The Transformation Occurrence With E.coli And The Ampicillin Resistance Gene.when Plasmids

As predicted the E. coli colony transformed with either the PUC18 or the lux plasmid developed an ampicillin resistance. Which made it easier for them to not only survive but also replicate in both the LB agar plates and the LB ampicillin rich agar plate. However the E. coli colony not treated with the plasmids could not survive and colonize in the LB ampicillin rich agar plates. The plate that had no ampicillin in its environment and no plasmid treated E. coli served as a positive control for this experiment because it demonstrated how the E. coli would colonize and grow in a normal setting. The cells in the positive control plate grew into lawn colonies because they were not placed into a selective environment or transformed, so they had no need to acquire ampicillin resistance. Two plates in the experiment contained E. coli cells that were transformed with either the PUC18 or the lux plasmid but were placed in an ampicillin free environment. These two colonies grew

The third and final factor in bacterial resistance to antibiotics is bacterial genetic exchange. It is very common for bacteria to share genetic info between cells even if the bacteria are not the same species. When this happens, genes and DNA get mixed into different species allowing more mutations against the antibiotics.

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).

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

In this investigation pUC19 plasmids were used as the vector due to its small size of 2686bp, high uptake efficiency by the host and fast replication time. Important features of this plasmid include the origin of replication and multiple cloning sites (MCS). The origin of replication allows the plasmid to replicate inside the host bacterium. The MCS is located within the lacZ gene and contains unique sites for the Xbal & EcoRI restirction enzymes to cut and produce sticky ends for the CIH-1 gene to bind to. Furthermore, the pUC19 plasmid also contains an ampiccilin resistance gene so only transforemed E.coli are able to remain viable when spread on the agar plates that also has the addition of ampiccilin. The lacZ gene encodes the β-galactosidase enzyme which aids in indentifying the recombinant E.coli from the non recombinant cells (Coventry University 2016).

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

Bacterial plasmids differ from bacterial chromosomes by not being essential to bacterial growth and metabolism. They are extracellular chromosomes that contain DNA. Bacterial chromosomes are essential for bacterial survival.

Our research on recombinant DNA mainly consisted of two experiments: Transformation and gel electrophoresis. In our first experiment, four microfuge tubes were given to us: pKAN DNA, pAMP DNA, unknown DNA, and a TE buffer without DNA. The two positive controls, pKAN and pAMP, consisted of an antibiotic resistance gene respectively to their name. The pKAN plasmid contained the gene resistance for kanamycin while pAMP carried the gene resistance for ampicillin. The negative control, TE, only contained buffer without DNA. The fourth tube was our unknown plasmid, which was either pKAN or pAMP; and by way of artificial transformation, we would be able to initiate the identification of our unknown plasmid.

Bacteria are the natural world 's unsung heroes. They receive bad rep due to the disease causing individuals like tuberculosis but the vast majority of bacteria on Earth are harmless if not beneficial to both the environment and humans. Take for example E. Coli a well known bacteria that lives within our intestines. This particular bacteria makes vitamins that we need in order to stay healthy. In this experiment we analyzed the changes seen in bacteria when adding to differing DNA plasmids; pUC18 and lux. To arrive to the most accurate results possible we had to have the E. Coli bacteria made permeable to the plasmids. So it was prepared with calcium chloride. One group of bacteria was given the pUC18 plasmid, and the other group was given the lux as well as the pUC18 plasmids. We then proceeded to incubate them in different containers. Some had ampicillin where as some had none. This was done in order to observe the effect of pUC18, and lux growth patterns. A control with neither plasmid was used to keep a basis for our experiments. Bacteria that had transformed and taken in the lux plasmid without ampicilin were able to illuminate, and only bacteria that gained the pUC18 were capable of surviving with ampicillin. The results observed reveal how foreign DNA can be adapted to fit into another cell 's DNA even incorporated to be an optimal part of the cell. This process makes it a very viable manner of being able to mass replicate advantageous genes, that would

Transformed bacteria cells would be found on plates that contain pGLO, so the LB/amp +pGLO plate and the LB/amp/ara +pGLO plate. The pGLO is a plasmid that expresses the gene for resistance to ampicillin. On the plates that contain pGLO, cells take up the pGLO plasmid and will be ampicillin resistant. When cells take in a foreign plasmid, they are transformed.

In this last situation, resistant genes become embedded in small units of DNA, called transpons, which can easily move into other DNA molecules. Making matters worse, many bacteria have specialized transpons called integrons, which act like flypaper when catching new genes (3).

For medical professionals, some of the hardest illnesses to cure are bacterial illness that will not respond to antibiotics. With this being said, antibiotic resistant bacteria are very dangerous. Some bacteria contain bacterial plasmids that contain genes, which function to improve the cell’s likelihood to survive under certain conditions. Plasmids are very tiny, circular DNA molecules within bacteria. They are passed on to daughter cells because enzymes can replicate them.

Some classes of plasmids encode the sex pilus necessary for their own transfer. Plasmid host-to-host transfer needs direct mechanical transfer by conjugation, or changes in incipient host gene expression allowing the intentional uptake of the genetic element by transformation. Microbial transformation with plasmid DNA is neither parasitic nor symbiotic in nature, because each implies the presence of an independent species living in a commensal or detrimental state with the host organism. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a

K. Pneumoniae is a gram negative opportunistic pathogen encapsulated in a polysaccharide capsule. K. Pneumoniae is frequently responsible for nosocomial infections. Typically K. Pneumoniae affects the respiratory tract but can also be found in urinary tract infections, wounds or in the blood stream. In recent years, multidrug resistant strains have been isolated from nosocomial infections. Bacteria have many ways to gain resistance. Plasmids are circular DNA strands separate from the host DNA. They are able to replicate independently of the host and are an important factor in horizontal gene transfer. Plasmids often encode resistance genes or other virulence factors. Transposable elements are another way bacteria can acquire resistance. Transposable elements are DNA sequences that have the ability to insert themselves into host DNA or plasmids. The transposing of a plasmid gene to the host can result in permanent genetic resistance. Integrons are mobile genetic structures with DNA genes that encode for an integrase site and attachment site. Integrons are able to acquire and incorporate gene cassettes which often contain resistance

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