...Cloning and expression of α-Amylase gene from Bacillus subtilis in Pichia pastoris and Escherichia coli. Introduction Enzyme is a type of catalyst that present in living organisms used for many biotechnological functions in various industrial processing. It has a special characteristic that allows the chemical reaction to speed up without being altered, thus significantly improve the industrial productivity (Roy et al. 2012). Among various enzymes available in market, α-amylase has received a special attention in commercial production due to its widely used applications. α-Amylase contributed to 50% of the world enzyme production and has a great importance in many industries such as in food processing, laundry and also in pharmaceutical (Asgher et al. 2007). α-Amylase enzyme acts on α-1,4 glycosidic bonds in starch substrate backbone leading to the formation of soluble maltodextrins, glucose and maltose (Vidyalakshmi et al. 2009). This characteristic is extremely useful especially in industries that require the hydroxylation of starch such as the production of sugar syrups. The α-amylase enzyme can be obtained from various sources such as plants animals and microbes (Ahmed et al. 2011). However, the naturally occurring enzyme is still insufficient to support all the industrial production and therefore, it is crucial to find a new alternative sources, which is cost-efficient and high yield capacity to meet the supply demand (Yin et al. 2003). In industries, the microbial...
Words: 1791 - Pages: 8
...Overview: Lab 2 The purpose of Lab 2 was to examine the role and nature of restriction enzymes as well to take the first steps in producing a recombinant DNA molecule. Plasmids are circular DNA pieces found in bacterial cells. Plasmids are used in genetic engineering to expedite gene cloning and gene expression (through protein production) in bacteria. Through cloning, antibiotic resistance genes have been placed inside of plasmids and the antibiotic resistance genes function as selectable markers – genes that allow scientists to select between bacteria that do and do not have the plasmid. If a bacterium does in fact possess a plasmid that has an antibiotic resistance gene then that particular bacterium will be able to reproduce in an environment with that specific antibiotic present. Bacteria that do not possess a plasmid with an antibiotic resistance gene will not be able to grow. Therefore, antibiotics can be used to pin point bacteria that are resistant and presumably contain a plasmid with the resistance gene from those that are not resistant...
Words: 1327 - Pages: 6
...products such as hybrid planting seeds and herbicides. Mechanism of Creating GMOs. The basis of creating these products lies on the fact that most living organisms are prone to pathogenic attacks. Scientists therefore design products which are immune to the attacks and which are resistant to unfavorable climatic conditions. The procedure long involves much scientific research. There is a wide range of organisms that scientists have subjected to genetic engineering. The two main methods employed in genetic engineering are Recombinant DNA Technology as well as Reproductive Cloning. The first case involves the scientist combining genetic materials from different organisms. This is based on the fact that DNA molecules from all organisms be it plant or animals have similar chemical composition. The resulting DNA sequence from recombinant DNA technology can be planted into any organisms with no biological implications apart from the intended use. The procedure utilizes a Cloning Vector obtained from Plasmids. These provide room for insertion of foreign DNA into the chosen DNA segment. Gibson Assembly is the most popular method used in the combination of the DNA segments. The resulting DNA is not necessarily expressed...
Words: 965 - Pages: 4
...of forensics, the scientific analysis of evidence for legal investigators * Has practical applications beyond its use in forensic science * Include the use of gene cloning in the production of medical and industrial products, the development of genetically modified organisms for agriculture, and even the investigation of genealogical questions * DNA evidence was used to solve a double murder in England * Showed that two murders could have been committed by the same person * Showed the innocence of someone who confessed to one of the murders * Showed the absence of a match in 5,000 men tested when the murderer persuaded another man to donate blood in his name * Showed a match with the murder and DNA found with both victims * Biotechnology: The manipulation of organisms or their components to make useful products * Grew out of discoveries made about 60 years ago by American geneticists Joshua Lederberg and Edward Tatum * Performed a series of experiments with E.coli that demonstrated that two individual bacteria can combine genes * Genetic engineering involves manipulating genes for practical purposes * Gene cloning leads to the production of multiple identical copies of a gene-carrying piece of DNA * Recombinant DNA is formed by joining DNA sequences from two different sources * One source contains the gene that will be cloned * Another source is a gene carrier, called...
Words: 3278 - Pages: 14
...Welcome to Biol 342 Molecular Biotechnology 1 Dr. Michael D.J. Lynch Biology 342 Molecular Biotechnology 1 Instructor: Dr. Michael D.J. Lynch Room: B2 - 249D e-mail: mdjlynch@uwaterloo.ca office hours: Thursdays 1:00 – 2:30 pm If you need to speak with me outside scheduled lecture time, please contact me via email to make an appointment – that way I can be sure to set aside time for you. Prerequisites: Biol 130, 239, 240, 309. Biol 241 recommended Required textbook: Glick & Pasternak Molecular Biotechnology 4th edition, 2010. ASM Press. Available from UWaterloo Bookstore. 2 copies on reserve at Davis library. Students find this textbook very useful, and I refer to it often for lectures. A worthwhile purchase. This text is also used in Biol 432. LEARN ● ● ● ● ● ● lecture notes (slides in .pdf) Podcasts (screencasts) of lectures course info, important dates tutorial information practice exams announcements Use your Quest/UWdir ID and password Accessing the podcasts…….. Check that you are using a LEARN-approved browser! Goals for this course: ● Understand the fundamentals of molecular biotechnology, primarily in the context of the methods that are employed in the field ● Develop skills in the designing of molecular approaches to biotechnology ● Develop critical thinking skills ● Effectively communicate concepts learned Assigned readings and student notes: Readings from the text will be assigned in lecture notes on...
Words: 4935 - Pages: 20
...Purposes of Gene Cloning To study genes in the laboratory, it is necessary to have many copies on hand to use as samples for different experiments. Such experiments include Southern or Northern blots, in which genes labeled with radioactive or fluorescent chemicals are used as probes for detecting specific genes that may be present in complex mixtures of DNA. Cloned genes also make it easier to study the proteins they encode. Because the genetic code of bacteria is identical to that of eukaryotes, a cloned animal or plant gene that has been introduced into a bacterium can often direct the bacterium to produce its protein product, which can then be purified and used for biochemical experimentation. Cloned genes can also be used for DNA sequencing, which is the determination of the precise order of all the base pairs in the gene. All of these applications require many copies of the DNA molecule that is being studied. Gene cloning also enables scientists to manipulate and study genes in isolation from the organism they came from. This allows researchers to conduct many experiments that would be impossible without cloned genes. For research on humans, this is clearly a major advantage, as direct experimentation on humans has many technical, financial, and ethical limitations. Importance for Medicine and Industry The ability to clone a gene is not only valuable for conducting biological research. Many important pharmaceutical drugs and industrial enzymes are produced from cloned...
Words: 5354 - Pages: 22
...IUBMB Life, 00(00): 000–000, Month 2012 Research Communication Fluorescent Protein Engineering by In Vivo Site-directed Mutagenesis Melvys Valledor1,2, Qinghua Hu3, Paul Schiller1,2,4, and Richard S. Myers1 1 Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, USA Geriatric Research, Education, and Clinical Center, and Research Service, Veteran’s Affairs Medical Center, Miami, FL, USA 3 Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, USA 4 Department of Orthopedics, University of Miami Miller School of Medicine, Miami, FL, USA 2 Summary In vivo site-directed mutagenesis by single-stranded deoxyribonucleic acid recombineering is a facile method to change the color of fluorescent proteins (FPs) without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the Escherichia coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the FP. Eight different FPs (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza, and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo sitedirected mutagenesis more efficient, the recombineering method was optimized using the...
Words: 4048 - Pages: 17
...Annotated Bibliography Barnes, Deborah E, Leland H Johnston, K-L Kodama, et al. 1990. Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences 87: 6679-6683. CDNA clones encoding for DNA ligase I were isolated. In one method, human cDNA was screened using oligonucleotides from partial amino acid sequence of purified bovine DNA ligase I. and the second approach the human cDNA library was screened for functional expression of a polypeptide able to complement of a DNA ligase mutant of Saccharomyces cerevisiae. The sequence found encodes a 102 kDa protein indistinguishable from DNA ligase I. It was also found that the amino acid sequence of the human ligase I is 40%...
Words: 1464 - Pages: 6
...named Dr. John Conrad Otto wrote a review about a hemorrhagic disposition that occurred in families affecting males. Following that, Nasse gave the first review in 1820 and Wright demonstrated evidence of laboratory defects in blood clotting in Aaron Chan Biology 330 06261560 1893. Our forces strengthened exponentially in 1937 after Patek and Taylor found the role of the soon to be named factor VIII describing its action in hemostasis. They characterized and named it an “antihemophilic globulin substance”. Not long after, the protein was purified and the gene was open to study for many scientists. The root of this evil The mastermind behind Hemophilia A is the gene coagulant factor VIII. The factor VIII gene sits on the long arm of chromosome 10 at location 28, more specifically, from base pairs 154,064,062 to 154,255,350. Its protein sequence is 2351 amino acids long transcribed from a 191,288 mRNA. This gene produces two alternatively spliced transcripts. Transcript variant 1 consists of 26 exons that encodes a large glycoprotein called isoform a, which circulates in the plasma and undergoes multiple...
Words: 2699 - Pages: 11
...introduced into an organism. (Theresa Phillips, 2008)Today, we can incorporate new genes from one species into a completely unrelated species through genetic engineering, optimizing agricultural performance or facilitating the production of valuable pharmaceutical substances. Some examples of genetically modified organisms are plants, animals and bacteria. Genetically modified organisms are all organism that has had a gene from one organism implanted into another organism in order to improve or change the genetic makeup of that organism. It can also be referred to as transgenic, due to the process being the transfer of genes from one organism to another. Other names also referred to are biotechnology, gene splicing, genetic engineering, or recombinant DNA technology, all of which meant the same thing as genetically modified crops. [ (Enquiries, 2007) ]Transferring genes from one organism to another, to enhance or improve that organism. Genetic Modification is done in a laboratory by extracting the desired genes from the organism and then implanting them into the other organism. When food is genetically modified the scientists use a process to change the crop’s genome makeup. The desired genes from the crop that has the desired information is taken and inserted into the other crop’s genetic makeup in order to make a better genetic makeup of a new crop. What happens when genetically modified crops are being produced is they are growing faster and mature faster and tolerate aluminum...
Words: 1184 - Pages: 5
...(Charles Taiwo) (Not Complete) 8. Moral and ethical implications (Layli Stroia) ……………………………………………………..… 30 Outline A brief description of the technology and an explanation of the associated science Definition: What is Genetic Engineering? Genetic engineering (GE) is the process of manipulation of an organism genome to create a new DNA. The new DNA might be implanted in a totally different DNA species. It is widely used to create hybrids (some species are not able to naturally breed), correct genetic flows in any type of being. It is applied in fields such as agriculture, industrial, medicine and pharmaceutical. (Aldridge, 1996). As Primrose explained, gene manipulation involves the creation and cloning of artificially created DNA (recombinant DNA) that provides with DNA sequences not found in nature. This created DNA is introduced in a host living cell. “…genetic engineering is a 'cut, paste, and copy' operation. The gene...
Words: 8761 - Pages: 36
...i50 Ann Rheum Dis 2000;59(suppl I):i50–i53 The pre-ligand binding assembly domain: a potential target of inhibition of tumour necrosis factor receptor function Francis Ka-Ming Chan Building 10, Room 11N311, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892–1892, USA Correspondence to: Dr Chan (fchan@nih.gov) Abstract Signalling by the tumour necrosis factor receptors (TNFR) is thought to be mediated by the binding of the trimeric ligand TNF to three monomeric subunits of the receptor. This ligand induced trimerisation model of TNFR signalling is mainly supported by crystallographic data of the p60 TNFR-1 and TNF complex in which the trimeric ligand interdigitates between the individual receptor chains and prevents the receptor subunits from interacting with each other. Recently, a domain NH2terminal to the ligand binding domain in the extracellular region of p60 TNFR-1, p80 TNFR-2 and Fas was identified that mediates receptor self association before ligand binding. This pre-ligand binding assembly domain or PLAD is critical for assembly of functional receptor complexes on the cell surface and may provide a potential target in the design of future novel therapeutics against diseases mediated by members of the TNFR family of receptors. (Ann Rheum Dis 2000;59(suppl I):i50–i53) Tumour necrosis factor (TNF) is a cytokine that plays an important regulatory part in both healthy and diseased...
Words: 3536 - Pages: 15
...INTRODUCTION Stem cells have the unique ability to differentiate into any cell in the body, which has made them a central focus for regenerative cell therapy for a vast number of human diseases ranging from cardiovascular to neurodegenerative disorders. A major example for the use of stem cell therapy is Parkinson’s disease (PD). Pharmacological treatments for PD have been developed; however, neuronal degeneration prevents these drugs from working for extended periods of time. Stem cell therapy does have the potential for providing a permanent treatment for this disorder. In order for stem cell therapy to be effective and safe, the stem cells must be at a certain differentiation state, which is currently defined by a combination of surface markers, prior to implantation. One major obstacle preventing translation of stem cell therapy into a clinical setting is the inability to determine and isolate stem cells at particular differentiation states. In the case of PD, the optimal stem cell for transplantation would be a dopaminergic neuroblast which is differentiated enough to allow neural integration thereby reversing disease progression. Additionally, optimal differentiation will prevent tumor formation resulting from an overly immature stem cell (Wernig et al, 2008). Thus, there is an urgent need to identify and track the differentiation state of cells in order to realize therapeutic potential. Our long term goal is to identify and purify appropriately differentiated stem cells...
Words: 4754 - Pages: 20
...Genetically Modified Organisms The world’s population is growing at a rapid pace. Advancements make possible for people to live longer lives as well as aiding infertile couples with creating children that otherwise would not have been born. Perhaps the biggest problem that humans face in their lives is how to quickly produce enough food to support a growing population. “The United Nations Food and Agriculture Organization estimate that the world will have to grow 70 percent more food by 2050 just to keep up with population growth. Climate change will make much of the world's land more difficult to farm” (Freedman, 2013). In addition to feeding the American people, farmers in the United States are also supplying food to the rest of the world. While Americans tend to over consume and waste much of the blessing that they have been given, there are thousands of people all over the world struggling to get enough food to keep themselves alive. While America has plenty of struggling citizens who find themselves homeless and in need of assistance in order to feed themselves, other countries have their population declining because the citizens in those countries have nowhere to turn for the help they need. “The United States is the world’s largest supplier of food aid, reaching fifty-five million people in forty-six countries last year” (Baragona, 2011). Genetically modified foods are foods derived from organisms whose genetic material has been modified in a way...
Words: 1594 - Pages: 7
...Te c h n i c a l M a n u a l Dual-Luciferase® Reporter Assay System INSTRUCTIONS FOR USE OF PRODUCTS E1910 AND E1960. INTEGRATED S OLUTI ON S u se m e w i th G LO M A X ® INST RUMENTS world-class INTEGRATED S O LUTIO N S SERV ICE & SUPPORT PRINTED IN USA. Revised 6/11 Part# TM040 Dual-Luciferase® Reporter Assay System All technical literature is available on the Internet at: www.promega.com/tbs/ Please visit the web site to verify that you are using the most current version of this Technical Manual. Please contact Promega Technical Services if you have questions on use of this system. E-mail: techserv@promega.com. 1. Description..........................................................................................................2 A. Dual-Luciferase® Reporter Assay Chemistry...................................................3 B. Format of the Dual-Luciferase® Reporter Assay .............................................5 C. Passive Lysis Buffer .............................................................................................6 2. Product Components and Storage Conditions ............................................8 3. The pGL4 Luciferase Reporter Vectors .........................................................9 A. Description of pGL4 Vectors ..............................................................................9 B. Important Considerations for Co-Transfection Experiments ........................9 4. Instrument Considerations...
Words: 9055 - Pages: 37
