For example, AAA is also a codon for lysine. In many cases, more than one codon can encode the same amino acid. For example, the codon TTG codes for the amino acid tryptophan, whereas the codon AAG codes for the amino acid lysine. A specific three nucleotide sequence that codes for a single amino acid is called a codon. They are made up of smaller subunits, amino acids, which are encoded by DNA nucleotides. Proteins have many different functions inside and cells. The DNA instructions can be transferred, and other organisms can express foreign traits. Genes from different organisms can be expressed in other organisms like bacteria since they are encoded in DNA. Recombinant plasmids and other forms of genetic engineering is possible because all living organisms use DNA as a platform to encode genetic information. Gene expression from a plasmid in the bacterial cell The expressed proteins may affect the visible traits when observing the bacteria colonies. 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). 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. Multiple copies of the recombinant plasmid can enable the bacterial cell to express large amounts of a protein. DNA polymerase locates the ori- the origin of replication, and starts to replicate the plasmid using the bacterial cell’s machinery. Bacterial transformation.Īfter a recombinant plasmid enters a bacterial cell, the cell begins to express the genes on it. 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. 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. For some plasmid DNA molecules, only about 1 in 10,000 cells will be transformed. However, even competent cells do not always uptake the plasmid. 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. The plasmid DNA can travel from the environment through these pores and enter the cell. By changing the temperature of the cells drastically from cold to warm, the plasma membranes become more fluid and create pores in them. The transformation efficiency can be further increased by stressing the cells in a heat shock. With the positive charge now coating the membrane, the inherently negatively charged DNA molecules will move through the plasma membranes and into the cell. Calcium ions are positively charged, and will neutralize the negatively charged outer membrane on the E. Competent cells can be made by treating the bacteria with a calcium solution. In addition, the cell wall is negatively charged and repels negatively charged DNA molecules.Ĭells that have been treated to become competent are more efficient at taking in DNA from their surrounding environment. 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. The uptake of DNA from the environment of a bacterial cell occurs with a very low efficiency in nature. 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. coli bacteria using a recombinant plasmid that contains a gene that produces colored proteins. In this exercise, you will carry out the transformation of E. If a gene of interest has been inserted into the plasmid vector, the bacteria produces the product encoded by that gene. The plasmid will be taken up by bacteria where it replicates, and its genes will be expressed using the bacterial cellular machinery. coli bacteria through a process that is called transformation, so named because it changes the DNA content of the bacteria. Therefore, in this lab we will put a recombinant plasmid into E. Both activities can only occur inside a cell. 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. Inserting a gene into a plasmid vector is an important first step in the gene cloning process. Transforming Bacteria with Recombinant Plasmid
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