The Mighty Bacteria: Nature's DNA Copiers!
Alteration via bacterial genetic modification
Bacteria, tiny miracles of nature, make perfect host cells for laboratory DNA replication because they are simple to cultivate in vast quantities. Their inherent cellular machinery is designed to perform DNA replication and protein synthesis, making them incredibly efficient copycats!
Miraculous Bacteria: The DNA Recyclers
Bacteria have an extraordinary ability to incorporate foreign DNA and replicate it, thus providing them with a competitive edge in evolving environments. This adaptability allows them to acquire DNA that gives resistance to antibiotics, for instance.
The bacterial genome is contained within a single, circular chromosome. Different from eukaryotic organisms, where genetic material is enclosed within a nuclear membrane, the bacterial genome floats freely within the cell. Occasionally, bacteria may also contain smaller circular DNA, called plasmids, comprising a much smaller gene set. These plasmids can be exchanged among bacteria through a process called conjugation.
Plasmids: Gene Carriers of the Lab
Plasmids can be utilized in the lab to transport foreign DNA into a cell. When inserted into the host cell, the plasmid is copied by the cell's DNA replication machinery. Specifically designed plasmids ensure the DNA introduced will be replicated by bacteria.
Plasmid Essentials
Laboratory-engineered plasmids contain essential genes, including:
- Origins of Replication: Specific sequences where DNA replication begins.
- Multiple Cloning Sites: Recognition sites for specific restriction enzymes, used to cut the plasmid for inserting foreign DNA.
- Resistance Genes: Genes coding for proteins that bacteria need to survive in specific growth media, such as antibiotics.
Inserting Genes into Plasmids
DNA Cloning
See more
A simplified overview of DNA cloning is shown below.
In DNA cloning, a piece of DNA or a gene of interest is cut using a restriction enzyme and then pasted into the plasmid via ligation. The transformed plasmid is now ready for insertion into bacteria.
Bacterial Transformation
Before transformation, bacteria are made swellable using calcium chloride, creating competent bacteria. Plasmid DNA containing the foreign DNA is mixed with competent bacteria and heated to allow for entry of the DNA into the bacteria through small pores in the cell membrane. Once inside, the new DNA replicates using the host's DNA replicating machinery.
Confirming the Success of Transformation
After transformation, bacteria are grown on agar rich in nutrients. Only bacteria containing a plasmid with antibiotic resistance will grow in the presence of antibiotics, such as ampicillin. Once selected, transformed bacteria can then be grown in large quantities, and the DNA of interest or the protein encoded by the DNA can be isolated and purified.
Transformation's Many Applications
Bacterial transformation is utilized:
- To make multiple copies of DNA, called DNA cloning.
- To produce large quantities of specific human proteins, for example, human insulin, useful in treating Type I diabetes.
- To genetically modify a bacterium or other cells.
Published: Referencing Hub media
Why stop there? Check out our additional enrichment data for a deeper understanding of laboratory plasmid manipulation:
Plasmid Isolation
- Harvesting Cells: Bacterial cells containing the plasmid are grown and collected via centrifugation.
- Cell Lysis: Cells are lysed using an alkaline lysis protocol, typically including sodium hydroxide (NaOH) and sodium dodecyl sulfate (SDS).
- Neutralization: Potassium acetate is added to precipitate proteins and genomic DNA, while plasmid DNA remains in solution.
- Purification: Plasmid DNA is purified using silica columns, alcohol precipitation, or similar methods.
- Elution: Pure plasmid DNA is recovered in water or buffer, ready for use in cloning[4].
DNA Fragmentation and Ligation
- Restriction Enzyme Digestion: The gene of interest and the plasmid are cut using the same restriction enzymes to create compatible ends.
- Ligation: The gene is inserted into the plasmid using DNA ligase, creating a recombinant plasmid.
Preparation of Competent Cells
- Chemical or Physical Treatment: Bacterial cells (e.g., E. coli) are treated to make them competent via chemical means like calcium ion treatment, or physical methods like heat shock or electroporation.
- Alternative Methods: Other simpler methods, such as using a transformation and storage solution (TSS), eliminate some steps and make cells ready for transformation with minimal preparation[5][1].
Transformation
- Mixing DNA and Cells: The recombinant plasmid is mixed with competent cells.
- Incubation: The mixture is incubated on ice, followed by a brief heat pulse or electroporation to enhance DNA uptake.
- Recovery: Cells are allowed to recover in nutrient broth, which helps them repair and express antibiotic resistance genes if present[2][5][1].
Selection and Screening
- Plating on Selective Media: Transformed cells are plated on agar containing antibiotics or other selective agents. Only cells that have successfully taken up the plasmid (with selectable markers) will grow.
- Colony Screening: Individual colonies are screened using PCR, restriction analysis, or sequencing to confirm the presence and correct insertion of the gene of interest[1][4].
Here's a summary table to help visualize the process:
| Step | Description ||------------------------|-----------------------------------------------------------------------------|| Plasmid Isolation | Extract and purify plasmid DNA from bacteria || DNA Fragmentation | Cut plasmid and gene of interest with restriction enzymes || Ligation | Insert gene into plasmid using DNA ligase || Preparation Competence | Make bacteria competent via chemical or physical means || Transformation | Mix plasmid with competent cells and induce DNA uptake || Selection/Screening | Grow on selective media and confirm successful transformants |
This process is fundamental for genetic engineering, mutagenesis, and various biotechnological applications[1][4].
Bacteria, genetically modified through technological means, can incorporate medical-condition related DNA sequences from foreign sources into their plasmids. This acquired DNA, resulting from the process of DNA cloning in a lab, can provide bacteria with resistance to certain medical-conditions, such as antibiotics.
The laboratory-engineered plasmids, essential for DNA cloning, contain multiple cloning sites that allow for the insertion of medical-condition related DNA, origins of replication for beginning DNA replication, and resistance genes that enable bacteria to survive in specific growth media, including antibiotics.
