pBR322

Introduction:

• pBR322 is a common DNA tool used for copying genetic material. 
• It was made in 1977 by scientists at the University of California, San Francisco. 
• The "p" stands for plasmid, a type of DNA. 
• "BR" comes from the names of the researchers who made it. 
• The "322" helps tell it apart from similar plasmids made in the same lab. 
• It's about 4363 pairs of DNA long.  

Here's what's inside pBR322:

• "Rep" helps the plasmid copy itself. It comes from another plasmid called pMB1.
• "Rop" makes a protein that helps keep the plasmid stable and controls how many copies there are. It's also from pMB1.
• "Tet" makes a protein that protects against tetracycline, an antibiotic. It's from a plasmid called pSC101.
• "bla" makes a protein called beta-lactamase, which stops ampicillin from working. It's from something called a "transposon Tn3."

Useful features of pBR322:

• Manageable Size: pBR322 is a plasmid vector that's about 4363 base pairs long. This size makes it easy to handle and purify both the vector itself and any new DNA pieces added to it. Even if you add more DNA, the resulting plasmid isn't too big to handle.
• Dual Antibiotic Resistance: pBR322 carries two different genes that make bacteria resistant to antibiotics: one for ampicillin and one for tetracycline. This is helpful because you can choose which antibiotic to use as a "marker" to identify bacteria that have taken up the plasmid. Each resistance gene also comes with its own special spots where you can easily insert new DNA during cloning experiments.

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• High Copy Number: When inside E. coli bacteria, pBR322 can multiply itself pretty well. Normally, there might be about 15 copies of pBR322 in each bacterial cell. But if you add a substance like chloramphenicol, which slows down protein production, you can get up to 1000 to 3000 copies per cell. This means you can get lots of copies of your DNA for experiments, which is super useful!

Recombinant Selection with pBR322

Insertional inactivation of an antibiotic resistance gene:

• PBR322 is like a toolbox scientists use to insert new pieces of DNA. It's special because it has certain spots where you can easily cut it open and add new DNA.
• Imagine PBR322 as a string of beads. BamHI is like a pair of scissors that can cut this string at a specific spot.
• Normally, PBR322 has genes that give bacteria resistance to tetracycline, an antibiotic. But if you use BamHI to cut PBR322 and insert a new piece of DNA there, it messes up one of these tetracycline resistance genes.
• So, when you put this changed PBR322 into bacteria, they can't resist tetracycline anymore. But they still resist another antibiotic called ampicillin.
• In simple terms, by adding new DNA into PBR322 with BamHI, you disable the bacteria's ability to resist tetracycline while keeping their resistance to ampicillin.

Screening of pBR322 was performed in the following ways:

• Plasmid Isolation: First, the plasmid DNA containing pBR322 is isolated from the bacterial cells that carry it. This step separates the plasmid DNA from the rest of the cellular components.
• Digestion with BamHI: The isolated pBR322 plasmid DNA is then cut using the enzyme BamHI. This enzyme specifically cuts the pBR322 at a particular site, usually within the region that codes for resistance to tetracycline.
• Insertion of Foreign DNA: A new piece of DNA, which could be from another source like a gene of interest, is inserted into the cut site of the pBR322 plasmid. This new DNA fragment can disrupt one of the genes responsible for tetracycline resistance.
• Transformation: The modified pBR322 plasmid, now carrying the foreign DNA fragment, is introduced into bacterial cells through a process called transformation. This allows the bacteria to take up the modified plasmid DNA.
• Selection: The transformed bacteria are grown on a culture medium containing both tetracycline and ampicillin. Bacteria that have successfully taken up the modified pBR322 plasmid will lose their ability to resist tetracycline due to the disruption of the tetracycline resistance gene. However, they will still be able to resist ampicillin.
• Screening: The bacterial colonies that grow on the culture medium are then screened to identify those that are sensitive to tetracycline but resistant to ampicillin. These colonies contain the modified pBR322 plasmid with the inserted DNA fragment disrupting the tetracycline resistance gene.

                This screening process allows scientists to identify bacteria that have successfully incorporated the desired DNA fragment into the pBR322 plasmid while disrupting the target gene, in this case, the gene responsible for tetracycline resistance.