Biology Essay

This pBlu lab had for purpose to present the changes of the strain of E. coli bacteria due to new genetic information being introduced into the cell. In this experiment we are freezing and heat shocking the E. Coli bacteria that is then forced to take the plasmid DNA. The E. coli then transforms the pBLu plasmid, which carries the genes coding for two identifiable phenotypes. After following the Carolina Biological steps our lab worked well and we able to see some colonies of bacteria on the plates. The x-gal plate showed a significant amount of bacteria to confirm that the pBlu plasmid took over the E. coli strain.

coli the bacteria that will be used to house the GFP is the competent cell. They are called competent cells because they are able to accept foreign DNA or plasmids. They are first observed in 1928 by Frederick Griffith where he proposed that bacteria are capable transferring genetic information (Havarstein, 2010). These cells such as E. coli are normally made competent by exposure to a rich calcium environment, where the mixture calcium chloride will be used in. Calcium chloride’s positive charges will cancel out the negative charge of the plasmid and E. coli’s cell wall weakening it, making it easy to pass.

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).

Purpose The purpose of this experiment is to demonstrate how genetic engineering works to manipulate the genetics to express certain traits. This can be seen with the insertion of the pGLO plasmid onto Escherichia coli. Introduction All of the things that is required to make a particular organism function properly, is called the genome. Eukaryotes have a genome that is composed of chromosomes, plasmids, and other specific organelles, such as mitochondria, chloroplasts, etc.

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.

The transposon in this experiment is contains kanR in between the inverted repeats on either end, which will be transposed from the plasmid pVJT128 to the chromosome of the recipient bacteria.

A basic method in which we get specific genes integrated with another organism’s chromosome is as follows: Isolate the DNA from which selected gene is to be taken from and treat it with enzymes that will cut out that specific gene. These genes are then inserted into bacteria and grown into colonies being

This was done by using DNA ligation and E.Coli transformation. We looked at agar plates to analyze which one of E.coli cell strains took up the vector alone of the vector containing the gene of interest. Four agar plates were used in this laboratory which were labeled Ligation, pGEM- T Positive Control, Competent Cells Negative Control, and Competent Cells Positive Control. Reagents were used such as DNA Ligase Buffer, SOC Media, Ampicillin, IPTG, and X-Gal. Ligation was performed using 2X ligation buffer, DNA Mix, T4 DNA Ligase and sterile dH2O. Then the transformation of E. coli occurred in which our 4 LB agar plates were prepared with the corresponding amount of IPTG, X-Gal and ampicillin gene. The next step was pipetting the content into the corresponding plates and SOC media was added.The transformation tubes consent were placed on the agar plates using a spreader. The plates were incubated at 37 Celsius and then will be stored at 4 degrees Celsius.The ligation plate was prepared by adding 100 µL of IPTG, 50 µL of X-Gal and Ampicillin. In the ligation plate, the expected color to be see was blue. However, in our ligation plate, we were seeing both blue and white colonies. Blue cells indicate that the cells take up the vector alone in the presence of IPTG and X-Gal due to Beta- Galactosidase expression. IPTG or

One of the four ways that bacteria can increase their genetic diversity is by conjugation. This is a process by which a bacterium can transfer genetic material to another bacterium of different mating type, through direct contact. During conjugation one bacterium is going to be the receptor of the genetic material while the other is going to be the donor. The donor bacterium is going to grow a tube-like structure called a pilus, which is going to be used to contact the other bacterium and to transfer the genetic material. Usually these genetic material is going to have the form of plasmid, which are small circular pieces of non-chromosomal DNA. Another method that bacteria have developed in order to acquire different DNA is transformation. These is a process by which some bacteria can get pieces of DNA from the external environment, under certain circumstances. Transformation occur naturally in Bacillus, and some Streptococcus and Staphylococcus species. The third means by which bacteria obtain new DNA is by the acquisition of plasmids. These plasmids can travel between bacteria of the same or different species, and the act like parasitic pieces of DNA that infect bacteria. Usually these plasmids are not required for the normal function of bacteria and is often expel by them, unless it contains material beneficial

Genetic modification is done through the recombination of DNA. Recombinant DNA is produced firstly by isolating a DNA sequence, then inserting it into the DNA of a different organism. Recombinant DNA is possible because the DNA of every organism is made of the same nucleotides5, recombinant DNA allows a gene from one organism to be moved into, and expressed in a different organism. Two important factors used to create recombinant DNA are restriction digestion6 using restriction enzymes7 and DNA ligation8 using the enzyme DNA ligase.

Scientist have harvested many different types of restriction enzymes. Each type of restriction enzyme has a distinct shape and attaches to a specific part of DNA where it eventually makes the cut. Through the use of specific restriction enzymes, scientists can cut out the exact part of the bacteria’s DNA that they would like to use. Once they have a small snippet of DNA from a bacteria that is responsible for what they want to get, like size or taste, they have to get it into the organism. In order to accomplish this a vector is used. The vectors that are used are essentially viruses that have the ability to go into a living thing and insert their own DNA into them. There are other types of vectors besides viruses, but they are the most commonly used. Usually these kind of viruses are very harmful to their hosts, but scientists have removed the harmful part of the virus and instead of it inserted the desired DNA section that they

New research techniques have made it possible to engineer and explore differences in the sets of chromosomes in organisms. This has been a technological revolution during the last decade. Allowing scientists to be able to explore DNA to a new extent. During the process of this research it has come apparent that foreign DNA inserted into self-replicating genetic elements such as bacteria plasmids can replicate. This breakthrough has also shown that the plasmids that have been used can also be used to change the genetic constitution of other organisms (1).

Genetic engineering uses three different techniques to rearrange the pattern of the DNA bases and form different genes. The first of these techniques is called either the plasmid method or recombinant DNA. Plasmids are small rings of DNA that are separate from the chromosomes, these are often found in bacteria and can be easily manipulated using other bacteria or viruses(Call). The DNA code is broken by the bacteria or virus and a new pattern is able to be inserted into the DNA. The cell then reads the information and begins to produce the new trait. Another way of changing the genes is to use a needle to inject the new code into the cell. This process is called microinjection, and it is most easily done with larger cells. The third technique is called bioballistics; it

Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial