Plasmids are small circular DNA molecules found in many bacteria, separate from the main bacterial chromosome. They often carry useful genes, such as antibiotic resistance genes, that help bacteria survive in certain environments. Scientists use plasmids as tools to move genes into bacteria because they are easy to copy and can be engineered in the lab.
Bacterial transformation matters because it allows cells to take up new DNA and gain new traits.
Understanding Biology: Plasmids and Bacterial Transformation
A useful plasmid for protein production needs more than a copied gene. The gene must sit beside a promoter, which is a DNA control region that tells bacterial enzymes where to begin reading. A strong promoter can lead to more messenger RNA, then more protein.
Many plasmids include a terminator that signals where reading should stop. They may have a short region with several cutting sites, so a chosen DNA fragment can be inserted in a planned location.
The direction of the inserted gene matters. If it faces the wrong way, the promoter may not produce the intended messenger RNA.
Bacterial cells do not usually accept large pieces of DNA easily. In the laboratory, cells are first made competent, meaning their outer boundary is temporarily easier for DNA to cross. A brief heat shock can help create this condition.
Electroporation uses a short electrical pulse to form tiny openings in the cell boundary. Both methods can stress or kill some cells, so the cells are given time in rich liquid nutrient before testing whether they received the new DNA. This recovery time lets cells repair damage and begin making the protein that protects them during selection.
Selection shows which cells probably took up a plasmid, but it does not prove that every plasmid has the correct inserted gene. A plasmid can close back together without the intended DNA fragment. It can contain an insert in the wrong direction or carry a damaged gene.
Scientists therefore screen individual bacterial colonies after selection. They may use colony PCR to check for a DNA fragment of the expected length. They can cut purified plasmid DNA with enzymes and compare the fragment sizes.
DNA sequencing gives the clearest confirmation because it reads the exact base order near the inserted gene. Good experiments include controls, such as cells treated without plasmid DNA, to reveal whether unexpected growth occurred.
Protein production depends on cell biology, not only on successful DNA delivery. Bacteria need nutrients, suitable temperature, oxygen conditions, and enough time to grow. Producing a foreign protein can slow growth because the cell must spend energy and raw materials making it.
Some proteins fold poorly in bacteria or form inactive clumps. Researchers can lower the growth temperature, use a weaker promoter, or choose a bacterial strain designed for protein expression. Students meet these ideas in the production of enzymes for food processing, proteins used in medical research, and fluorescent proteins that make cells visible.
The important habit is to separate each stage of the work. First confirm the DNA sequence.
Then confirm that the cells carry it. Finally test whether the expected protein is present and working.
Key Facts
- A plasmid is circular DNA that replicates independently of the bacterial chromosome.
- Bacterial transformation is the uptake of foreign DNA by a bacterial cell.
- Selectable markers, such as antibiotic resistance genes, help identify bacteria that received a plasmid.
- Only transformed bacteria grow on agar containing the matching antibiotic.
- A recombinant plasmid contains DNA from two or more sources, often written as vector DNA + inserted gene.
- Protein yield can be estimated with total protein = protein per cell x number of cells.
Vocabulary
- Plasmid
- A plasmid is a small circular DNA molecule that can replicate separately from the main bacterial chromosome.
- Transformation
- Transformation is the process in which a bacterial cell takes up DNA from outside the cell.
- Selectable marker
- A selectable marker is a gene that lets scientists identify cells that contain a desired plasmid.
- Recombinant DNA
- Recombinant DNA is DNA made by combining genetic material from different sources.
- Gene expression
- Gene expression is the process by which information in a gene is used to make RNA or protein.
Common Mistakes to Avoid
- Assuming every bacterium takes up the plasmid is wrong because transformation efficiency is usually low, so only some cells become transformed.
- Forgetting the selectable marker is wrong because scientists need a way to separate plasmid-containing bacteria from bacteria that did not take up DNA.
- Thinking antibiotic resistance means the bacteria make the desired protein is wrong because resistance only shows plasmid uptake, not necessarily strong expression of the inserted gene.
- Confusing plasmids with bacterial chromosomes is wrong because plasmids are smaller, usually circular DNA molecules that replicate independently from the main chromosome.
Practice Questions
- 1 A transformation experiment plates 200 microliters of cells from a 1.0 mL recovery culture and 80 colonies grow. Estimate the total number of transformed cells in the full recovery culture.
- 2 A plasmid has 4000 base pairs and an insulin gene insert has 900 base pairs. What is the total size of the recombinant plasmid in base pairs?
- 3 A student plates transformed bacteria on agar with ampicillin, but no colonies grow. Give two possible biological or experimental reasons for this result.