The Discovery of Transformation by Frederick Grifith in Streptococcus Pneumonia

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

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.

The purpose of this lab is to use genetic engineering to transform E. coli bacteria by inserting the plasmid pGLO, and to then see if the bacteria was transformed by using the antibiotic, ampicillin.

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 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.

This experiment focuses on genetic engineering and transformation of bacteria. The characteristics of bacteria are altered from an external source to allow them to express a new trait, in this case antibiotic resistance. In is experiment foreign DNA is inserted into Escherichia coli in order to alter its phenotype. The goal of the experiment is to transform E. coli with pGLO plasmid, which carries a gene for ampicillin resistance, and determine the transformation efficiency. The bacteria are transformed by a combination of calcium chloride and heat shock. When the bacteria are incubated on ice, the fluid cell membrane is slowed and then the heat shock

In this experiment, the E. coli sample used harbors a genetic mutation on the repressor gene of the lac operon, resulting in the constitutive enzyme production of -galactosidase. Therefore, the expectation of this strain of E. coli should produce higher yields of -galactosidase, as so much as 1000 fold in comparison to wildtype, non-mutated strains (Mowery and Seidman, pg. 3).

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

Depending on the bacteria species, the manner in which transformation occurs varies. Haemophilus influenza is an example that utilises membrane-bound vesicles to capture double-stranded DNA 3. On the other hand, S. pneumoniae can uptake single-stranded DNA molecules after its cells express competency factors 3. Alternatively, scientists in laboratories transform cells, including ones that are not naturally competent, by inducing them to uptake DNA 3. These transformed cells are now known as competent cells for they have been rendered able to uptake DNA from the environment 18. This artificial transformation process is achieved by using a procedure called the heat shock method 18. This transforming method includes adding DNA to cells in the

Introduction Antibiotic resistance can be introduced into bacteria by a chromosome, a phage or a plasmid (Gilbride et al. 2016). The gram negative E.coli was the bacterial strain for isolation, cloning and transformation (Gilbride et al. 2016). pUC 18 and pKan plasmids were used to carry antibiotic resistance in this experiment (Gilbride et al. 2016). pUC 18 is a 2686 base pairs long, small circular DNA vector in E.coli that carries a gene for ampicillin resistance (Gilbride et al. 2016) . The antibiotic resistance that has pBR322 origin can bind to ∞-complementing restriction site of LacZ gene in E.coli and disrupt the production of β-galactosidase by this genes (Gilbride et al. 2016).

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).

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

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].

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