Insert The Plasmid Ring

134). They are loops of DNA that are separate from the chromosomal DNA and can self-replicate in a cell, found mostly in bacteria (Brown, 2011; Addgene, 2015). Lederberg and William Hayes discovered that plasmids were being transferred from one cell to another, not the chromosomal DNA (Brown, 2011, p. 135). This discovery lead to plasmids being an essential tool for scientists. Scientists can engineer plasmids to have specific genes to introduce into new cells (Brown, 2011, p. 134). On a plasmid loop there will be an origin of replication (ORI) and a multiple cloning site (MCS) where the gene of interest is inserted (Bio-Rad, 2015). This region has specific restriction enzyme recognition sites, which are cut by the enzymes to open up the DNA where the new gene will be inserted (Jove Science Education Database, 2015). Most plasmids will also contain an antibiotic resistance gene allowing cell survival in environments containing antibiotics (Jove Science Education Database, 2015).

This lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which causes them to glow a green color under a ultraviolet light.

The plasmid pGLO contains an antibiotic-resistance gene, ampR, and the GFP gene is regulated by the control region of the ara operon. Ampicillin is an antibiotic that kills E. coli, so if E. coli, so if E. coli cells contain the ampicillin-resistance gene, the cells can survive exposure to ampicillin since the ampicillin-resistance gene encodes an enzyme that inactivates the antibiotic. Thus, transformed E. coli cells containing ampicillin-resistance plasmids can easily be selected simply growing the bacteria in the presence of ampicillin-only the transformed cells survive. The ara control region regulates GFP expression by the addition of arabinose, so the GFP gene can be turned on and

After reading the first half of chapter two, The Analytic Project, I was most interested in the section titled, The Discovery of Insulin. I furthered my research of this area after reading and was able to better understand insulin and why it was such an important discovery. With that being said, diabetes was the result of malfunctions of the pancreas. Before the discovery of insulin, diabetes was a disease that usually caused death, especially during the late nineteenth century. Many feared this disease, and according to A Philosophy of Science, A new Introduction, most people lost their life within a year of being diagnosed with the disease, even with the best treatment available at the time.

First, the human gene that codes for insulin production is isolated and then copied numerous times. Restriction enzymes are used to cut out a section of the plasmid DNA from E. coli bacteria. Then, the human insulin gene is inserted into the section of the E. coli plasmid that was cut out. These are then inserted into a viable host, where it will grow and multiply. The bacterial cell processes ‘turn on’ the human insulin gene allowing for it to be produced within that cell wall.

Insulin heroes was an idea that was conceived in my freshman biomedical sciences class, and was later developed during my junior year in HOSA as part of my group’s medical innovation project. Insulin Heroes was basically lotion that contained insulin. It could be used for Type 1 diabetics who didn’t like needles and would be used instead of an insulin shot.

For this experiment, E. coli was best for genetic engineering because of their size, and their fast reproduction (Spilios, 2017). E. coli will be genetically transformed using an engineered plasmid. A plasmid is a circular piece of DNA which independently replicates and multiplies because it has its own origin of replication (Spilios, 2017). The pGLO is the plasmid used in this experiment. Plasmids are used as vectors and they contain manipulated genes such as genes coding for antibiotic resistance for drugs like ampicillin. This antibiotic resistance of such serves as the selectable marker in genetic transformation and for genetic transformation to proceed, the cell must reach competency which is the physiological state that is required for the vector plasmid to get into the cell for transformation (Spilios, 2017). While competency can be reached naturally in some organism, it must be reached artificially in E. coli through treatment with CaCl2 and exposing them to heat shock using incubation (Spilios, 2017).

This process uses enzymes to cut and insert piece of DNA into a plasmid vector. This vector is then transfected into cells. The different pieces of DNA that were being inserted into this plasmid were a control and three different mutants (an alpha-5-tailless mutant, full-length alpha-5, and full-length alpha-2). These pieces of DNA code for integrins. Once these pieces of DNA were inserted into the plasmid vector, the plasmid needed to be inserted into a cell where it would be able to replicate. In these tests, rat intestinal epithelial wild-type cells (cells that would be found in nature, not cells grown in the laboratory) were used. To insert the plasmid into these cells lipofectamine plus was used. Lipofectamine plus acts like a detergent and opens the membrane of the cell so the plasmid can enter. Also, the plasmids that were transfected into cells were all treated with G-418, an antibiotic. When the cells were plated and allowed to multiply, only those containing the plasmid with the antibiotic would live. This made it possible to know which cells actually received the plasmid and properly underwent mitosis, and to eliminate those cells that never took up the plasmid in the first place.

Insulin has been used for diabetes since 1922. “Leonard Thompson, a 14-year-old boy dying from diabetes in a Toronto hospital, became the first person to receive an injection of insulin” (“The History of a Wonderful Thing We Call Insulin” 1). Without insulin, thousands of people with diabetes would die. Insulin is available for people who need it because it was initially tested on animals. Oskar Minkowski and Joseph von Mering removed a pancreas gland from a dog in 1889, and it ended up dying later (“The History of a Wonderful Thing We Call Insulin” 1). Animals like dogs, have hormones in their blood so the experiment worked on them. It wasn’t safe for humans yet (Parry 1). Although putting an animal’s life at risk for medical research might

Diabetes affects 9.3% of the population in America. It is the 7th leading cause of death in the United States [1]. Diabetes is a disease where the body is unable to produce insulin in sufficient amounts. The insulin breaks down glucose, so without the insulin, the glucose is unable to be broken down which leads to the buildup in the blood. Without glucose metabolizing, the body will not have energy to function [2]. Treatments for diabetes has varied throughout the years. However, the one thing they all have in common is insulin replacement. Previously, animal insulin was the only solution. Now, we are able to administer human insulin using a recombinant DNA technology [4]. It is most commonly administered with a subcutaneous injection.

Plasmids are small double stranded circular non chromosomal DNA molecules containing their own origin of replication. Hence, they are capable of replication independent of the chromosomal DNA in bacteria. Plasmids present in one or more copies per cell, can carry extra chromosomal DNA from one cell to another cell and serve as tools to clone and manipulate genes. Plasmids used exclusively for this purpose are known as vectors. The genes of interest can be inserted into these vector plasmids creating a recombinant plasmid. Recombinant plasmids can play a significant role in gene therapy, DNA vaccination, and drug delivery [Rapley, 2000].

It is a cold, snowy day in early 1918. Your parents have been conversing for the last hour. They think that you are in bed, but you are not. You have heard every word that has come from their mouths, and you are petrified with what you have learned. You have juvenile diabetes. You are going to die. A great thirst overtakes you, and you stumble up the steps. The countdown has begun!

Insulin is a major metabolism regulating hormone. It is secreted by beta cells of the

In anatomy we are learning about the human body which is including the function and structure of cells and how the cells are regulated. In the case of diabetes there is a problem with the hormone insulin which results in irregular metabolism of carbohydrates and prominent levels of glucose in the blood and urine. There are two types of diabetes: type 1 and type 2. Type 1 diabetes is sometimes known as juvenile diabetes, this type is where the body does not produce insulin. Type 2 is due to obesity which can be regulated and controlled by diet and exercise.

Darwin’s ideal played a major role in driving the scientific community to begin looking into the causes of characteristics, eventually leading to the discovery of DNA, and ultimately genes. It is through this interest that pharmaceutical applications of genetic engineering were further researched; a major example being the transition from developing insulin for diabetic patients from the pancreas of swine to using bacterial genetic modification to develop the same results in a cheaper and more efficient manner. Now, centuries after Darwin, scientific and technological advances have given scientists the ability to use restriction enzymes in order to successfully alter the order of traits in a chromosome. The restriction enzymes splice, or cut apart, the desired gene out of the donor chromosome, taken from a human cell. Because genes cannot be directly inserted into the recipient organism, plasmids—circular DNA molecules separate from the chromosomal DNA and usually found in bacteria—allow this transaction to occur. The small molecular plasmids transfer the new DNA strand into various cells in the body. Ligase then repairs the breaks in the organism’s DNA and after the reproduction process, the new DNA segment is found in the majority of the cells in the body, thus altering the phenotypic representation of the gene.