The Use Of Bacteria For Recombinant Protein Production

Escherichia coli, or E. coli, is a common bacterium that can be found in diverse environments all over the planet, including the gastrointestinal tracts of animals and humans. Many of these strains of E. Coli are essential mechanisms in the digestive tract, while others are pathogens that can cause complications in urinary and intestinal tracts. (Payne & Sparks) In research, E. Coli is commonly used as a model organism, meaning they are widely studied by scientists for a variety of purposes due to their experimental advantages. E. Coli is comparatively simple, and there are many advantages to studying these prokaryotic cells in the fields of biochemistry and molecular biology. E. Coli has this simplicity and is relatively easy to propagate in a lab environment. Their genome has been completely sequenced and many things we know about DNA, protein synthesis, and gene linkage have been derived from studies regarding this particular organism. (Cooper)

In our hypothesis we stated that only the container containing all of the components +pGLO, LB broth, ampicillin, and arabinose would be the one that genetically transformed. In order for the bacteria to grow at a rapid pace all it needed was LB broth but when you added ampicillin, an antibiotic, it killed off all of the bacteria. +pGLO has the gene to resist the antibiotic so when that was added it was allowed to grow but there was no sugar to turn on the glowing protein. Finally, after arabinose, a sugar, was added it turned on the switch located in the +pGLO for the fluorescence and enabled to grow and glow.

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.

Escherichia Coli is a rod-shaped bacterium that measures approximately 0.5 μm in width by 2 μm in length. It is a Gram-negative bacterium. Its cells stain gram negative because they have a thin cell wall with only one to two layers of peptidoglycan. They live in environments with higher temperatures rather than cooler temperatures. E. coli is said to be the “model organism”. Many microbiologists use these bacteria as a resource for understanding other prokaryotic life and are the most carefully studied life form on the planet. It was discovered by German-Austrian pediatrician Theodor Escherich in 1885.

Bacteria like E. coli contain an operon, or a group of genes that have a promoter, which is transcribed as a single mRNA and is a good representation of this inducible system (KhanAcademy). When looking at

Escherichia coli, (E. coli) 0157 is a gram negative bacillus rod shaped bacterium it belongs to the Enterobacteriacea family. (Gould, 2011) E. coli 0157 is a member of a large group of bacterial germs that inhibit the intestinal tract of humans and other warm – blooded animals. (Marler, 2011). This bacterium can live in an environment with or without the presence of oxygen. E. coli has been around since the 1800’s. There is one good benefit to E. coli. Studies have found that E.- coli, colonizes the human bowel within a few weeks of birth and is beneficial to the human body because it reduces the risk of over growth by other bacteria that is more than likely to cause gastrointestinal infection. It was first detected in the stools of babies in 1885. (Gould, 2011) It is named after Esherich, the scientist who 1st isolated it. E. coli is the organism most known for causing urinary tract infections and is especially known for being a main contributor of diarrhea for individuals traveling overseas.

This creates a long chain of amino acids and will soon become a protein. Another system you must know about is the Operon system, which can either be repressible or inducible. If inducible the the inducer will cause the repressor to inactive and the RNA polymerase will be able to move through protein synthesis and create a protein, if repressible the RNA polymerase will not be able to move through. The last topic you need to know is biotechnology. This is the process in which a gene is isolated through different genetic techniques that then insert the bacteria into another organism. In the pGLO lab we used a clone GFP gene (green fluorescent protein) from jellyfish that was then used to make a plasmid called pGLO. I predict that bacteria with +pGLO plasmids which are resistant to the antibiotic ampicillin and have the gene for GFP, will survive and grow if they have LB and AMP. Also +pGLO bacteria on a plate with LB, AMP, and ARA will grow and glow under a UV light because of the arabinose. Finally the plate with -pGLO bacteria that are not resistant to ampicillin will not grow on the LB and AMP plates. This is important to know for genetic

This was done by using DNA ligation and E.Coli transformation. We looked at agar plates to analyze which one of E.coli cell strains took up the vector alone of the vector containing the gene of interest. Four agar plates were used in this laboratory which were labeled Ligation, pGEM- T Positive Control, Competent Cells Negative Control, and Competent Cells Positive Control. Reagents were used such as DNA Ligase Buffer, SOC Media, Ampicillin, IPTG, and X-Gal. Ligation was performed using 2X ligation buffer, DNA Mix, T4 DNA Ligase and sterile dH2O. Then the transformation of E. coli occurred in which our 4 LB agar plates were prepared with the corresponding amount of IPTG, X-Gal and ampicillin gene. The next step was pipetting the content into the corresponding plates and SOC media was added.The transformation tubes consent were placed on the agar plates using a spreader. The plates were incubated at 37 Celsius and then will be stored at 4 degrees Celsius.The ligation plate was prepared by adding 100 µL of IPTG, 50 µL of X-Gal and Ampicillin. In the ligation plate, the expected color to be see was blue. However, in our ligation plate, we were seeing both blue and white colonies. Blue cells indicate that the cells take up the vector alone in the presence of IPTG and X-Gal due to Beta- Galactosidase expression. IPTG or

According to Medicine.net (2012), genetic transformation is a process by which the genetic material carried by an individual cell is altered by the incorporation of foreign/exogenous DNA into its genome. Competent cells are able to accept DNA presented by experimental influence or manipulation, and the application of genetic engineering with bacteria can aid in the fight against diseases, allowing individuals to maintain their lifestyles without the threat of certain illnesses like heart disease, cancer or hereditary disorders (McPhersson 2008). Plasmid DNA are small circular double-stranded helixes, and present in the plasmid are ampr (selectable marker gene) and GFP (Green Florescent Protein), (BIO-RAD 2010).

Industrial applications include transforming microorganisms like bacteria or yeast, or insect cells with genes for creating a useful protein.

Abstract:Conjugation is a natural occurring process that involves the transfer of DNA from one cell into another through a physical connection between the cells. In the following experiment, two strains of Escherichia coli bacterial cells (donor F'lac+strs and recipient F-lac-strr) underwent conjugation to produce a transconjugant strain (F'lac+strr). MAC plates and streptomycin were utilized to determine if conjugation had occurred. When plated, the donor colonies appeared red and the recipient colonies appeared white. The transconjugant plates showed red and white colonies. Using alkaline lysis miniprep, a DNA plasmid was isolated from the donor and transconjugant strains and FIGE electrophoresis was used to determine the size of the

In order to manipulate, clone and express certain genes it is essential that a greater understanding of how the gene in question operates, in order to isolate and potentially recreate that particular characteristic in a host organism (Strand ect al. 2003) Escherichia coli is almost the most used organisms of preference in lab based experumentation for the manufacturing of recombinant proteins. Its used as a mobile factory because it is stable and it has become the most famous expression platform organism to use. (Lee ect al.

Escherichia coli, is a common bacterium found in the human small intestine that aids in digestion and the absorption of vitamins. (Reference about E. coli) E. coli is a model organism for molecular biology research and has played a crucial role in the development of recombinant DNA technology. The basic concepts behind this biotechnology is the insertion of a desired gene into a circular bacterial plasmid DNA using restriction enzymes, transforming the plasmid DNA into living cells, and growing the cells on a culture plate with the desired gene isolated.

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

Now the T-DNA is delivered as a single stranded DNA molecule coupled with the VirD2 protein in the plant cell. The T-DNA is then integrated into the plant chromosome and are expressed by plants transcriptional machinery.