The Discovery of Transformation by Frederick Grifith in Streptococcus Pneumonia

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.

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).

This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.

One of the most imperative functions in maintaining the development of evolution is the frequency of genetic transformation: the injection of foreign DNA into another organism’s DNA. This term is defined by the actions of a vector, but more specifically by the actions of plasmids and phages. However, in this experiment we are primarily focused on the effect of the pGLO plasmid transformation of GFP on the E. coli bacteria by introducing a second chromosome or a plethora of cloned plasmids. (Bassiri 2011)

The field of biotechnology involves the concept of genetic engineering, altering the DNA/genetic material of an organism using information from a different one. The process in which bacteria can obtain this manipulated genetic information from another source is called genetic transformation. The goal of this experiment was to genetically transform Escherichia coli bacteria’s DNA by inserting the vector pGLO plasmid which codes for ampicillin resistance as well as the green fluorescent protein, GFP. For the experiment, the E. coli bacteria were separated into two groups; control and

Streptococcus is becoming more resistant to antibiotics. Over a period of three years from September 1, 1993 to August 31, 1996 a study was done on children suffering from the resistant group A streptococcus (Dr. Arditi, 1999). This new group A strain caused children pneumococcal meningitis. Researches looked at eight different hospitals in the United States and one hundred and eighty cases within these three years. The study was designed to look at clinical presentation, hospital course, and the outcome from the children being cured. As they watched and monitored the children's progress on the different antibiotics used to cure streptococcus. Also the researchers has to take into consideration if the streptococcus was becoming resistant, which antibiotics were given as treatment, and from those antibiotics which streptococcus became

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].

Streptococcus pneumoniae (S. pneumoniae) is an important human pathogen. It causes severe and invasive infections, such as pneumonia, septicemia, otitis media, and meningitis, especially in children, the elderly, and immuno-compromised patients [1,2,3]. S. pneumoniae initially colonize the nasopharynx and may persist for months without causing illness, forming specialized structures called biofilms [4,5]. Pneumococci from these biofilms can migrate to other sterile anatomical sites, causing severe biofilm associated infections such as pneumonia and otitis media [6,7,8]. The planktonic bacteria from these biofilm-associated infections can migrate to other sterile sites, such as the blood stream, causing bacteremia, or to the brain,

Streptococcus pneumonia is a pathogen with a niche in the upper respiratory tract of the human that has a two component signaling system. The two component signaling system has CPS accumulate extracellular to the cell, CPS eventually binding to the comD receptor, which stimulates auto-phosphorylation of comE, the response regulator(1). This is a strictly fermenting microbe that tolerates oxygen’s presence, also known as an aerotolerant pathogen (3). This microbe ferments glucose, a sugar, into lactic acid, in the lactic acid fermentation cycle. It also can ferment other sugars like sucrose or fructose. 20% of the strains that are clinical samples only grow anaerobically (3). When placed in the proper multifaceted medium S. pneumonia can replicate in 20-30 minutes. Since there are some many strain of streptococcus with different sugar branches it makes the microbe hard to be overcome by antibiotics (3).

Frederick Griffith conducted one of the first experiments showing bacteria can get DNA by a process called “transformation.” He used two types of bacteria that infect mice-- S strain and R strain. The S strain covers itself with a polysaccharide capsule that protects it from the host's immune system, causing the mice to dice. The R strain does not have that protective shield around it and is killed by the host's immune system. The S strain was heated to kill it, and then injected into mice, it produced no bad effects. But, when dead S strain mixed with live R strain was injected into the mouse, the mouse died. the combination of the two was able to kill it. He concluded that the R strain had been "transformed" into the S strain by a "transforming

3. Oswald Avery and his colleagues showed that the transforming substance that enables Streptococcus to produce a capsule and be virulent is DNA. They discovered that:

The expression of lac operon in each tube equals the amount of beta-galactosidase produced. Therefore, by looking at the amount of beta-galactosidase under different conditions collectively is a good way to understand the function of inducers and repressors in supervising the expression of lac operon and the control of gene expression generally. Throughout this experiment, CTAB was used to kill the E. coli celles at specific time sets so that the cell lyses and releases its content. This is very important as if the cells do not lyse, the beta galactosidase would remain in the cells and there would be no way to measure the amount of beta galactosidase produced. We can measure the amount of beta galactosidase produced in each tubes by knowing the

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