Plasmid Lab Report

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.

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 pGLO plasmid is engineered to express green fluorescent protein (GFP) in the presence of the sugar arabinose as well as the ampicillin resistance gene β-lactamase (bla) (Brown, 2011). Original E. coli HB101 do not have ampicillin resistance or the GFP gene allowing them to glow under UV light. In this experiment, we transformed E. coli HB101 with the pGLO plasmid by heat shock to make the bacterial cells competent, allowing the plasmid to enter the cell (Brown, 2011). The mixture of bacteria with pGLO plasmid were given recovery time after heat shock, then spread on LB/amp and LB/amp/ara agar plates. The bacteria mixture with no plasmid added were spread on LB and LB/amp agar plates and all four plates were incubated at 37°C for

Control plasmids lux and pUC18 were introduced into E. Coli through a process of transformation.

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

Plasmid map of pRSETB. Primers were designed to amplify via PCR to cDNA. The PCR product was digested with XhoI and EcoRI enzymes and ligated into the pRSETB plasmid. The pRSETB plasmid contains a T7 promoter region, is ampicillin resistant, inducible with Isopropyl β-D-1-thiogalactopyranoside IPTG, a molecular mimic of a lactose metabolite that triggers transcription of the lac operon, and has XhoI and EcoRI cut sites. The pRSETB plasmid is transformed into dH5α E. coli and plated on carbenicillin plates. Colonies are selected and grown on a carbenicillin plate while PCR is used to check that the plasmid that was up-taken was not

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].

Currently, our society is at a point, where our knowledge and understanding of human, nature, and science are very controversial, yet many promote advancement in scientific technology like cloning as a benefit for humans. Human beings becoming Gods, and manipulating the DNA of animals, plants, bacteria, and humans, so large corporate developers can profit from hybrid creations. Eating from the metaphorical tree of the Knowledge of good and evil, listening to the evil one’s whispers that you shall not die. In addition, people have become smart enough to cause major problems to our society and future as living beings. I believe it is not coincidence that DNA and cloning began advancing around the same time as the Atom Bomb, because both the

Cloning is defined as the “creation of an exact copy of a living matter, such as a cell or organism” according to Encarta encyclopedia. The copies produced through cloning have identical genetic makeup and are known as clones. Scientists use cloning techniques in the laboratory to create copies of cells or organisms with valuable traits.

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

The possibility of stem cell research exploded with momentum when a certain sheep named Dolly was born. The reason why this was such a breakthrough is because Dolly was the first mammal ever cloned. Although this all began in 1996, the study into stem cell research has been documented since the 1960’s, where Joseph Altman and Gopal Das brought forward new evidence of adult neurogenesis ( the ongoing stem cell activity in the brain). The birth of Dolly brought many breakthroughs for the scientific community but it also created an avalanche of concern because of the ethical implications. With all of the reporting on Dolly, the news media only spoke of one type of cloning and that is reproductive cloning. This may be the most popular

DNA cloning is the process of creating a multitude of copies of isolated DNA fragments; DNA cloning can be carried out via in vitro or in vivo methods. One can clone a specific DNA sequence or entire gene fragments. There are a multitude of procedures to carry out DNA cloning, but the major steps are the same for all types. To begin the process, one must isolate a DNA fragment from the chromosomal DNA. This is done by using a restriction enzyme. One could also use gel electrophoresis and polymerase chain reaction (PCR). The next step involves introducing the fragment into a DNA sequence that can replicate itself along with the DNA fragment. The restriction enzyme will cut a DNA molecule that can self-replicate, and then the isolated DNA fragment will be inserted by ligation, which connects the fragment to the larger DNA molecule. This new artificially joined DNA is called recombinant DNA.

DNA is also known Deoxyribonucleic acid. It is the carrier of genetic info. The distinctive characteristics or qualities of someone or something come from DNA. It is regarded as unchangeable. Nearly every cell in a person’s body has the same DNA. A small tiny amount of DNA can also be found in the cell’s mitochondria. Dna is made up of four chemical bases: A,T,C,G. Human DNA consists of 3 million different bases. Each base is also attached to a sugar molecule and a phosphate molecule. Dna can also copy and replicate itself. One strand of DNA is known as the double-helix. It has rungs, just like a ladder. When cells divide, each new cell needs a copy of DNA from the old cell.

In addition to the mph gene, few gene regulations are implemented as safety assessment for the product. In the recombination process of making this product, Ecoli plasmid has been used as a vector to insert mph gene into host bacteria. We use a plasmid vector from E.coli that has conserved sequence for Plac promoter and gef suicide gene. Plac promoter is positioned upstream of mph gene to regulate its expression. Later, Plac promoter will be activated by induction with IPTG.

Imagine a future where humans are manufactured, a future where humans are created by science, a future where humans are the new lab specimen. Human cloning is like opening Pandora's Box, unleashing a torrent of potential evils but at the same time bringing a small seed of hope. No matter how many potential medical and scientific benefits could be made possible by human cloning, it is unethical to clone humans.