Chromosomes vs. Plasmids:

In biology, the chromosome is the main genetic blueprint of a cell. In bacteria, this typically takes the form of a single circular chromosome, which carries all the essential genes required for survival, such as those involved in DNA replication, cell division, metabolism, and stress response. In contrast, eukaryotic cells—like those in humans, animals, or plants—have multiple linear chromosomes housed in a membrane-bound nucleus. For example, human cells contain 46 chromosomes (23 pairs), which are tightly packed using histone proteins to form chromatin. During cell division, this chromatin condenses and becomes visible under a light microscope, which is a hallmark of mitosis and meiosis.

In addition to chromosomes, many cells—especially bacteria—also contain plasmids. Plasmids are small, circular, double-stranded DNA molecules that replicate independently of the chromosome. While not essential for basic survival, plasmids can carry advantageous genes, such as those for antibiotic resistance, virulence, or metabolic specialization. For instance, some plasmids enable bacteria to break down unusual sugars or resist antibiotics like tetracycline. Unlike chromosomes, which exist in one or two copies per cell, plasmid copy number varies—natural plasmids usually have fewer than 30 copies, while engineered plasmids (used in labs) can reach hundreds of copies per cell, allowing high levels of gene expression.

For a plasmid to be maintained in a cell, it must be able to replicate. This begins at a specific sequence known as the origin of replication (ori). The ori is where proteins like DNA helicase (which unwinds the double helix) and DNA polymerase (which synthesizes new DNA) bind and initiate the replication process. The efficiency of plasmid replication depends on the type of ori and its compatibility with the host. For instance, ColE1 is a common ori used in E. coli plasmids, enabling multiple rounds of replication and high plasmid yield. This feature is incredibly useful in genetic engineering and biotechnology, where researchers introduce foreign genes into plasmids and use bacterial hosts to replicate and express these genes. But getting a plasmid into a bacterium is not trivial—this is where the process of transformation comes in.

Transformation is the process of introducing foreign DNA (like a plasmid) into a bacterial cell. However, this process is naturally inefficient—only about 1 in every 10,000 bacterial cells will successfully take up a plasmid during a standard chemical transformation (e.g., using calcium chloride and heat shock). To identify which cells have been successfully transformed, researchers use selectable markers, most commonly antibiotic resistance genes. For example, the ampicillin resistance gene (bla) encodes the β-lactamase enzyme, which breaks down ampicillin. When bacteria are plated on agar containing ampicillin, only the transformed cells carrying the plasmid survive and grow, forming colonies. Untransformed cells are killed by the antibiotic, making selection straightforward and efficient.

Plasmids used in molecular biology are designed for flexibility and ease of manipulation. One key feature is the polylinker, also called the multiple cloning site (MCS). This is a short synthetic DNA segment that contains several unique restriction enzyme recognition sites, allowing scientists to insert genes of interest at precise locations. These sites are typically palindromic sequences—meaning they read the same from 5' to 3' on both strands. For example, the sequence GAATTC (cut by EcoRI) reads the same on the complementary strand: 5'-GAATTC-3' and 3'-CTTAAG-5'. Restriction enzymes recognize these palindromes and cut them, creating sticky or blunt ends that facilitate gene insertion during cloning.

Expression of Cytochrome P450 in Transformed E. coli

In the lab described, two strains of E. coli are transformed with plasmids to study the expression of Cytochrome P450 BM3, a protein that is not naturally found in E. coli but comes from Bacillus megaterium. One strain receives an empty vector (pET3a), while the other receives pET3a with the P450BM3 gene inserted (pT7BM3). These plasmids carry the ampicillin resistance gene for selection and are designed to express the P450 gene only under certain conditions.

The pET3a vector contains a T7 promoter, which is recognized only by T7 RNA polymerase, not by E. coli's natural polymerase. The E. coli strain used here has been genetically modified to contain T7 RNA polymerase integrated into its chromosome, under control of an inducible promoter (like the lac operon). When researchers add IPTG, a molecule that mimics lactose, it induces T7 polymerase production. This polymerase then binds to the T7 promoter on the plasmid and transcribes the P450 gene at very high rates—up to five times faster than native E. coli RNA polymerase.

This regulated, high-efficiency system allows researchers to "turn on" protein expression only when needed, avoiding the potential toxicity or resource drain of constant overexpression. Once expressed, the Cytochrome P450 BM3 enzyme can be isolated and studied for its role in drug metabolism, biotransformation, or other biochemical processes.

Introduction to Cytochrome P450_BM3:

Cytochrome P450 enzymes (often abbreviated as CYPs) form a vast and highly conserved superfamily of heme-thiolate proteins found across all domains of life—from bacteria to humans. These enzymes catalyze a wide array of oxidation reactions, making them crucial for various biological functions. In humans, for instance, P450s are vital in drug metabolism, cholesterol synthesis, and detoxification of xenobiotics (foreign substances). For example, human CYP3A4 metabolizes more than 50% of pharmaceuticals, impacting drug dosing and drug-drug interactions. In this course, however, our focus will be on the prokaryotic enzyme cytochrome P450BM3, originally isolated from the bacterium Bacillus megaterium. This bacterial enzyme is among the best-characterized P450s and serves as an excellent model system due to its high activity and simplicity of expression in E. coli.

All cytochrome P450 enzymes contain a heme prosthetic group, which is responsible for their catalytic activity and gives them their characteristic reddish-brown color. This heme contains a central iron atom (Fe) coordinated to four nitrogen atoms of a porphyrin ring and covalently bound to a cysteine residue from the protein. The heme iron cycles between Fe³⁺ (ferric) and Fe²⁺ (ferrous) states during catalysis. Importantly, it is this iron atom that binds oxygen and facilitates its activation for incorporation into substrates. For example, when lauric acid (a 12-carbon saturated fatty acid) is used as a substrate in the lab, one of the oxygen atoms from molecular O₂ is inserted into the fatty acid, forming hydroxy-lauric acid, while the second oxygen atom is reduced to water.

The primary reaction catalyzed by P450 enzymes is hydroxylation, where a hydrogen atom in a substrate is replaced with a hydroxyl group (-OH). P450BM3, specifically, is a fatty acid hydroxylase—meaning it hydroxylates medium- to long-chain fatty acids like lauric acid. This type of enzyme is also called a monooxygenase, because it incorporates one atom of molecular oxygen into the substrate and reduces the other to water. This distinguishes it from dioxygenases, which insert both oxygen atoms into the product. This kind of reaction is highly valuable in biotechnology and pharmaceutical industries, where selective oxidation is difficult to achieve with synthetic catalysts. For instance, engineered P450s can be used to produce drug metabolites or to hydroxylate complex molecules like steroids and vitamins.

For P450 enzymes to function, electrons must be transferred to the heme iron to enable oxygen activation. These electrons are typically supplied by NADPH, which gets oxidized to NADP⁺ in the process. In class I P450 systems (mostly bacterial and mitochondrial), electrons flow from NADPH to ferredoxin reductase (which contains FAD), then to ferredoxin (an iron-sulfur protein), and finally to the P450 heme. Class II systems (more common in eukaryotes), on the other hand, transfer electrons via cytochrome P450 reductase, a protein containing both FAD and FMN domains. What's unique about P450BM3 is that it is a rare bacterial class II enzyme and is fused into a single polypeptide chain—combining the heme, FAD, and FMN domains. This structural integration allows for rapid intramolecular electron transfer, making P450BM3 one of the fastest P450 enzymes known. In contrast, most P450 systems are multipart, requiring complex protein-protein interactions to complete the electron transfer chain.

The catalytic cycle of P450 enzymes is a multi-step process beginning with substrate binding. Initially, the heme iron is in the ferric state (Fe³⁺) and coordinated to a water molecule. When a substrate (like lauric acid) binds to the enzyme’s active site, it displaces this water, which triggers a conformational change and allows the heme to accept an electron from NADPH via the FAD/FMN cofactors. This reduces Fe³⁺ to Fe²⁺, enabling it to bind molecular oxygen (O₂). A second electron is transferred, reducing O₂ to a superoxide, which undergoes protonation and O–O bond cleavage. One oxygen atom is released as water, and the other is converted into a highly reactive iron-oxo species capable of abstracting a hydrogen from the substrate. This results in the insertion of an OH group into the substrate—a process central to drug metabolism and biosynthesis. The cycle then resets as the hydroxylated product leaves the active site.

P450_BM3 in the Lab

In this lab course, you will work with E. coli cells transformed with either an empty plasmid (pET3) or a recombinant plasmid (pT7BM3) carrying the gene encoding for P450BM3 under control of a T7 promoter. This system utilizes E. coli strains with a chromosomally integrated T7 RNA polymerase gene, which allows for inducible, high-level expression of the P450 gene. This is because the T7 RNA polymerase, unlike native E. coli RNA polymerase, only transcribes genes downstream of a T7 promoter and does so at a significantly faster rate (up to five times faster). The pET3 plasmid also contains an ampicillin resistance gene, which facilitates selection of successfully transformed E. coli using antibiotic-containing media.

Each group in the lab receives two cultures: one with the empty vector (control) and one with the vector containing the P450BM3 gene. Your objective is to induce expression of the P450 protein, purify it, and study its enzymatic kinetics using lauric acid as a substrate. These experiments allow you to explore fundamental concepts in molecular cloning, protein expression, and enzymology—all through the lens of a real, biotechnologically relevant enzyme.

Cytochrome P450BM3 is not just another enzyme—it is a biocatalytic powerhouse. Its efficiency, simplicity, and amenability to engineering make it a model enzyme for studying P450 catalysis and for developing new biotechnological applications. Because all the electron transfer components are within a single polypeptide, there are fewer bottlenecks in the catalytic cycle. This makes it ideal for research into enzyme kinetics, biocatalyst engineering, and drug metabolism modeling. Its native substrate, lauric acid, provides a straightforward system to monitor activity, and mutations in the active site have allowed scientists to alter substrate specificity—leading to engineered variants that can hydroxylate drugs, pollutants, or complex natural products.