Blogs: Creation of A Plasmid
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Genetic engineering continues to grow with technological advancements, reshaping the landscape of scientific research in molecular biology. A process that once took almost two years now takes only weeks, depending on the plasmid. Creating a plasmid involves a multitude of intricate stages of plasmid creation; instead, we've narrowed it down to the four main steps in creating a plasmid: identification, gene sequencing, transfer to the expression vector, and the experiment itself.
Step 1: Back to the Basics
Before diving into the creation process, it's essential to understand what plasmids are. Researchers and scientists worldwide use plasmids to further their research to advance the knowledge of living organisms, biotechnology and medicine, including infectious diseases, immunology, and ecology. Plasmids, small circular pieces of DNA, act as an open reading frame (a portion of a DNA sequence with the information to become a protein and a start codon but no stop codon), allowing scientists to insert genes from various organisms.1 This unique characteristic enables scientists to clone and express proteins of interest.
Step 2: Decoding the Gene Sequence
The next step of the process starts by knowing the gene sequence of the gene of interest. "If you know the gene sequence, that's 90% of the battle. If you don't know the gene sequence, it's a much more difficult path," Mitch Magee, a researcher at DNASU, shared. Organizations such as the National Center of Biotechnology Information now house the resources to share this information with the public, expediting the process by listing if a gene sequence is available. After obtaining this information, researchers can go to a company like GenScript and inform them what plasmid to put it in and the nature of the planned experiment. In about a month, the company will send you a plasmid containing the sequenced gene of interest.
Step 3: Plasmid Relay Race
Through the gateway system, researchers can create a master donor vector, which allows easy transfer from the donor vector to an expression vector. Researchers use the expression vector in their experiment. Then, researchers can make a fusion partner by adding a tag protein, so when expressed, it produces two proteins (the gene of interest and tag protein) with the fusion tag attached to the protein.2 The tag protein aids the experiment by improving detection expression, purification, solubility, and localization. These improvements make it much simpler to identify and study the target protein. "We print an antibody to the tag, and when the protein is produced, the antibody grabs the tag and then displays our protein of interest so that we can measure if a patient's antibody sticks to it," Magee said.
Step 4: Breaking through Bacterium
The expression vector can go through the transformation process upon creation. The process starts by inserting the plasmid into the host cell, commonly done in E. Coli bacteria4 The DNA polymerase, a molecule that recognizes the origin of the replication of a plasmid, locates the origin and then starts replicating the plasmid using the bacterial or host cell's machinery.5
Step 5: Survival of the Fittest
Only bacteria containing a plasmid will grow because the plasmid holds the antibiotic resistance gene, which allows the bacteria with plasmid DNA to grow in the presence of antibiotics. Antibiotics function as a screening mechanism, allowing only bacteria carrying plasmid DNA to survive. So when all the bacteria are placed on an antibiotic plate to select for ones that took up a plasmid, the cells that took the plasmid DNA will show, but bacteria without a plasmid will die.6 Every bacterium containing a plasmid will create clusters of small, dot-like groups of identical bacteria, all possessing the same plasmid. The small dots, called a colony, will replicate growing the plasmids or protein in large amounts to distribute.7
References
Bacterial transformation & selection (article). (n.d.). Khan Academy. Retrieved October 5, 2023,from https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/bacterial-transformation-selection
Bacterial Transformation Workflow 4 Main Steps. (n.d.). Thermo Fisher Scientific. Retrieved October 5, 2023,from https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/bacteria-transformation-workflow.html
Fusion tags. (n.d.). Abcam. Retrieved October 12, 2023, from https://www.abcam.com/proteins/fusion-tagsNational Human Genome Research Institute. (2023, October 9).Plasmid. National Human Genome Research Institute. Retrieved October 12, 2023, from https://www.genome.gov/genetics-glossary/Plasmid
1.13: Transformation. (2021,September 9). Biology LibreTexts. Retrieved October 5, 2023, from https://bio.libretexts.org/Bookshelves/Biotechnology/Lab_Manual%3A_Introduction_to_Biotechnology/01%3A_Techniques/.13%3A_Transformation
Overview: DNA cloning (article). (n.d.). Khan Academy. Retrieved October 12, 2023, from https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/biotechnology/a/overview-dna-cloning
