Ligation of EGFP into pET41a(+) vector transformed into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step.
Introduction
Enhanced green fluorescent protein (EGFP) was originally isolated from a bioluminescent jellyfish called Aequorea victoria. As suggested by the name, this protein fluoresces green when exposed to light in the ultraviolet range. The ultimate goal of the following experiment was to successfully create a pET41a(+)/EGFP recombinant plasmid that was transformed into live E. coli cells. The success of this transformation could be evaluated based on whether EGFP’s fluorescence properties were displayed by the colony in question. The protein’s fluorescence properties “triggered the widespread and growing use of GFP as a reporter for gene expression and protein localization in a broad variety of organisms” (Ormo, et. al., 1996). Although EGFP and GFP differ for a few amino acids that make EGFP’s fluorescence mildly stronger, the basic principle that such a protein allows for the evaluation of transformation success remains intact.
The first step of the experiment was ligation, and the objective was to insert EGFP cDNA into a restriction cut pET41a(+) vector to obtain a recombinant plasmid that would express green fluorescent gene. pET41a(+) was the choice of vector to ligate the EGFP into. Its structural design and genomic sequential properties render it especially well-suited for cloning and high-level expression of peptide sequences. This 5933 bp circular vector contains a built in sequence for Kanamayacin resistance gene. “Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin” (Montserrat, et. al., 2001). This allowed the growth of recombinant and non-recombinant colonies of E. coli, all of which contained the vector insert.
Once the recombinant plasmid was obtained, it was then inserted into E. coli cells through transformation. From a successful transformation, we expected the bacterial cells to translate the inserted EGFP sequence into its protein form. The bacteria cultures were plated on petri dishes containing growth supplement, Luria Broth (LB), an antibiotic: Kanamycin, and IPTG which induced the fluorescence property within successfully transformed bacterial colonies. Different variants of the petri dishes were also included as control and unknown.
The miniprep consisted of isolating the DNA plasmid from the bacterial cells. This was used to identify the success of EGFP ligation into pET41a(+) vector upon restriction digest and gel electrophoresis. Additionally, Polymerase Chain Reaction (PCR) was run on the isolated DNA plasmids with one of the primers specifically annealing to a part of pET41a(+) sequence and the other annealing to the EGFP gene.
Recombinant DNA technology: Sub cloning of cDNA molecule CIH-1 into plasmid vector pUC19, transformation of XLI-Blue Ecoli & restriction mapping.
ABSTRACT: Water samples from local ponds and lakes and snow runoff were collected and tested for coliform as well as Escherichia coli. Humans as well as animals come into contact with these areas, some are used for recreational activities such as swimming and some are a source of drinking water for both animals and humans The main goal of this experiment was to see which lakes, snow run off and ponds tested positive for coliform or Escherichia coli and to come up with some reasoning as to why. It was found that the more remote pond with less contact contained the most Escherichia coli. However, another lake that many swim in and use as their drinking water indeed tested positive for a small amount of Escherichia coli. The two samples from the snow showed negative results for both coliform and Escherichia coli and the two more public ponds that aren’t as commonly used as a source of human drinking water but animal drinking water tested in the higher range for coliforms but in the little to no Escherichia coli range. It was concluded that the remote pond should be avoided as it’s not a safe source of drinking water for humans or animals. Other than that, the the other ponds are likely to be safe from Escherichia coli, but coliforms are a risk factor.
Transformation of T87 cells was done by culturing the cells in B5 medium supplemented with 1 μM 1-naphthaleneacetic acid (NAA) and 40 g L-1 sucrose. The cells were cultured for one day at 22°C with continuous illumination and shaking at 120g. Next, 10 μL of overnight cultured Agrobacterium transformed with respective vectors were added into the cell suspension and cultured for an additional two days. After co-cultivation, the cell suspension was washed thrice with 10 mL of JPL3 medium supplemented with Carbencilin (250 μg mL-1) by centrifuging at 100g for two minutes. Finally, the cells were resuspended and spread onto JPL3 selection agar plate supplemented with Carbencilin (250 μg mL-1), Kanamycin (30 μg mL-1) an...
Escherichia coli is a member of the family Enterobacteriaceae. It is a bacterium with a cell wall that has many components. Escherichia coli can live without oxygen which means that it is a facultative anaerobe. It is also capable of fermenting lactose under anaerobic conditions, and in the absence of alternative electron acceptors. There are effects and various factors that limit its growth rate. Its morphology consists of a rod-shaped gram negative bacteria that is commonly found in soil, water, vegetation, human intestines, as well as the intestines of animals. Its presence can be good or bad.
An example of bioluminescence is a firefly. The production of light in bioluminescent animals is caused by converting chemical energy to light energy (Bioluminescence, 1 of 1). In a firefly, oxygen, luciferin, luciferase (an enzyme), and ATP combine in the light organ in a chemical reaction that creates cold light (Johnson, 42). This bright, blinking light helps the male firefly attract female fireflies as a possible mate. Other examples of bioluminescent organisms are fungi, earthworms, jellyfish, fish, and other sea creatures (Berthold Technologies, 1 of 2).
Goldfish, like other cold-blooded animals, have pigment cells that are called chromatophores. Inside chromatophores are chromatosomes, which are the organelles which hold the pigment. The chromatosomes can absorb or reflect light. The color of a fish is dictated by what kinds of chromatosomes are in its cells, how many chromatosomes there are, and where in the cell the chromatosomes are located; these, in sum, control which chromatosomes absorb light and which ones reflect it, therefore affecting what color we see when we look at the fish. Chromatophores can change color in two ways: by the chromatosomes spreading apart inside the cells, making the color more apparent to the eye; or by the chromatosomes changing color, prompting a visual difference in color throughout the entire organism.
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
The synthetic A and B chains are then inserted into the bacteria’s gene for B-galactosidase, which is carried in the vectors plasmid. The vector for the production of insulin is a weakened strain of the common bacteria Escherichia coli, usually called E. coli. The recombinant plasmids are then reintroduced to the E. coli cells. As the B-galactosidase replicates in a cell undergoing mitosis the insulin gene is expressed. To yield substantial amounts of insulin millions of the bacteria possessing the recombinant plasmid are required.
Optogenetics is a new field of study that’s based upon “the integration of optics and genetics to achieve gain or loss-of-function of well-defined events within specific cells in living tissue.”(Davidson et al. 9) Scientists have now been able to genetically modify virus’ that express their genes only within specific tissues in the body. Once these viruses are inserted into the cell, specific molecules activate the transcription of cell receptors called opsins. Opsins are membrane-bound ion pumps or channels that are sensitive to specific wavelenths of light. They are typically categorized into three groups: Halorhodopsins are chloride pumps that are used to hyperpolarize, or inhibit, the cell membrane; bacteriorhodopsi...
"Fluorescence in Situ Hybridization (FISH) Fact Sheet." National Human Genome Research Institute. 15 Nov. 2007. National Institutes of Health. .
Fulda, K. G., and K. Lykens. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 25 Aug. 0005. Web. 18 Mar. 2014. .
Every year, the rate of mortality increasing because most diseases may lead to death if not treated early. One of the methods that can be used to cure some diseases is by using the treatment known as gene therapy. Based on Pruitt’s (2008) study, numbers of inherited and acquired diseases were reduced since gene therapy has the ability to provide new treatments to cure them. According to Shi and Zou (2008), gene therapy is defined as expression of protein or interrupts the synthesis of protein in cell by transferring the genetic material into a host in order to treat or prevent a disease. Besides that, Kelly (2007) stated that an “abnormal” hereditary disease-causing gene in an individual’s cells and tissues is treated and used gene therapy by to replace them with a “normal” gene. Around 1970’s, idea to use “genes” as “drugs” for human therapy was originally from United States (Giacca, 2010). Moreover, there are some objectives in using the gene therapy as a treatment. First, gene therapy is used to cure or slow the progression of disease by introducing the genetic material into target cells and next objective is to aim at the direct correction of endogenous genetic defects by delivered some additional copies of a gene (Pruitt, 2008; Giacca, 2010). Furthermore, Yadav and Tyagi (2008) found that there are two types of gene therapy which are germline gene therapy and somatic cell therapy. As stated by Shi and Zou (2008), therapy that involved modification of any cells in a patient’s body is called as somatic cell gene therapy while germ line gene therapy is therapy that involved modifying of human eggs or sperms that pass genes on to future generations. Other than that, animal tissue culture is used to test the effective...
Current research methods of transfection, delivering foreign DNA into cells, have capitalized on using non-viral vectors because of the recent advantages researchers have been able to exploit. The process of transfecting cells runs into a number of problems by way of the cell’s own defense mechanisms. Vectors must be able to not only enter the cell past the cell’s membrane but also must be able to make its way into the cell’s nucleus to access the targeted genetic material. The problem with traditional transfection methods is that they are not able to enter the cell in high efficiency without triggering an immune response. This, coupled with the inability for prolonged gene expression in vivo even once transfected, results in a very expensive and ineffective method for introducing a foreign plasmid into the cell. In the past viral vectors had been used with a degree of success in vitro, but because they lack a high degree transfection efficiency and duration of gene expression using them for transfection could not produce substantial practical applications. Another problem is that these laboratory-engineered viruses had low success rates in vivo due to activating an immune response. New techniques are being discovered by modifying non-viral vectors in novel ways, producing increasingly effective methods for transporting DNA into cells with the hope of clinical application and advancing gene therapy.
Distinct characteristics are not only an end result of the DNA sequence but also of the cell’s internal system of expression orchestrated by different proteins and RNAs present at a given time. DNA encodes for many possible characteristics, but different types of RNA aided by specialized proteins sometimes with external signals express the needed genes. Control of gene expression is of vital importance for an eukaryote’s survival such as the ability of switching genes on/off in accordance with the changes in the environment (Campbell and Reece, 2008). Of a cell’s entire genome, only 15% will be expressed, and in multicellular organisms the genes active will vary according to their specialization. (Fletcher, Ivor & Winter, 2007).
The Use of Recombinant DNA Technology Recombinant DNA technology is the technology of preparing recombinant DNA in vitro by cutting up DNA molecules and splicing together fragments from more than one organism.(1) This is the process of using recombinant DNA technology to enable the rapid production of human protein from a single gene of insulin. Firstly the single gene required must be isolated. This can be done three ways: Either by working backwards from the protein- Finding the amino acid sequence for the protein needed, the order of bases can be established using known genetic code. New DNA can be made from this sequence of bases resulting in artificial gene made from complementary DNA.