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Explores the process of creating recombinant DNA using bacterial plasmids as vectors for gene cloning.
What if you could turn a common bacterium into a microscopic factory that pumps out life-saving human medicine? By 'hijacking' a small circle of DNA, scientists have done exactly that to produce the insulin used by millions of diabetics today.
In biotechnology, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell. The most common vector is the plasmid, a small, circular, extrachromosomal DNA molecule found in bacteria. For a plasmid to be useful in the lab, it must contain three key features: 1) The **Origin of Replication (), which ensures the plasmid is copied by the host cell's machinery; 2) A Selectable Marker**, usually an antibiotic resistance gene like (ampicillin resistance), which allows researchers to kill off bacteria that didn't take up the plasmid; and 3) A Multiple Cloning Site (MCS), a short region containing several restriction sites where foreign DNA can be inserted.
Quick Check
Why is the 'Origin of Replication' () critical for gene cloning?
Answer
It allows the plasmid to be independently replicated by the host bacterium, ensuring that as the bacteria divide, the recombinant DNA is passed on to all daughter cells.
To insert a human gene into a plasmid, we use restriction endonucleases (restriction enzymes). These enzymes act like molecular scissors, cutting DNA at specific palindromic sequences. Most enzymes create sticky ends—short, single-stranded overhangs. If we cut both the human DNA and the plasmid with the same enzyme, their sticky ends will be complementary. We then use DNA Ligase, the molecular glue, to catalyze the formation of phosphodiester bonds, permanently joining the two DNA fragments into a single recombinant DNA molecule.
Step-by-step process of ligation: 1. Identify a restriction site (e.g., for EcoRI) on both the plasmid and the human gene. 2. Incubate both DNA samples with EcoRI to create complementary sticky ends. 3. Mix the cut plasmid and the human gene fragment together. 4. Add DNA Ligase and to seal the sugar-phosphate backbone. 5. Result: A closed circular plasmid now containing the human gene.
Quick Check
What would happen if you cut the human gene with Enzyme A but the plasmid with Enzyme B?
Answer
The sticky ends would likely not be complementary, meaning they would not base-pair correctly, and DNA ligase would be unable to join them efficiently.
Once the recombinant plasmid is ready, it must be moved into a living bacterium through transformation. This is often achieved via heat shock or electroporation, which makes the bacterial cell membrane temporarily permeable. However, transformation is inefficient; only a small fraction of bacteria actually take up the DNA. To find them, we grow the bacteria on agar plates containing an antibiotic. Only cells with the selectable marker () survive. To further distinguish between 'empty' plasmids and those that actually contain our gene, we often use Blue-White Screening, where successful insertion disrupts a reporter gene (), turning the resulting bacterial colonies white instead of blue.
Imagine you are screening for a recombinant plasmid using ampicillin resistance and the (Blue-White) system: 1. Plate A (No Antibiotic): All bacteria grow (a lawn). This is your control. 2. Plate B (With Ampicillin): Only transformed bacteria grow as individual colonies. Non-transformed bacteria are dead. 3. Observation: On Plate B, you see 50 blue colonies and 10 white colonies. 4. Conclusion: The 10 white colonies are the 'winners'—they contain the recombinant plasmid where the gene insertion disrupted the gene.
A circular plasmid is base pairs (bp) long. It has two restriction sites for the enzyme HindIII located at position and position . If you digest the plasmid with HindIII and run it on a gel, what fragments will you see? 1. The first cut is at , the second at . 2. The distance between them is bp. 3. The remaining distance around the circle is bp. 4. Result: You will see two bands on the gel, one at bp and one at bp.
Which component of a plasmid ensures that the inserted gene is copied every time the bacterium divides?
In Blue-White screening, why are the 'white' colonies the ones researchers want?
DNA Ligase forms hydrogen bonds between the nitrogenous bases of the sticky ends.
Review Tomorrow
In 24 hours, try to sketch a plasmid map from memory, labeling the , the antibiotic resistance gene, and the MCS. Explain to yourself why each is necessary.
Practice Activity
Research the 'pUC19' plasmid online. Look at its map and identify which specific antibiotic resistance it provides and which restriction enzymes can be used in its MCS.