BIOS 205: Plasmid Transformation

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| By Jakethesnake69

Jakethesnake69

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Quizzes Created: 2 | Total Attempts: 912

Questions: 14 | Attempts: 113 | Updated: Mar 21, 2023

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1.

What cells were used in plasmid transformation?

Explanation
E. coli competent cells were used in plasmid transformation. Competent cells are cells that have been treated to increase their ability to take up foreign DNA, such as plasmids. E. coli is a commonly used bacteria in molecular biology research, and its competent cells are often used for the introduction of foreign DNA into the cells through transformation. This allows for the replication and expression of the plasmid DNA within the E. coli cells.

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2.

"Competent" indicates that the cells were treated with

Explanation
The term "competent" in this context refers to the treatment of cells with calcium chloride. This treatment makes the cell membrane more permeable, meaning that it allows substances, such as DNA, to pass through more easily. By making the cell membrane more permeable, the cells become capable of taking up DNA, which is an essential step in many genetic engineering techniques.

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3.

1st step of Transformation (and purpose)

Explanation
The treatment with calcium chloride and incubation on ice slows down the fluidity of cell membranes. Calcium chloride is known to stabilize cell membranes by forming cross-links between phospholipids, reducing their movement. Incubation on ice further reduces the kinetic energy of the molecules, making the membranes less fluid. This step is commonly used in cell transformation protocols to increase the efficiency of introducing foreign DNA into cells. Slowing down the fluidity of cell membranes helps to enhance the uptake and integration of the foreign DNA into the host cells.

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4.

2nd step of transformation (and purpose)

Explanation
Heat shock at 42°C causes an increase in the permeability of the cell membrane. This means that the cell membrane becomes more porous, allowing substances to pass through more easily. This increase in permeability can be beneficial in certain transformation processes, such as introducing foreign DNA into the cell. The heat shock helps to create temporary openings in the cell membrane, allowing the DNA to enter the cell more efficiently. This step is crucial in genetic engineering techniques like bacterial transformation, where the goal is to introduce new genetic material into the host cell.

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5.

At which step is the plasmid DNA actually taken up by the cell?

Explanation
During heat shock, the plasmid DNA is actually taken up by the cell. Heat shock involves subjecting the cells to a sudden increase in temperature, typically around 42°C. This increase in temperature causes the cell membrane to become more permeable, allowing the plasmid DNA to enter the cell. The heat shock method is commonly used in molecular biology techniques such as bacterial transformation, where foreign DNA is introduced into bacterial cells.

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6.

3rd Step of Transformation (and purpose)

Explanation
During the incubation in LB nutrient broth at 37°C for one hour, the genes on the plasmid are expressed, particularly the beta lactamase gene. This gene is responsible for providing resistance to the antibiotic ampicillin. Incubating the plasmid in the nutrient broth and at the optimal temperature allows for the activation of the gene, enabling the bacteria to survive in the presence of ampicillin.

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7.

In the presence of arabinose, araC

Explanation
In the presence of arabinose, the araC protein facilitates the binding of RNA polymerase to the DNA, resulting in the production of GFP. This suggests that araC acts as a transcriptional activator, enhancing the expression of the GFP gene in the presence of arabinose.

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8.

When arabinose is absent, araC

Explanation
In the absence of arabinose, the araC protein does not facilitate the binding of RNA polymerase. This means that RNA polymerase cannot bind to the promoter region of the GFP gene, leading to the absence of GFP production.

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9.

E.coli cells are sensitive to ampicillin and

Explanation
E.coli cells are sensitive to ampicillin, which means that they are unable to survive or grow in the presence of this antibiotic. Ampicillin targets and inhibits a specific enzyme in the bacterial cell wall synthesis, leading to cell death. Therefore, when ampicillin is present, E.coli cells are unable to withstand its effects and will ultimately die.

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10.

How does ampicillin affect the bacterial cells?

Explanation
Ampicillin affects bacterial cells by interfering with their ability to synthesize the cell wall. The cell wall is crucial for maintaining the structural integrity of bacterial cells, and its synthesis is necessary for bacterial growth and survival. Ampicillin works by inhibiting the enzymes involved in cell wall synthesis, leading to weakened cell walls and ultimately cell death. This mechanism of action makes ampicillin an effective antibiotic against bacterial infections.

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11.

The pGLO plasmid carries the bla gene

Explanation
The pGLO plasmid carries the bla gene, which is responsible for producing beta lactamase. Beta lactamase is an enzyme that breaks down ampicillin, an antibiotic, and provides resistance to it. Therefore, the presence of the bla gene in the pGLO plasmid confers ampicillin resistance to the organism that carries it.

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12.

If E. coli uptakes the plasmid, it is

Explanation
If E. coli uptakes the plasmid, it will become resistant to the antibiotic ampicillin (LBamp plates) and will be able to grow on these plates. This is because the plasmid likely carries a gene that confers resistance to ampicillin, allowing the bacteria to survive and grow in the presence of this antibiotic.

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13.

If E. coli uptakes only water (control plate) or no plasmid,

Explanation
When E. coli uptakes only water or no plasmid, it means that it does not have any resistance genes against the antibiotic ampicillin. LBamp plates contain ampicillin, which inhibits the growth of bacteria that are not resistant to it. Therefore, E. coli without any plasmid or only water uptake will not be able to survive on LBamp plates and will die. This indicates that it is sensitive to ampicillin and lacks the ability to survive in the presence of this antibiotic.

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14.

How is plasmid DNA separated from chromosomal DNA?