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What is the purpose of this experiment?

What is the purpose of this experiment?

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
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What is the purpose of this experiment?
Transform bacteria with the recombinant PARA-R plasmid that contains the gene of interest, red fluorescent protein (rfp). Once transformed, the rfp gene can be expressed using the bacteria's own machinery. The PARA- R plasmid also contains a gene for ampicillin resistance (ampR), which allows us to identify transformed bacteria by their ability to grow in the presence of ampicillin. The PARA-R plasmid also contains an origin of replication (ori) to ensure the plasmid is maintained in the bacterial population, as well as the PBAD promoter and araC gene, which regulate expression of the rfp gene. How do bacteria take up DNA during transformation? Some bacteria are naturally competent, meaning they are able to take up DNA from their environment. However, not all bacteria are naturally competent, and naturally occurring transformation can be inefficient. In the lab, cells are chemically treated, and techniques like heat shock or electroporation are used to enhance the uptake of plasmids by a variety of bacteria. Cell membranes and DNA are both negatively charged and, therefore, normally repel each other. Adding calcium chloride neutralizes these charges and helps render the cells competent. Heat shock refers to incubating bacteria at elevated temperatures, typically 42°C, which creates holes in the membrane through which DNA can enter the cell. Electroporation uses electric current to accomplish a similar result. In this simulation, the heat shock protocol will be used. DNA plasmids are taken up Bacteria express plasmid genes and replicate Bacterial DNA -> heat Transformed bacteria are antibiotic resistant shock
Transcribed Image Text:Transform bacteria with the recombinant PARA-R plasmid that contains the gene of interest, red fluorescent protein (rfp). Once transformed, the rfp gene can be expressed using the bacteria's own machinery. The PARA- R plasmid also contains a gene for ampicillin resistance (ampR), which allows us to identify transformed bacteria by their ability to grow in the presence of ampicillin. The PARA-R plasmid also contains an origin of replication (ori) to ensure the plasmid is maintained in the bacterial population, as well as the PBAD promoter and araC gene, which regulate expression of the rfp gene. How do bacteria take up DNA during transformation? Some bacteria are naturally competent, meaning they are able to take up DNA from their environment. However, not all bacteria are naturally competent, and naturally occurring transformation can be inefficient. In the lab, cells are chemically treated, and techniques like heat shock or electroporation are used to enhance the uptake of plasmids by a variety of bacteria. Cell membranes and DNA are both negatively charged and, therefore, normally repel each other. Adding calcium chloride neutralizes these charges and helps render the cells competent. Heat shock refers to incubating bacteria at elevated temperatures, typically 42°C, which creates holes in the membrane through which DNA can enter the cell. Electroporation uses electric current to accomplish a similar result. In this simulation, the heat shock protocol will be used. DNA plasmids are taken up Bacteria express plasmid genes and replicate Bacterial DNA -> heat Transformed bacteria are antibiotic resistant shock
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