Plasmids can be used as vectors to carry foreign DNA into a cell. In the lab, plasmids are specifically designed so that the DNA they contain will be copied by bacteria. Laboratory-designed plasmids contain a small number of genes that help transformation.
These include:. The piece of DNA or gene of interest is cut from its original DNA source using a restriction enzyme and then pasted into the plasmid by ligation.
The plasmid containing the foreign DNA is now ready to be inserted into bacteria. This process is called transformation. Before transformation, bacteria are treated with a chemical called calcium chloride, which causes water to enter into the cells and makes them swell.
These swollen bacteria are then known as competent bacteria. The plasmid DNA enter the bacteria through small pores created in the cell membranes. These novel genes were cloned into a plasmid and transformed into E. When the cells were examined, the synthetic biologists had created a wide variety of fluorescent proteins that.
Interestingly, the scientists had also created several chimeric genes that produced highly pigmented cells. These colorful, chromogenic proteins were visible by the naked eye, meaning that a UV light source or fluorescent microscope was not necessary for visualization Figure 3. Chromogenic proteins are already used in biotechnology as controls for protein expression and as visual markers for protein purification. As the technology becomes more common, they may become important markers for in vivo gene expression studies.
Skip to content Close Menu. What is transformation? Like this: Like Loading How will you know if you are successful? In the examples for plasmids we have recommended for this exercise, the recombinant bacteria will have a new and highly visible trait: It will now produce colored protein, which makes the cells themselves colored!
As the bacteria multiply on the media, they form visible collections of cells called colonies. Each colony represents the decedents of the original bacterial cell that landed on that spot on the medium and began to replicate. Thus colonies are clones exact copies of the cell that began the replication process.
The relevant components of your plasmid are the gene for the colored protein, the inducible promoter, and the ampicillin resistance gene ampR. The ampR gene confers resistance to the antibiotic ampicillin.
Biotechnologists call these genes selectable markers because only bacteria having the gene will survive in the presence of an antibiotic. This will allow protein to be produced. Discuss the following amongst yourselves. Be ready to share your thoughts with the rest of the class.
Read through the Procedures below and outline the steps, using words and a flowchart in your lab notebook. Check your protocol and follow all safety measures and wear proper attire prior to conducting the experiment.
Practice aseptic technique while using E. Please note the following:. Introduction Genetic engineering or DNA technology has been useful for producing large quantities of a specific protein to treat human diseases. Transforming Bacteria with Recombinant Plasmid Inserting a gene into a plasmid vector is an important first step in the gene cloning process.
Bacterial Transformation Once a recombinant plasmid is made that contains a gene of interest, such as insulin, the plasmid can enter bacterial cells by a process called transformation. Figure 1. Bacterial transformation.
From Plasmid DNA to Protein After a recombinant plasmid enters a bacterial cell, the cell begins to express the genes on it. Figure 2. Gene expression from a plasmid in the bacterial cell Recombinant plasmids and other forms of genetic engineering is possible because all living organisms use DNA as a platform to encode genetic information.
Transforming Bacteria with Plasmids In this laboratory experiment you will transform E. After the cells are heat-shocked, they will be grown under various testing conditions: The control group on nutrient agar a type of growth media that bacteria thrive on.
The control group on nutrient agar with an antibiotic added. The experimental group on nutrient agar. The experimental group on nutrient agar with an antibiotic. The experimental group on nutrient agar, antibiotic and an inducer such as IPTG.
Prelab Questions Discuss the following amongst yourselves. Ampicillin is a derivative of the antibiotic penicilliin. It disrupts cell wall formation in bacterial cells which kills the cells.
However, our recombinant plasmid contains a gene that provides antibiotic resistance by producing a protein that breaks down ampicillin. Why do we include ampicillin in the test medium? What will happen if the transformed cells do not grow in the presence of the chemical inducer? In the experiment, you will add the control and experimental groups of cells onto different media combinations.
What do you predict for each condition? Fill in Table 1 by indicating if you predict growth or no growth, and if growth, will there be minimal growth or lots of growth.
Table 1. Predictions for your experiment; transformation of E. Transforming E. Please note the following: Wear gloves when working with bacteria. Avoid touching contaminated areas which includes anything that has touched bacteria. Notify your instructor ASAP if an accident takes place, such as a spill. Put all supplies that have been exposed to bacteria into either a biohazard bag, or a designated biowaste container.
These contaminated supplies may include pipette tips, cell spreaders, and microfuge tubes. Keep agar plates closed at all times after removing them from the incubator. Always wash your hands for 20 seconds with soap and water before leaving the lab.
Retrieve a chilled CC tube and put in the cup of crushed ice. Keep the competent cells cold at all times. Hold the tube by the rim, not the bottom. Figure 3. To ensure the best results possible, it is crucial that each step is followed exactly.
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