what is the new and best techniques to knockout gene from bacteria?
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I can give you the reference for the procedure that is easy and very effective. Use this article to get an idea:\nhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10829079&query_hl=1&itool=pubmed_docsum
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Do not go there.\nLeave the microorganisms in peace.\n\nIf you have not got the entire DNA structure mapped and well understood, you are asking for nothing but trouble.  You might end up with a pathogen which targets human nerve cells or muscle nerve discharges.  Write a sci-fi book about your idea and leave the experiments until you finish a college degree and have a much better idea of just how complicated the Big Picture really is.
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finding out ways to produce enzymes quickly and efficiently
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Genetic engineering, genetic modification (GM) and gene splicing are terms for the process of manipulating genes, usually outside the organism's normal reproductive process.\n\nIt involves the isolation, manipulation and reintroduction of DNA into cells or model organisms, usually to express a protein. The aim is to introduce new characteristics or attributes physiologically or physically, such as making a crop resistant to a herbicide, introducing a novel trait, or producing a new protein or enzyme. Examples can include the production of human insulin through the use of modified bacteria, the production of erythropoietin in Chinese Hamster Ovary cells, and the production of new types of experimental mice such as the OncoMouse (cancer mouse) for research, through genetic redesign.\n\nSince a protein is specified by a segment of DNA called a gene, future versions of that protein can be modified by changing the gene's underlying DNA. One way to do this is to isolate the piece of DNA containing the gene, precisely cut the gene out, and then reintroduce (splice) the gene into a different DNA segment. Daniel Nathans and Hamilton Smith received the 1978 Nobel Prize in physiology or medicine for their isolation of restriction endonucleases, which are able to cut DNA at specific sites. Together with ligase, which can join fragments of DNA together, restriction enzymes formed the initial basis of recombinant DNA technology.\n\nGenetic Engineering and Research\n\nAlthough there has been a tremendous revolution in the biological sciences in the past twenty years, there is still a great deal that remains to be discovered. The completion of the sequencing of the human genome, as well as the genomes of most agriculturally and scientifically important plants and animals, has increased the possibilities of genetic research immeasurably. Expedient and inexpensive access to comprehensive genetic data has become a reality, with billions of sequenced nucleotides already online and annotated. Now that the rapid sequencing of arbitrarily large genomes has become a simple, if not trivial affair, a much greater challenge will be elucidating function of the extraordinarily complex web of interacting proteins, dubbed the proteome, that constitutes and powers all living things. Genetic engineering has become the gold standard in protein research, and major research progress has been made using a wide variety of techniques, including:\n\nLoss of function, such as in a knockout experiment, in which an organism is engineered to lack the activity of one or more genes. This allows the experimenter to analyze the defects caused by this mutation, and can be considerably useful in unearthing the function of a gene. It is used especially frequently in developmental biology. A knockout experiment involves the creation and manipulation of a DNA construct in vitro, which, in a simple knockout, consists of a copy of the desired gene which has been slightly altered such as to cripple its function. The construct is then taken up by embryonic stem cells, where the engineered copy of the gene replaces the organism's own gene. These stem cells are injected into blastocysts, which are implanted into surrogate mothers. Another method, useful in organisms such as Drosophila (fruit fly), is to induce mutations in a large population and then screen the progeny for the desired mutation. A similar process can be used in both plants and prokaryotes. \nGain of function experiments, the logical counterpart of knockouts. These are sometimes performed in conjunction with knockout experiments to more finely establish the function of the desired gene. The process is much the same as that in knockout engineering, except that the construct is designed to increase the function of the gene, usually by providing extra copies of the gene or attracting more frequent transcription. \n'Tracking' experiments, which seek to gain information about the localization and interact
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In order to perform in vitro analyses, a protein must be purified away from other cellular components. This process usually begins with cell lysis, in which a cell's membrane is disrupted and its internal contents released into a solution known as a crude lysate. The resulting mixture can be purified using ultracentrifugation, which fractionates the various cellular components into fractions containing soluble proteins; membrane lipids and proteins; cellular organelles, and nucleic acids. Precipitation by a method known as salting out can concentrate the proteins from this lysate. Various types of chromatography are then used to isolate the protein or proteins of interest based on properties such as molecular weight, net charge and binding affinity. The level of purification can be monitored using gel electrophoresis if the desired protein's molecular weight is known, by spectroscopy if the protein has distinguishable spectroscopic features, or by enzyme assays if the protein has enzymatic activity.\n\nFor natural proteins, a series of purification steps may be necessary to obtain protein sufficiently pure for laboratory applications. To simplify this process, genetic engineering is often used to add chemical features to proteins that make them easier to purify without affecting their structure or activity. Here, a "tag" consisting of a specific amino acid sequence, often a series of histidine residues (a "His-tag"), is attached to one terminus of the protein. As a result, when the lysate is passed over a chromatography column containing nickel, the histidine residues ligate the nickel and attach to the column while the untagged components of the lysate pass unimpeded.\n\n\n[edit] Cellular localization\n \nProteins in different cellular compartments and structures tagged with green fluorescent protein.The study of proteins in vivo is often concerned with the synthesis and localization of the protein within the cell. Although many intracellular proteins are synthesized in the cytoplasm and membrane-bound or secreted proteins in the endoplasmic reticulum, the specifics of how proteins are targeted to specific organelles or cellular structures is often unclear. A useful technique for assessing cellular localization uses genetic engineering to express in a cell a fusion protein or chimera consisting of the natural protein of interest linked to a "reporter" such as green fluorescent protein (GFP). The fused protein's position within the cell can be cleanly and efficiently visualized using microscopy, as shown in the figure opposite.\n\nThrough another genetic engineering application known as site-directed mutagenesis, researchers can alter the protein sequence and hence its structure, cellular localization, and susceptibility to regulation, which can be followed in vivo by GFP tagging or in vitro by enzyme kinetics and binding studies.\n\n\n[edit] Proteomics and bioinformatics\nMain articles: Proteomics and Bioinformatics\nThe total complement of proteins present in a cell or cell type is known as its proteome, and the study of such large-scale data sets defines the field of proteomics, named by analogy to the related field of genomics. Key experimental techniques in proteomics include protein microarrays, which allow the detection of the relative levels of a large number of proteins present in a cell, and two-hybrid screening, which allows the systematic exploration of protein-protein interactions. The total complement of biologically possible such interactions is known as the interactome. A systematic attempt to determine the structures of proteins representing every possible fold is known as structural genomics.\n\nThe large amount of genomic and proteomic data available for a variety of organisms, including the human genome, allows researchers to efficiently identify homologous proteins in distantly related organisms by sequence alignment. Sequence profiling tools can perform more speci
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You need a DNA plasmid with a few things.  First a origin of replication, so the plasmid can be replicated.  Second, an antibiotic resistance marker to issolate only bacteria containing the plasmid.  Third you need a bacterial promoter with a consensus kozak sequence followed by your gene of interest in frame.  Usually this gene is followed by or preceded by glutathione-S-transferase (GST) to facilitate purification of the protein).  Also usually the promoter contains a repressor region LacI, which means the gene is turned off normally, unless you add lactose or IPTG (a synthetic lactose), which turns on gene transcription and translation.  This is routinely done in labs nowadays.
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Ok, what I normally do is to digest the plasmid with RE, digesting as much as possible for long time (even over-night). Then, without any purification I treat my digestion with CAP (calf alkaline phosphatase) for one hour at 37C. This phosphatase is generally versatile and works in any buffer, but you might want to check with the manufacturer's instructions for buffer compatibility.\nAfter de-phosphorylation, I make a 1% agarose in TAE with a comb with big wells. If you do not have one, you can tape 2 "teeth" together to make a bigger well. Make sure you add Ethidium Bromide to the gel. I run all my digest on the gel for 1 hour or so at medium volt (so that the gel does not become hot) and then cut the band  and purify with a gel purification kit (Qiagen has a very good one). The kit has columns that bind DNA from which your plasmid can be eluted - If you don't have a lot, elute with a small volume of H2O (DNAse-free!).\n\nThe advantage of running the gel and purifying the band is that you get rid of all un-digested plasmid which would be useless in the ligation but that would definitely be transformed into your E.coli (quite well, as well!).\nAlso, if you are cutting and purifying an insert from a different plasmid, you can run vector and insert side by side to see in which proportion to organise your ligation. If you don't have a lot material, try "Rapid Ligation Kit" by Roche. It works at room temperature within 1 hour.\n\nHope this helps!\nGood luck
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You can find practical information from Qiagen website if you use their kits which are quite popular these days.  \n\nIt depends on which methods you use for isolation.  The alkali lysis (see link below) was commonly used to rupture bacteria.  Plasmid DNA (smaller in size and more soluble) is separated from baterial genomic DNA (larger and less soluble).  The plasmid DNA is then separate from proteins by phenol extraction where the proteins are removed to the phenol phase while plasmid DNA stays in the aquous phase.  The plasmid DNA is then precipated with ethanol to complete the purification.\n\nQiagen's kit and others overcome the use of phenol (which is toxic and smelly) by using a solid phase that bind DNA to seperate the contaminating proteins.  The DNA is later eluted from the solid phase to complete the purification.\n\nHere are additional links:\nhttp://biotech.biology.arizona.edu/labs/DNA_isolation_plasmid.html\nThe concept of alkali lysis method of plasmid DNA isolation is shown here:\nhttp://a32.lehman.cuny.edu/molbio_course/plasmid_protocol.htm
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1. Isolate the genome of organism on which the desired gene sits.\n2. Cut it with restriction enzyme with the desired recognition site.\n3. Perform polymerase chain reaction procedure with oligomer primers which have the suspected complimentary sequence with your gene\n4. Perform agarose gel electophoresis to isolate the multiplied sequences\n5. Separate the duplex structure of primer:DNA in alkaline medium.\n6. Perform Southern Blot Transfer (could be found in any molecular biology laboratory manual)\n7. Transform the target organism with the resulting gene fragments from the southern blot. (transformation procedure vary with the varying target organism)
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Medical Therapy.\n1- Cells can be engineered to produce a pathogen's surface protein, which can be used as a subunit vaccine. \n\n2-Synthetic genes linked to the β-galactosidase gene in a plasmid vector were inserted into E. coli, allowing E. coli to produce and secrete the two polypeptides used to make human insulin. \n\n3-DNA probes can be used as rapid diagnostics to identify a pathogen quickly in body tissue or food. \n\n4-Genetic screening uses Southern blotting to look for mutations responsible for inherited disease in humans.\n\nAny Help?
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http://www.microbesonline.org/operons/gnc205922.html
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i dont think you'll be able to access this without logging in to my school, so i'll paste it below in case. try publications at your school as well, pubmed is always good, as ia google.\n\nTransgenics\nThe production of transgenic mice is a gain-of-function approach to manipulation of the mouse genome (Clarke, 2002; Nagy et al., 2003; Hedrich, 2004). Transgenic mice can be designed for numerous purposes including: (1) identification and characterization of specific regulatory sequences; (2) ubiquitous or tissue-specific overexpression of a transgene; and (3) cell lineage ablation achieved by coupling a tissue-specific promoter to a sequence encoding a toxin which kills cells that express it. Transgenic mice are typically generated by microinjection of the transgene construct into the male pronucleus of fertilized oocytes, which are then reimplanted into the oviducts of foster females. Expression of the transgene is dependent on its site of integration, with individual insertion sites typically containing multiple copies of the transgene. It is important to keep in mind that transgene copy number and expression levels of the transgene do not necessarily correlate. When chromosomal integration occurs in the fertilized oocyte, the developing animal will be fully transgenic, since all nucleated cells in the animal will contain the transgene; however, if integration occurs later, at a postzygotic stage, the animal will be a mosaic, with some cells containing the transgene and others lacking it. Founders are identified through analysis of tail biopsies, and copy number and transgene integrity are then evaluated. To establish a transgenic line, a founder is bred to mice of a defined genetic background and offspring are characterized for expression of the transgene. Generally at least two founders should be maintained for experiments. Due to the nature of random integration, no two transgenic lines will be identical. Details on the identification and analysis of founders and the establishment of transgenic lines can be found in UNIT 23.9.\n\nTransgenics carrying large segments flanking a gene of interest isolated as bacterial artificial chromosomes (BACs) have been used to minimize the abovementioned variation due to the effects of different integration sites (UNIT 23.1). The versatility of this strategy has been significantly increased by the development of homologous recombination–based strategies to mutagenize and manipulate the BAC constructs (UNITS 1.16 & 23.11) and the use of regulated transgenics (e.g., with tetracycline; UNIT 23.12). In the GENSAT project (http://www.ncbi.nlm.nih.gov/projects/gensat; Gong et al., 2003), for example, an atlas of gene expression in the mouse central nervous system is based on large numbers of BAC transgenics in which the protein-coding sequences of genes expressed in the brain are replaced by an EGFP reporter.
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DNA from organisms displaying both normal and abnormal phenotypes. Mutation studies can be done, comparing the sequences of normal and abnormal, isolating the gene in question and identifying the protein it encodes. "Knock-out" experiments can also be done, wherein the effect of deleted genes will be observed. Other things to consider would be promoters, suppressors and regulators of the gene.
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If you didn't have to physically show it, the best way would be introducing a virus into the bacteria and force it to transplant some of its own genetic material into the bacteria. (Cancer Cells)
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FIrst, using redundant coding, you find the gene by using redundant PCR primers which have the codons coding for the amino acid sequence.  Actually, nowadays, because the human genome is known, you can input the amino acid sequence to the NIH site (and others) and the codon sequence (and gene) which codes for that protein will be determined.  You use specific primers to pull out copies of the gene, and insert them into bacterial vectors- insert coding vectors with the gene into bacteria, and allow the bacteria to produce it in quantity-  Purify it through HPLC, and there you go.
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Protoplast fusion: a tool for intergeneric gene transfer in bacteria.\n\nGokhale DV, Puntambekar US, Deobagkar DN.\n\nDepartment of Biochemical Sciences, National Chemical Laboratory, Pune, India.\n\nProtoplasts can be isolated from bacterial cells by digestion of the cell wall with the help of lysozyme in presence of osmotic stabilizers. Fusion of protoplasts can be induced by chemical fusogens like polyethylene glycol. The electrofusion technique has been reported in bacteria in which the fusion frequency is much higher than that obtained by PEG induced protoplast fusion. This technology allows recombination to take place not only between related species but also between unrelated genera and is of great potential in the breeding and improvement of industrial strains.
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cloning\nstem cells\ngenetic manipulation