Characteristics And Mechanisms Of Agrobacterium Tumefaciens

Stanley N. Cohen is perhaps one the few men who have had to greatly influence America. Mr. Cohen is an American biochemist who alongside Herbert Boyer “…led to the development of methods to combine and transplant genes…”, the beginning of bio engineering, and would eventually become the first to create a genetically modified organism. This man and his team began the first GMOs in 1972 with bacteria. Cohen and Boyer found that DNA could be changed within bacteria using enzymes to “…counter invasion by viral organism…” that would harm bacteria. This demonstrated “…that these genes were active…” after insertion and could be passed on when bacteria replicated.

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

This experiment was performed to assess the efficacy of genetic transformations on bacteria via plasmid DNA coding for ampicillin resistance and green fluorescent protein. Genetic transformation was studied by taking transformed and untransformed Escherichia Coli (E. coli) and placing them on various media to observe gene expression via growth and color under UV light. The transformed E. coli were able to grow on ampicillin while the untransformed E. coli, which lacked the plasmid genes for ampicillin resistance, only grew on nutrient broth. In the presence of arabinose, the transformed E. coli glowed green. These results support the previous scientific understanding of bacterial competency, vectors, and gene expression and support gene transformations as an effective method to transfer the desirable DNA of one organism into another organism’s DNA. These results can be applied to real world issues such as medical treatments, food production, and environmental conservation.

Genetic transformation occurs when genes are inserted into another gene to change the organism’s trait (Weedman2016). In this experiment, we proceeded to transform the E. coli bacteria with a gene that contained green fluorescent protein. The green fluorescent protein is used in experiments because it beams a green color under a UV light (Chalfie2008). Typically, it is used to mark the expression of genes, which is why it serves as the symbol for all gene expressions (Tsien1998). In the experiment, we will be using pGLO as the organisms that will transmit the disease, otherwise known as a vector. The pGLO in the experiment

The idea of changing natural processes to produce an “ideal” crop raises concern amongst environmentalists. Extracting bacteria from soil and inserting it into an entirely different species may appear unethical because of the changing of natural processes. Genetic engineering promotes producing a type of crop that is not susceptible to pests, but carries the risk of decreasing genetic variation (Green Peace). Once a crop is perfected, farmers then mass produce that specific crop, and the different variations of the crop are lost (Green Peace). Another concern regarding the use of the Bt protein is the increased risk of allergies (Smith). The transgenic protein comes from bacteria in the soil, and thus raises the concern of allergic reactions to the corn. Consumers are worried that the cry protein injected into Bt corn could have allergies unknown to them because of the cry protein’s characteristic of an extended digestion time. Consumers are also skeptical that genes of this antibiotic resistance could somehow be introduced to other bacteria in the soil, and possibly infect consumers with this bacteria. A major ethical concern of genetically modified crops, specifically Bt corn, is the lack of knowledge of the long term effects these crops have on humans and other organisms. Consumers are uninformed about the

The purpose of the Gram Stain test was to identify the morphology and gram nature of the unknown bacteria (Delfiner, Martinez, & Pavia, 2016). First, a mixed smear from the unknown bacterium was placed on a microscope slide. Once the mixed smear had air dried it was then heat fixed to adhere the bacterial cells to the slide. After the heat fixation, a series of stains/solutions were added to complete the Gram stain test. The series of stains/solutions included Crystal violet (1min), Iodine (1min), ethanol (2-3 drops), and Safranin (30 sec). Also, after the addition of the Crystal violet and Safranin, DI water was used to remove the stains. Lastly, bibulous paper was used to blot dry the microscope slide and the slide was placed under the microscope. To clearly identify the morphology and gram nature of the unknown, a drop of oil immersion was placed on the slide to utilize the oil immersion

Results of the “-DNA” plate coincide with the expectation of lawn growth due to the non-selective Luria broth and agar plate which allows for almost anything to grow. The “-DNA/+AMP” labeled plate also yields expected results with no bacterial cell growth. The cells do not contain DNA plasmid that encode for the antibiotic resistant gene, preventing the formation of the bacterial cell wall. There is a discrepancy in the agar plate labeled “+DNA/+AMP” because a blue colony is observed and the plate contains bacterial cells. This unexpected growth could be due to cross contamination with the “+DNA/+AMP/+IPTG” plate. For the last plate (+DNA/+AMP/+IPTG), the observations solidify the expectancy of minimal bacterial growth with production of pigmentation. One colony is observed with satellite cells, in addition to a miniscule spot of blue pigmentation. The “Competent” cells are capable of resisting ampicillin. IPTG, the inducible promoter, results in the production of color. With gene regulation, “competent” cells can acclimate to their environment due to methods of turning genes on and off5.

They are non-spore forming bacteria that are nonmotile and aerobic. F. tularensis grow best at thirty-five to thirty-seven degrees Celsius in the lab setting, while in nature, it can survive up to weeks on end at low temperatures. Fortier states that F. tularensis also contains siderophores, which are molecules that have the ability to bind to iron, that grow under iron-limiting conditions. This feature is crucial to the bacterium “because intracellular replication of F. tularensis is iron-dependent, as shown with deferroxamine having inhibitory effects in a tissue during infection” (Fortier et

These chemical signals set off a cascade of gene activity in the A. tumefaciens which direct a series of events required for the transfer of tDNA from the plasmid into the plant’s cells through the wounds of the plant. The tDNA then moves into the nucleus of the plant cell and becomes integrated into the plant chromosome (Understanding GMOs).

Experiments introduce the idea that the uptake of DNA by competent bacteria can be the result for survival reasons such as need for food or evolution purposes (5). The difference between natural and artificial gene transformation in a bacteria, however, with those who are incompetent versus competent allow he or she to conduct an experiment with artificial transformation. By doing this one may be able to determine and observe the formation of biofilms or mutations by transferring a gene to another organism for beneficial or harmful purpose. This could lead to finding resistants or if a gene introduced has any effect on the bacteria. In the experiment, the pGLO plasmid contained encodes the gene for GFP as well as a gene for resistance to an antibiotic allowing a transformation to take place when adding a carbohydrate such as arabinose to the medium. Research has been found on the horizontal gene transfer on how virulence factors are acquired as well as genes resistant to antibiotics spread with microorganisms (5). By conducting a similar experiment he or she can observe similar findings or observe the results and conduct some sort of understanding of these ideas. The findings in this experiment are to show a positive result in what causes growth and the glowing when the plasmid pGLO is added to the positive as well as the difference in the medium. Understanding the way artificial

Professor Mitra emphasizes on how science is ever-changing and how major leaps in knowledge are made regularly. She believes the today’s advancements in genome sequencing and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology will dramatically change the field of cellular and molecular biology moving forward. On a smaller-scale, she believes the focus that some of the biologist, including herself, make on mastering mechanisms and disease in “easy-to-study” systems will eventually equip them with the knowledge and skills needed to leverage them to more complicated systems, which will allow them to solve agricultural problems, such as determining what causes pathogens to affect plants at the roots and reduce food

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

Hypothesis: If genetic transformation involves the insertion of a gene into an organism in order to change the organism’s trait where genes can be moved from one organism to another with the aid of a plasmid and pGLO plasmid encodes for a gene resistant to the antibiotic ampicillin as well as the gene GFP which allows the bacteria, E. coli, to express the glowing gene by producing the fluorescent protein if switched on after having undergone transformation by adding sugar arabinose – which is a nutrient for the cell -, then when the procedure of the pGLO transformation lab is completed as instructed in the student manual, the agar plate +pGLO Lb/ampicillin will produce colonies of bacteria that do not glow, -pGLO LB/ampicillin will not produce any bacteria, and –pGLO LB (constant) will produce a lawn of bacteria that does not glow (instead appearing as a

The coding region of the gene is usually fused to a promoter, most commonly used is the 35S promoter from cauliflower mosaic virus (CMV), in order to promote higher expression levels. (Snow et. al, 1997) The popular method for genetic engineering of crop plants is natural gene transfer via an Agrobacterium tumefaciens vector, a bacterium normally found in soils. The transfer-DNA (T-DNA) vector is made by inserting the desired gene fragment in between specific 25bp repeat domains in the bacterium. The vector is then inserted into the Agrobacterium and "the virulence gene products of Agrobacterium actively recognize, excise, transport, and integrate the T-DNA region into the host plant genomes." (Conner et. al, 1999) The amount of DNA transferred is only about 10kb and the nature of the gene is usually well understood. The expression of the gene introduced can also be controlled by adding additional sequences that might allow the gene to be constitutively expressed, expressed only in certain cell types, or expressed as a result of different environmental changes. This method of gene transfer, however, will only work for the natural host range of the bacterium and therefore other methods are used for additional crop plants. Such methods are uptake of naked DNA by electroporation or particle gun bombardment. The use of genetic markers, as mentioned previously, allows for the preferential growth of cultures that contain the new genetic

Bacterial transformation is a versatile tool of central importance to molecular biology (The American Phytopathological Society, 2016; The Biotechnology Education Company, 2003). It is a process whereby bacterial cells take up and express exogenous DNA, resulting in the expression of a newly acquired genetic trait that is both stable and heritable (The Biotechnology Education Company, 2003; Bruce Roe, n.d.). For transformation to take place, the bacterial cells must enter a particular physiological state, called ‘competency’, which will accommodate transformation (The Biotechnology Education Company, 2003). Bacteria are single celled organisms that can easily pass information between one another, and thus, changes in