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Goals:
Student Learning Outcomes: Upon completion of this lab, students will be able to:
IntroductionGenetic engineering or DNA technology has been useful for producing large quantities of a specific protein to treat human diseases. For example, patients with diabetes, hemophilia, or anemia require treatments with insulin, clotting factor, and growth factor proteins. Targeted genes (DNA) can be cut with restriction enzymes and joined with other DNA with the enzyme ligase. A cloning vector is used to carry the recombinant DNA into living cells, so that the cells can synthesize the encoded proteins. The best cloning vectors are small in size, able to replicate its DNA, contain restriction enzyme recognition sites, and have a marker gene (usually antibiotic resistance gene). In this lab, we will use a recombinant plasmid as the cloning vector. This recombinant plasmid contains (1) a promoter that enables transcription of desired gene, (2) a sequence for the initiation of DNA replication (ori site), and (3) an antibiotic resistance gene. Transforming Bacteria with Recombinant PlasmidInserting a gene into a plasmid vector is an important first step in the gene cloning process. However, if the ultimate goal is to produce a large amount of a particular protein, the plasmid must replicate to make sure that there are many copies of the gene and the gene of interest must be expressed, meaning the gene is utilized to produce the encoded protein. Both activities can only occur inside a cell. Therefore, in this lab we will put a recombinant plasmid into E. coli bacteria through a process that is called transformation, so named because it changes the DNA content of the bacteria. The plasmid will be taken up by bacteria where it replicates, and its genes will be expressed using the bacterial cellular machinery. If a gene of interest has been inserted into the plasmid vector, the bacteria produces the product encoded by that gene. In this exercise, you will carry out the transformation of E. coli bacteria using a recombinant plasmid that contains a gene that produces colored proteins. Bacterial TransformationOnce 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 13.1 illustrates transformation. The uptake of DNA from the environment of a bacterial cell occurs with a very low efficiency in nature. E. coli bacteria have complex plasma membranes that separate the external environment from the internal environment of the cell and carefully regulate which substances can enter and exit the cell. In addition, the cell wall is negatively charged and repels negatively charged DNA molecules. Cells that have been treated to become competent are more efficient at taking in DNA from their surrounding environment. Competent cells can be made by treating the bacteria with a calcium solution. Calcium ions are positively charged, and will neutralize the negatively charged outer membrane on the E. coli bacteria. With the positive charge now coating the membrane, the inherently negatively charged DNA molecules will move through the plasma membranes and into the cell. The transformation efficiency can be further increased by stressing the cells in a heat shock. By changing the temperature of the cells drastically from cold to warm, the plasma membranes become more fluid and create pores in them. The plasmid DNA can travel from the environment through these pores and enter the cell. The cells are then plunged back into a cold temperature, which causes the pores to close and the plasmid DNA to remain inside the cell. However, even competent cells do not always uptake the plasmid. For some plasmid DNA molecules, only about 1 in 10,000 cells will be transformed. When so few cells have taken in the plasmid, how will you be able to identify transformed cells? When designing a recombinant plasmid, one of the requirements is to add a gene for an antibiotic resistance. This way, the bacteria can be grown in the media with an antibiotic added to it, and only cells that have the resistance gene, such as those that express the recombinant plasmid, will be able to grow. Figure 1. Bacterial transformation.From Plasmid DNA to ProteinAfter a recombinant plasmid enters a bacterial cell, the cell begins to express the genes on it. DNA polymerase locates the ori- the origin of replication, and starts to replicate the plasmid using the bacterial cell’s machinery. Multiple copies of the recombinant plasmid can enable the bacterial cell to express large amounts of a protein. Usually, a bacterial cell will only make the protein of interest, after it is induced to do so by adding a chemical which will promote the transcription of the gene. Recall that to express the gene encoding the protein on the recombinant plasmid, DNA is transcribed to mRNA, which is then translated to protein (Figure 13.2). The expressed proteins may affect the visible traits when observing the bacteria colonies. Figure 2. Gene expression from a plasmid in the bacterial cellRecombinant plasmids and other forms of genetic engineering is possible because all living organisms use DNA as a platform to encode genetic information. Genes from different organisms can be expressed in other organisms like bacteria since they are encoded in DNA. The DNA instructions can be transferred, and other organisms can express foreign traits. Proteins have many different functions inside and cells. They are made up of smaller subunits, amino acids, which are encoded by DNA nucleotides. A specific three nucleotide sequence that codes for a single amino acid is called a codon. For example, the codon TTG codes for the amino acid tryptophan, whereas the codon AAG codes for the amino acid lysine. In many cases, more than one codon can encode the same amino acid. For example, AAA is also a codon for lysine. In addition, there are informational codons, such as the start codon (ATG) and the stop codon (TTA), which show where in the DNA sequence the code for the protein begins and ends. Transforming Bacteria with PlasmidsIn this laboratory experiment you will transform E. coli bacteria cells with plasmids. You will be using E. coli that has been made competent with a calcium chloride treatment, and form two different testing groups: a negative control cell group that does not have plasmids added to it, and the experimental group that has the plasmids added. After the cells are heat-shocked, they will be grown under various testing conditions:
By examining the growth of bacteria under these conditions, you can verify that your procedure worked, and you can identify the bacteria transformed with the added plasmid. 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.) If the inducer is present in the bacteria, the promoter will be “turned on” so RNA polymerase can transcribe the gene of interest. This will allow protein to be produced. Prelab QuestionsDiscuss 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. Table 1. Predictions for your experiment; transformation of E. coli
Transforming E. coliMATERIALSReagents
Plate 1: NA Plate 2: NA/amp Plate 3: NA/amp/ind Supplies and Equipment
SAFETYCheck your protocol and follow all safety measures and wear proper attire prior to conducting the experiment. Practice aseptic technique while using E. coli or other live specimens in a laboratory setting. Aseptic technique is the practice of taking precautions to limit potential contamination to both the person performing the experiment, and to the sample/s. Please note the following:
PROCEDURE
Analysis
Study Questions
Which of the following best predicts why the recombinant bacteria will fail to produce the eukaryotic protein?Which of the following best predicts why the recombinant bacteria will fail to produce the eukaryotic protein? Introns must be removed from eukaryotic DNADNA before the gene is inserted into the plasmid.
Which of the following best predicts the result of the bacteria failing to uptake the recombinant plasmid?Which of the following best predicts the result of the bacteria failing to uptake the recombinant plasmid? The bacterial cell will stop producing bacterial proteins.
Which of the following is most likely the primary cause of the pattern of frequency of trisomy 21 births?The majority of full trisomy 21 is caused by chromosomal nondisjunction occurring during maternal meiotic division (∼90%). Errors occur more frequently in the first maternal meiotic division than the second (73% vs.
Which of the following best explains the cause of phenotypic variation observed in butterflies?Which of the following best explains the cause of the phenotypic variation observed in the butterflies? Different mutations occurred in the caterpillars that were exposed to different colors of light. The energy used to grow a larger body results in butterflies with lighter colored wings.
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