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The antibiotic 'kanamycin' in the growing of culture E. coli

The antibiotic 'kanamycin' in the growing of culture E. coli



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I'm a first year student and I'm not sure if I understand correctly the role played by the antibiotic 'kanamycin' in the growing of culture E. coli (Rosetta).

Can anybody explain me why we use 'kanamycin' antibiotic and kanamycin resistance gene during that process?


Absolutely, Positive Selection

You are looking for the bacteria which have the gene (probably a plasmid) you (or someone else for you) put into the bacteria. That gene lets them live in the presence of kanamycin, and any bacteria that don't have it, or that try to get rid of it, will die. Thus you can select for the bacteria you want by killing everything else off (hopefully your sample is not contaminated with other bacteria also resistant to kanamycin).

I totally thought there would be a good wiki article on positive selection but there isn't. The reason it's positive selection is that you are selecting for bacteria that have something; instead of negative selection where you would be killing them for having something.

You can find plenty more if you search "positive selection" or more generally "artificial selection."


According to this data sheet, the genotype of the strain RosettaTM(DE3)pLysS is F- ompT hsdS (r- m-) gal dcm (DE3) pLysSRARE (CamR).

This strain carries certain tRNA genes on the same plasmid as the LysS gene, and these tRNAs boost expression from genes containing rare (in E. coli) codons. The plasmid encodes chloramphenicol resistance (CamR).

If you have a protocol that suggests growing your strain under kanamycin selection then presumably you have an additional plasmid present which carries your gene of interest. Then, as the accepted answer says, you are selecting positively for the correct strain.

I don't know how stable the pLysSRARE plasmid is, but I would have expected that you should also maintain selection for chloramphenicol resistance.


Testing Antibiotic On Bacteria

Introduction Problem To determine the effect of ten different antibiotics on two different types of bacteria. I will test six antibiotics on Escherichia coli, and six antibiotics on Bacillus subtilis. On Escherichia coli I will test tetracycline, chloramphenicol, furadantin, nalidixic acid, triple sulfa, and kanamycin. On Bacillus subtilis I will test streptomycin, erythromycin, novobiocin, tetracycline, chloramphenicol, and penicillin. As a side observation, I would also like to see if Bacillus subtilis shows resistance to penicillin. The use of penicillin is being reduced because of the resistance many types of bacteria are developing against it.

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Hypothesis My hypothesis is that penicillin will inhibit the most growth against Bacillus subtilis, and that tetracycline will stop Escherichia coli more effectively than the others. Rationale I feel that this experiment is valid because it shows how different antibiotics react to different types of bacteria. It also points out the fact that not all antibiotics work the same or that they work at all on all types of bacteria. Materials 1 bottle of tryptic soy agar 1 culture of Escherichia coli 1 culture of Bacillus subtilis 4 sterile petri dishes 1 pack of test discs 2 sterile pipettes 2 tubes of physiological saline 1 forceps 1 wax pencil 2 sterile swabs Method 1. Loosen the cap of the tryptic soy agar to allow it to vent.

Place the bottle in a water bath at 100 degrees Centigrade so that the water level reached the level of the medium. The agar will melt in about 20 minutes. 2. Gradually cool the medium to about 45 degrees Centigrade by letting the water bath cool.

The Review on Antibiotic

When citing these abstracts please use the following reference: Author(s) of abstract. Title of abstract [abstract]. Int J Infect Dis 201014S1: Abstract number. Please note that the official publication of the International Journal of Infectious Diseases 2010, Volume 14, Supplement 1 is available electronically on http://www.ijidonline.com/ Final Abstract Number: 23.001 Session: Antibiotic .

Then let the medium sit for 10 minutes. 3. Pour two plates for each species of bacteria. The agar should be approximately 5 mm deep. Cover each petr dish immediately after poring to prevent contamination. 4.

After pouring the plates, allow the agar a few minutes to solidify. You can check this by tilting the plate. If the agar flows to one side, more cooling time is needed. 5. Add a tube of physiological saline to each of the bacteria culture tubes.

Replace the top on the saline tube. Swirl the culture tube to disperse the bacteria in the saline. 6. Using a sterile pipette, transfer 0.

25 ml of saline from one bacterial tube onto each of the two plates. With a sterile swab, spread the saline in a thin layer over the entire agar surface. Repeat with the other bacterial tube and plates, using a pipette and swab. Allow 5 to 10 minutes for the liquid to be absorbed into the agar. 7. Use the wax pencil to label each dish so that you will know what species it contains.

8. When the agar has solidified, the antibiotic test discs can be added. Using a forceps flamed between each operation place one of each type of test disc on the surface of each agar plate. The discs should be placed about 3 cm apart.

The antibiotic on each disc should be identified by the following abbreviations: S = Streptomycin, E = Erythromycin, N = Novobiocin, T = Tetracycline, C = Chloramphenicol, P = Penicillin, F = Furadantin, Na = Nalidixic acid, Ts = Triple sulfa, K = Kanamycin 9. After the cultures have been in the incubator for 48 hours. Remove them and check around each disc for an area where the bacterium could not grow. Measure each grow area in millimeters and record them on a table (see table 1).

The Essay on Agar Plate and Cobra Vine Plant

I. Product Background A. Introduction In this highly luxurious extravagant world, food and other beverages has always been one of the things which maintain the mainstream of life. Aside from being one of the basics for survival, gatherings are also made perfect by food preparation and stress is now often associated with food. This activities show that food intake nowadays is far different from the .

Literature Search Escherichia is a genus of rod-shaped bacteria, in the family Enterobacteriaceae. Named for Theodor Escher ich (1857-1911), a German bacteriologist, the only species, Escherichia coli, is found in large numbers as a normal inhabitant of the large intestine of warm-blooded animals.

Whenever they leave their usual habitat, these organisms can cause urinary-tract infections, peritonitis, endocarditis, and other diseases. Some strains cause severe gastroenteritis. E. coli has been widely used as a model in molecular biology studies. Certain rare strains of the bacteria Escherichia coli can cause food poisoning in young children, the elderly, and people with impaired immune systems.

E. coli 0157: H 7, normally found in the intestines and fecal matter of humans and animals, can survive in meat if the meat is not cooked past 155 degrees F. A 1993 U. S. outbreak of this type of food poisoning, which affected over 450 people, was attributed to contaminated hamburgers that were cooked rare. In 1928, Alexander Fleming noticed that growth of the pus-producing bacteria, Staphylococcus aureus, had stopped around an area in which an airborne mold contaminant, Penicillium no tatum, had begun to grow.

Fleming determined that a chemical substance had diffused from the mold, and named it penicillin. The small, impure amounts he initially extracted lacked potency, yielding disappointing results in early attempts to treat human infections with penicillin. In 1939, Ernst Boris Chain, Howard Walter Florey, and Edward Penney Abraham at Oxford University began to study the possibility that purer, more stable penicillin preparations might be effective. In 1941 the partially purified material was administered to a policeman suffering from osteomyelitis. Dramatic improvement ensued, but the supply was exhausted before a cure could be effected, and the patient died. Nonetheless, the matter obviously deserved further exploration, and the outbreak of World War II added an element of urgency.

The war, however, interfered with attempts to make penicillin in England on a large scale. Chain therefore hand-carried a vial of the mold to the United States, where the necessary industrial capacity was available for mass production. Application of beer-brewing technology yielded large amounts of mold liquor, from which partially purified penicillin could be laboriously recovered for clinical use. The first batches became available for military use in 1943.

The Term Paper on Side Effects Antibiotics Bacteria Antibiotic

Antibiotics have played a major role in our society thanks to Sir Alexander Fleming's careful observations in 1928. Without it, many lives would be in danger due to infectious diseases. Antibiotics are chemical substances produced by various species of microorganisms and other living systems that are capable in small concentrations of inhibiting the growth of or killing bacteria and other .

The material was so scarce that patients’ urine was collected and the excreted penicillin recrystallized to be used again. Meanwhile, Rene Dubois at the Rockefeller Institute had been pursuing Pasteur’s original train of thought. Observing that microbial populations in soil held one another in check, he isolated and purified an antibiotic from a soil bacterium in 1939. It and similar substances subsequently isolated were effective when applied to superficial wounds but proved too toxic for systemic administration.

By 1944, Selman Abraham Waksman and his colleagues had isolated streptomycin from a soil microbe and proved its effectiveness against the tubercle bacillus. Between 1945 and 1960, a systematic search was carried on for antibiotics derived from bacteria and molds found all over the world. Many hundreds of antibiotics were discovered, and dozens were screened for antibiotic activity and toxicity. Many were eventually marketed, and prescription use accounted for hundreds of tons annually. In 1957, penicillin was synthesized in the laboratory.

Complete synthesis of penicillins proved prohibitively expensive, but harvesting the basic molecules of penicillin from Penicillium molds and then tacking on diverse molecules proved feasible and led to a large number of tailor-made penicillin variants. The 1960 s witnessed a veritable explosion of so-called semisynthetic (part mold-made, part synthetic) penicillins, each designed to deal with the increasing problem of penicillin-resistant bacteria, to achieve better absorption and higher concentrations in the body, or to broaden the penicillins’ effective antimicrobial spectrum. Conclusion My conclusion is that my hypothesis was only partially correct. In my hypothesis I stated that penicillin would inhibit bacterial growth best in Bacillus subtilis. This section of the hypothesis was correct. However, I also stated that tetracycline would inhibit bacterial growth best in Escherichia coli.

This section was incorrect. The antibiotic that worked the best on Escherichia coli was nalidixic acid. Results In my experiment I received the following results: Bacillus subtilis (gram positive) Growth Area: Tetracycline: 12 mm Novobiocin: 9 mm Chloramphenicol: 18 mm Strepomycin: 11 mm Penicillin: 21 mm Erythromycin: 15 mm Escherichia coli (gram negative) Growth Area: Chloramphenicol: 17 mm Kanamycin: 11 mm Triple sulfa: 9 mm Nalidixic acid: 17 mm Furadantin: 14 mm Table 1 Escherichia coli Bacillus subtilis Antibiotic Width in mm Antibiotic Width in mm Chloramphenicol 12 Tetracycline 12 Kanamycin 11 Novobiocin 9 Triple sulfa 9 Chloramphenicol 18 Nalidixic acid 17 Strepomycin 11 Furadantin 14 Penicillin 21 Tetracycline 10 Erythromycin 15 Bibliography United States Pharmacopeia. Complete Drug Reference. Consumer Reports Books, Yonkers, New York. Copyright 1994.

The Essay on India, China Economic Growth

India with about 1. 2 million populations and china with about 1. 3 billon population are two big demographic and emerging countries in the world . Over a past few decade India’s combination into the economic has been accompanied by remarkable economic growth (World Bank 2011¬). India is having the 3th position on the economy in purchasing power parity (PPP) terms (The Economic Times, 2012). .

pp 45, 970, 1489, 1686 Facts and Comparisons. Drug Facts and Comparisons. Facts and Comparisons Inc, St. Louis.

Copyright 1991. pp 120, 1374, 1598, 1895 USP DI. Health Care Provider. United States Pharmacopeia l Convention. Rockville, Maryland. Copyright 1985.

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Side Effects Antibiotics Bacteria Antibiotic

. bacteria was a species of Penicillium, he named the germ-killing substance penicillin. The first use of an antibiotic, . to sunlight, these drugs are given with caution. Tetracyclines. Tetracyclines are effective against pneumonia, typhus, and other .

Bacterial Dna Antibiotics Bacteria Antibiotic

. antibiotics. There are many incidences of multi-drug resistance in bacteria, but the most notorious was the triumph of Staph. Even though Penicillin . a bacterium known to be resistant to ampicillin, carbenicillin, tetracycline, streptomycin, .

Penicillin Deadly Bacteria

. Problems One of the problems with penicillin and other newer antibiotics is that bacteria are becoming resistant to them because . been dying from deadly bacteria. Until penicillin was discovered there was no known form of antibiotic to cure infectious .

The Effects Of Antibiotics On Bacterial Growth

. antibiotic in the bacteria culture: Tetracycline Chloramphenicol Kanamycin Neomycin Penicillin Streptomycin Erythromycin B. Cerus 5. 5 9 56. 6 1 7 13 E. Coli . surface. Escherichia coli is a disease causing gram-negative bacillus. These bacteria are .


Introduction

Escherichia coli is a common inhabitant of the human and animal gut, but can also be found in water, soil and vegetation. It is the leading pathogen causing urinary tract infections 1 , 2 , 3 and is among the most common pathogens causing blood stream infections 4 , wounds, otitis media and other complications in humans 5 , 6 . E. coli is also the most common cause of food and water-borne human diarrhea worldwide and in developing countries, causing many deaths in children under the age of five years 7 .

Antimicrobial resistance in E. coli has been reported worldwide and increasing rates of resistance among E. coli is a growing concern in both developed and developing countries 8 , 9 . A rise in bacterial resistance to antibiotics complicates treatment of infections. In general, up to 95 % of cases with severe symptoms are treated without bacteriological investigation 10 . Occurrence and susceptibility profiles of E. coli show substantial geographic variations as well as significant differences in various populations and environments 11 . In Ethiopia, a number of studies have been done on the prevalence and antimicrobial resistance patterns of E. coli from various clinical sources 5 , 12 , 13 . The aim of this study was to determine antimicrobial susceptibility of E. coli from clinical sources at Dessie Regional Health Research Laboratory.


MATLAB code used for the analysis of the experiments is available from the corresponding author upon request.

Blankenship, R. E. et al. Comparing photosynthetic and photovoltaic efficiencies and recognizing the potential for improvement. Science 332, 805–809 (2011).

Scheffe, J. R. & Steinfeld, A. Oxygen exchange materials for solar thermochemical splitting of H2O and CO2: a review. Mater. Today 17, 341–348 (2014).

Snoeckx, R. & Bogaerts, A. Plasma technology—a novel solution for CO2 conversion? Chem. Soc. Rev. 46, 5805–5863 (2017).

Zhang, Q., Kang, J. & Wang, Y. Development of novel catalysts for Fischer–Tropsch synthesis: tuning the product selectivity. ChemCatChem 2, 1030–1058 (2010).

Jouny, M., Luc, W. & Jiao, F. General techno-economic analysis of CO2 electrolysis systems. Ind. Eng. Chem. Res. 57, 2165–2177 (2018).

Yishai, O., Lindner, S. N., Gonzalez de la Cruz, J., Tenenboim, H. & Bar-Even, A. The formate bio-economy. Curr. Opin. Chem. Biol. 35, 1–9 (2016).

Szima, S. & Cormos, C. C. Improving methanol synthesis from carbon-free H2 and captured CO2: a techno-economic and environmental evaluation. J. CO2 Util. 24, 555–563 (2018).

Bertsch, J. & Muller, V. Bioenergetic constraints for conversion of syngas to biofuels in acetogenic bacteria. Biotechnol. Biofuels 8, 210 (2015).

Bennett, R. K., Steinberg, L. M., Chen, W. & Papoutsakis, E. T. Engineering the bioconversion of methane and methanol to fuels and chemicals in native and synthetic methylotrophs. Curr. Opin. Biotechnol. 50, 81–93 (2017).

Muller, J. E. et al. Engineering Escherichia coli for methanol conversion. Metab. Eng. 28, 190–201 (2015).

Dai, Z. et al. Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae. Bioresour. Technol. 245, 1407–1412 (2017).

Yu, H. & Liao, J. C. A modified serine cycle in Escherichia coli coverts methanol and CO2 to two-carbon compounds. Nat. Commun. 9, 3992 (2018).

Meyer, F. et al. Methanol-essential growth of Escherichia coli. Nat. Commun. 9, 1508 (2018).

Woolston, B. M., King, J. R., Reiter, M., Van Hove, B. & Stephanopoulos, G. Improving formaldehyde consumption drives methanol assimilation in engineered E. coli. Nat. Commun. 9, 2387 (2018).

Bennett, R. K., Gonzalez, J. E., Whitaker, W. B., Antoniewicz, M. R. & Papoutsakis, E. T. Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph. Metab. Eng. 45, 75–85 (2017).

Gonzalez, J., Bennett, R. K., Papoutsakis, E. T. & Antoniewicz, M. R. Methanol assimilation in Escherichia coli is improved by co-utilization of threonine and deletion of leucine-responsive regulatory protein. Metab. Eng. 45, 67–74 (2017).

Rohlhill, J., Sandoval, N. R. & Papoutsakis, E. T. Sort-Seq approach to engineering a formaldehyde-inducible promoter for dynamically regulated Escherichia coli growth on methanol. ACS Synth. Biol. 6, 1584–1595 (2017).

Woolston, B. M., Roth, T., Kohale, I., Liu, D. R. & Stephanopoulos, G. Development of a formaldehyde biosensor with application to synthetic methylotrophy. Biotechnol. Bioeng. 115, 206–215 (2018).

Whitaker, W. B. et al. Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli. Metab. Eng. 39, 49–59 (2017).

Lu, X. et al. Constructing a synthetic pathway for acetyl-coenzyme A from one-carbon through enzyme design. Nat. Commun. 10, 1378 (2019).

Wang, X. et al. Biological conversion of methanol by evolved Escherichia coli carrying a linear methanol assimilation pathway. Bioresour. Bioprocess. 4, 41–46 (2017).

Anthony, C. The Biochemistry of Methylotrophs (Academic Press, 1982).

Drake, H. L., Kirsten, K. & Matthies, C. Acetogenic Prokaryotes. in The Prokaryotes (eds., Stanley Falkow, Eugene Rosenberg, Karl-Heinz Schleifer, Erko Stackebrandt) 354–420 (Springer, 2006).

Bar-Even, A., Noor, E., Flamholz, A. & Milo, R. Design and analysis of metabolic pathways supporting formatotrophic growth for electricity-dependent cultivation of microbes. Biochim. Biophys. Acta 1827, 1039–1047 (2013).

Bar-Even, A. Does acetogenesis really require especially low reduction potential? Biochim. Biophys. Acta 1827, 395–400 (2013).

Noor, E. et al. Pathway thermodynamics highlights kinetic obstacles in central metabolism. PLoS Comput. Biol. 10, e1003483 (2014).

Figueroa, I. A. et al. Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathway. Proc. Natl Acad. Sci. USA 115, E92–E101 (2018).

Kawasaki, H., Sato, T. & Kikuchi, G. A new reaction for glycine biosynthesis. Biochem. Biophys. Res. Commun. 23, 227–233 (1966).

Motokawa, Y. & Kikuchi, G. Glycine metabolism by rat liver mitochondria. Reconstruction of the reversible glycine cleavage system with partially purified protein components. Arch. Biochem. Biophys. 164, 624–633 (1974).

Pasternack, L. B., Laude, D. A. Jr. & Appling, D. R. 13 C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae. Biochemistry 31, 8713–8719 (1992).

Tashiro, Y., Hirano, S., Matson, M. M., Atsumi, S. & Kondo, A. Electrical-biological hybrid system for CO2 reduction. Metab. Eng. 47, 211–218 (2018).

Yishai, O., Bouzon, M., Doring, V. & Bar-Even, A. In vivo assimilation of one-carbon via a synthetic reductive glycine pathway in Escherichia coli. ACS Synth. Biol. 7, 2023–2028 (2018).

Crowther, G. J., Kosaly, G. & Lidstrom, M. E. Formate as the main branch point for methylotrophic metabolism in Methylobacterium extorquens AM1. J. Bacteriol. 190, 5057–5062 (2008).

Tishkov, V. I. & Popov, V. O. Catalytic mechanism and application of formate dehydrogenase. Biochem. (Mosc.) 69, 1252–1267 (2004).

Wenk, S., Yishai, O., Lindner, S. N. & Bar-Even, A. An engineering approach for rewiring microbial metabolism. Methods Enzymol. 608, 329–367 (2018).

Bassalo, M. C. et al. Rapid and efficient one-step metabolic pathway integration in E. coli. ACS Synth. Biol. 5, 561–568 (2016).

Gleizer, S. et al. Conversion of Escherichia coli to generate all biomass carbon from CO2. Cell 179, 1255–1263.e12 (2019).

Claassens, N. J., Cotton, C. A., Kopljar, D. & Bar-Even, A. Making quantitative sense of electromicrobial production. Nat. Catal. 2, 437 (2019).

Nicholls, P. Formate as an inhibitor of cytochrome c oxidase. Biochem. Biophys. Res. Commun. 67, 610–616 (1975).

Warnecke, T. & Gill, R. T. Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications. Micro. Cell Fact. 4, 25 (2005).

Dragosits, M. & Mattanovich, D. Adaptive laboratory evolution—principles and applications for biotechnology. Micro. Cell Fact. 12, 64 (2013).

Wytock, T. P. et al. Experimental evolution of diverse Escherichia coli metabolic mutants identifies genetic loci for convergent adaptation of growth rate. PLoS Genet. 14, e1007284 (2018).

Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

Gutheil, W. G., Kasimoglu, E. & Nicholson, P. C. Induction of glutathione-dependent formaldehyde dehydrogenase activity in Escherichia coli and Hemophilus influenza. Biochem. Biophys. Res. Commun. 238, 693–696 (1997).

Kotrbova-Kozak, A., Kotrba, P., Inui, M., Sajdok, J. & Yukawa, H. Transcriptionally regulated adhA gene encodes alcohol dehydrogenase required for ethanol and n-propanol utilization in Corynebacterium glutamicum R. Appl. Microbiol. Biotechnol. 76, 1347–1356 (2007).

Wu, T. Y. et al. Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1. Appl. Microbiol. Biotechnol. 100, 4969–4983 (2016).

Roth, T. B., Woolston, B. M., Stephanopoulos, G. & Liu, D. R. Phage-assisted evolution of Bacillus methanolicus methanol dehydrogenase 2. ACS Synth. Biol. 8, 796–806 (2019).

Zhang, W. et al. Expression, purification, and characterization of formaldehyde dehydrogenase from Pseudomonas aeruginosa. Protein Expr. Purif. 92, 208–213 (2013).

Cotton, C. A., Claassens, N. J., Benito-Vaquerizo, S. & Bar-Even, A. Renewable methanol and formate as microbial feedstocks. Curr. Opin. Biotechnol. 62, 168–180 (2020).

Thoma, S. & Schobert, M. An improved Escherichia coli donor strain for diparental mating. FEMS Microbiol. Lett. 294, 127–132 (2009).

Thomason, L. C., Costantino, N. & Court, D. L. E. coli genome manipulation by P1 transduction. Curr. Protoc. Mol. Biol. https://doi.org/10.1002/0471142727.mb0117s79 (2007).

Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006–2008 (2006).

Nyerges, A. et al. A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. Proc. Natl Acad. Sci. USA 113, 2502–2507 (2016).

Zelcbuch, L. et al. Spanning high-dimensional expression space using ribosome-binding site combinatorics. Nucleic Acids Res. 41, e98 (2013).

Sambrook, J. & Russell, D. W. Molecular Cloning: A Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, 2001).

Braatsch, S., Helmark, S., Kranz, H., Koebmann, B. & Jensen, P. R. Escherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine-tuning. Biotechniques 45, 335–337 (2008).

Giavalisco, P. et al. Elemental formula annotation of polar and lipophilic metabolites using 13 C, 15 N and 34 S isotope labelling, in combination with high‐resolution mass spectrometry. Plant J. 68, 364–376 (2011).

Liu, A., Feng, R. & Liang, B. Microbial surface displaying formate dehydrogenase and its application in optical detection of formate. Enzym. Microb. Technol. 91, 59–65 (2016).

Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 − ΔΔCT method. Methods 25, 402–408 (2001).

Zhou, K. et al. Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. BMC Mol. Biol. 12, 18 (2011).


Contents

Spectrum of activity Edit

Kanamycin is indicated for short-term treatment of bacterial infections caused by one or more of the following pathogens: E. coli, Proteus species (both indole-positive and indole-negative), Enterobacter aerogenes, Klebsiella pneumoniae, Serratia marcescens, and Acinetobacter species. In cases of serious infection when the causative organism is unknown, Kanamycin injection in conjunction with a penicillin- or cephalosporin-type drug may be given initially before obtaining results of susceptibility testing.

Kanamycin does not treat viral infections. [7]

Pregnancy and breastfeeding Edit

Kanamycin is pregnancy category D in the United States. [7]

Kanamycin enters breast milk in small amounts. The manufacturer therefore advises that people should either stop breastfeeding or kanamycin. The American Academy of Pediatrics considers kanamycin okay in breastfeeding. [8]

Children Edit

Kanamycin should be used with caution in newborns due to the risk of increased drug concentration resulting from immature kidney function. [7]

Serious side effects include ringing in the ears or loss of hearing, toxicity to kidneys, and allergic reactions to the drug. [9]

Other side effects include: [7]

Kanamycin works by interfering with protein synthesis. It binds to the 30S subunit of the bacterial ribosome. This results in incorrect alignment with the mRNA and eventually leads to a misread that causes the wrong amino acid to be placed into the peptide. This leads to nonfunctional peptide chains. [10]

Kanamycin is a mixture of three main components: kanamycin A, B, and C. Kanamycin A is the major component in kanamycin. [11] The effects of these components do not appear to be widely studied as individual compounds when used against prokaryotic and eukaryotic cells.

While the main product produced by Streptomyces kanamyceticus is kanamycin A, additional products are also produced, including kanamycin B, kanamycin C, kanamycin D and kanamycin X.

The kanamycin biosynthetic pathway can be divided into two parts. The first part is common to several aminoglycoside antibiotics, such as butirosin and neomycin. In it a unique aminocyclitol, 2-deoxystreptamine, is biosynthesized from D-glucopyranose 6-phosphate in four steps. At this point the kanamycin pathway splits into two branches due to the promiscuity of the next enzyme, which can utilize two different glycosyl donors - UDP-N-acetyl-α-D-glucosamine and UDP-α-D-glucose. One of the branches forms kanamycin C and kanamycin B, while the other branch forms kanamycin D and kanamycin X. However, both kanamycin B and kanamycin D can be converted to kanamycin A, so both branches of the pathway converge at kanamycin A. [12]

Kanamycin is used in molecular biology as a selective agent most commonly to isolate bacteria (e.g., E. coli) which have taken up genes (e.g., of plasmids) coupled to a gene coding for kanamycin resistance (primarily Neomycin phosphotransferase II [NPT II/Neo]). Bacteria that have been transformed with a plasmid containing the kanamycin resistance gene are plated on kanamycin (50-100 ug/ml) containing agar plates or are grown in media containing kanamycin (50-100 ug/ml). Only the bacteria that have successfully taken up the kanamycin resistance gene become resistant and will grow under these conditions. As a powder, kanamycin is white to off-white and is soluble in water (50 mg/ml).

At least one such gene, Atwbc19 [13] is native to a plant species, of comparatively large size and its coded protein acts in a manner which decreases the possibility of horizontal gene transfer from the plant to bacteria it may be incapable of giving resistance to bacteria even if gene transfer occurs.

The selection marker kanMX is a hybrid gene consisting of a bacterial aminoglycoside phosphotransferase (kan r from transposon Tn903) under control of the strong TEF promoter from Ashbya gossypii. [14] [15]


LB Agar Kanamycin-50, Plates

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The antibiotics in the LB-agar plates are not stable. The antibiotic degrades over time, making it ineffective at selecting transformed bacteria grown on the plate. Because of this, we recommend the plates are used as soon as possible once they are received. For labs with low use, the plates may expire quickly, so they may benefit from pouring their own plates using the LB - Miller powder with agar formulation (L3147).


References

Gerdes K, Rasmussen PB, Molin S: Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells. Proc Natl Acad Sci USA. 1986, 83: 3116-20. 10.1073/pnas.83.10.3116.

Handa N, Kobayashi I: Post-segregational killing by restriction modification gene complexes: observations of individual cell deaths. Biochimie. 1999, 81 (8): 931-938. 10.1016/S0300-9084(99)00201-1.

Degryse E: Stability of a host-vector system based on complementation of an essential gene in Escherichia coli. J Biotechnol. 1991, 18: 29-39. 10.1016/0168-1656(91)90233-L.

Cranenburgh RM, Hanak JA, Williams SG, Sherratt DJ: Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration. Nucleic Acids Res. 2001, 29: E26- 10.1093/nar/29.5.e26.

Hagg P, de Pohl JW, Abdulkarim F, Isaksson LA: A host/plasmid system that is not dependent on antibiotics and antibiotic resistance genes for stable plasmid maintenance in Escherichia coli. J Biotechnol. 2004, 111: 17-30. 10.1016/j.jbiotec.2004.03.010.

Fiedler M, Skerra A: ProBA complementation of an auxotrophic E. coli strain improves plasmid stability and expression yield during fermenter production of a recombinant antibody fragment. Gene. 2001, 274: 111-8. 10.1016/S0378-1119(01)00629-1.

Vidal L, Pinsach J, Striedner G, Caminal G, Ferrer P: Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli. J Biotechnol. 2008, 134: 127-36. 10.1016/j.jbiotec.2008.01.011.

Pfaffenzeller I, Mairhofer J, Striedner G, Bayer K, Grabherr R: Using ColE1-derived RNA I for suppression of a bacterially encoded gene: implication for a novel plasmid addiction system. Biotechnol J. 2006, 1: 675-81. 10.1002/biot.200600017.

Mairhofer J, Pfaffenzeller I, Merz D, Grabherr R: A novel antibiotic free plasmid selection system: advances in safe and efficient DNA therapy. Biotechnol J. 2008, 3: 83-9. 10.1002/biot.200700141.

Luke J, Carnes AE, Hodgson CP, Williams JA: Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system. Vaccine. 2009, 27: 6454-6459. 10.1016/j.vaccine.2009.06.017.

Goh S, Good L: Plasmid selection in Escherichia coli using an endogenous essential gene marker. BMC Biotechnol. 2008, 11: 61-10.1186/1472-6750-8-61. 10.1186/1472-6750-8-61.

Soubrier F, Cameron B, Manse B, Somarriba S, Dubertret C, Jaslin G, Jung G, Caer CL, Dang D, Mouvault JM, Scherman D, Mayaux JF, Crouzet J: pCOR: a new design of plasmid vectors for nonviral gene therapy. Gene Ther. 1999, 6: 1482-8. 10.1038/sj.gt.3300968.

Szpirer CY, Milinkovitch MC: Separate-component-stabilization system for protein and DNA production without the use of antibiotics. Biotechniques. 2005, 38: 775-81. 10.2144/05385RR02.

Palmeros B, Wild J, Szybalski W, Le Borgne S, Hernández-Chávez G, Gosset G, Valle F, Bolivar F: A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria. Gene. 2000, 247: 255-64. 10.1016/S0378-1119(00)00075-5.

Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000, 97: 6640-5. 10.1073/pnas.120163297.

Mizoguchi H, Tanaka-Masuda K, Mori H: A simple method for multiple modification of the Escherichia coli K-12 chromosome. Biosci Biotechnol Biochem. 2007, 71: 2905-11. 10.1271/bbb.70274.

Summers DK, Sherratt DJ: Multimerization of high copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability. Cell. 1984, 36: 1097-103. 10.1016/0092-8674(84)90060-6.

Balding C, Blaby I, Summers DA: Mutational analysis of the ColE1-encoded cell cycle regulator Rcd confirms its role in plasmid stability. Plasmid. 2006, 56: 68-73. 10.1016/j.plasmid.2005.12.001.

Stirling CJ, Stewart G, Sherratt DJ: Multicopy plasmid stability in Escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination. Mol Gen Genet. 1988, 214: 80-4. 10.1007/BF00340183.


Isolation of Microorganisms from Food Materials

Nutrient agar plates (prepared by students)

Normal saline (0.85%% sodium chloride solution)

Sterile L-shaped spreaders

Food samples used were minced meat (10 g in 90 mL sterile saline), milk (10 mL in 90 mL sterile saline), and soft-rot vegetables (10 g in 90 mL sterile saline). They were blended in saline with a Waring blender, giving 10 −−1 dilution of the original concentration.

Experiments carried out within the 5 days.

Day . Experiments .
1 Preparation of culture media (agar plates)
Isolation of microorganisms from food samples by serial dilution and plating
2 Enumeration of microorganisms isolated from food
Pure culture techniques (streak plate method)
Antimicrobial susceptibility test by disk diffusion method
Minimum inhibitory concentration (MIC) of antibiotics
3 Gram staining of bacterial isolates and observation of bacterial morphology
Streaking of isolates on selective and differential media
Oxidase test for bacterial isolates
Read results of disk diffusion and MIC tests
Minimum bactericidal concentration (MBC) of antibiotics
4 Assessment of growth of isolates on selective and differential media
Identification of bacterial isolates using the API20E system
Read results of MBC test
5 Read results of API20E test strip
Day . Experiments .
1 Preparation of culture media (agar plates)
Isolation of microorganisms from food samples by serial dilution and plating
2 Enumeration of microorganisms isolated from food
Pure culture techniques (streak plate method)
Antimicrobial susceptibility test by disk diffusion method
Minimum inhibitory concentration (MIC) of antibiotics
3 Gram staining of bacterial isolates and observation of bacterial morphology
Streaking of isolates on selective and differential media
Oxidase test for bacterial isolates
Read results of disk diffusion and MIC tests
Minimum bactericidal concentration (MBC) of antibiotics
4 Assessment of growth of isolates on selective and differential media
Identification of bacterial isolates using the API20E system
Read results of MBC test
5 Read results of API20E test strip

Procedure

The students did serial tenfold dilutions and plating in groups of three. Using a sterile 10-mL pipette, 9 mL of saline was placed in each of four sterile tubes labeled 10 −−2 , 10 −−3 , 10 −−4 , and 10 −−5 . Using a sterile micropipette tip, 1 mL of the 10 −−1 food suspension was transferred into 9 mL of saline to give a 10 −−2 dilution and mixed well. Serial dilution was carried out until 10 −−5 . Then, 0.1 mL each of the 10 −−4 and 10 −−5 dilutions were plated on nutrient agar plates in triplicates. The plates were incubated in an inverted position in the incubator at 37°°C overnight.

Results

The following day, the students counted the number of colonies. The number of colony-forming units per milliliter was calculated as follows: mean number of colonies per plate ×× dilution factor ×× 10. Plates containing unknown bacteria remained sealed during the counting process, and the bacteria were not subcultured further. They were autoclaved after counting was done.


The Effects Of Antibiotics On Bacterial Growth

The Effects of Antibiotics on bacterial growth Biology II 1996 Bacteria are the most common and ancient microorganisms on earth. Most bacteria are microscopic, measuring 1 micron in length. However, colonies of bacteria grown in a laboratory petri dish can be seen with the unaided eye. There are many divisions and classifications of bacteria that assist in identifying them. The first two types of bacteria are. Both groups have common ancestors dating to more than 3 billion years ago.

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Archaebacteria live in environments where, because of the high temperature, no other life can grow. These environments include hot springs and areas of volcanic activity. They contain lipids but lack certain chemicals in their cell wall. Eu bacteria are all other bacteria. Most of them, i. e.

they use the sun’s energy as food through the process of photosynthesis. Another classification of bacteria is according to their need of oxygen to live. Those who do require oxygen to live are considered aerobes. The bacteria who don’t use oxygen to live are known as anaerobes. The shape of specific bacteria provides for the next step in the identification process. Spherical bacteria are called cocci the bacteria that have a rod like shape are known as bacilli corkscrew shaped bacteria are spirilla and filamentous is the term for bacteria with a threadlike appearance.

The Essay on Bacteria Live Survive Cell Food

Bacteria live almost everywhere, even where other forms of life can? t. The only places? where they can? t survive is in sanitized places. Some bacteria need oxygen to survive, and others don? t need any. Also some can survive with both, but some can? t survive with oxygen. They protect themselves by forming a thick cell membrane inside the old one. Bacteria get food by feeding off of other tiny .

Hans Christian Joachim Gram, a Danish microbiologist, developed a method for distinguishing bacteria by their different reaction to a stain. The process of applying Gram’s stain is as follows: the bacteria are stained with a violet dye and treated with Gram’s solution (1 part iodine, 2 parts potassium iodide, and 300 parts water).

Ethyl alcohol is then applied to the medium the bacteria will either preserve the blue color of the original dye or they will obtain a red hue. The blue colored bacteria are gram-positive the red bacteria are identified as gram-negative.

Bacteria contain DNA (acid) just like all cells. However, in bacteria the DNA is arranged in a circular fashion rather than in strands. Bacteria also contain ribosomes which, like in eukaryotic cells, provide for protein synthesis. In order for a bacterium to attach itself to a surface, it requires the aid of pili, or hairlike growths. Bacteria, just like sperm cells, have flagella which assist in movement. But, sperm cells only have one flagellum, whereas bacteria contain flagella at several locations throughout their body surface.

Although most bacteria are not harmful, a small fraction of them are responsible for many diseases. These bacterial pathogens have affected humans throughout history. The “plague”, an infamous disease caused by bacteria, has killed millions of people. Also, such a disease as tuberculosis, a disease responsible for the lives of many, is caused by bacterial pathogens ingested into the body. Bacteria affect everyone in their daily life because they are found nearly everywhere. They are found in the air, in food, in living things, in non-living things, and on every imaginable surface.

Escherichia coli is a disease causing gram-negative bacillus. These bacteria are commonly found within the intestines of humans as well as other vertebrates. This widely spread bacteria is known to cause urinary tract infections as well as diarrhea. Microcococcus Luteus are gram-positive parasitic spherical bacteria which usually grows in grape like clusters. This species is commonly found in milk and dairy products as well as on dust particles. Bacillus Cereus are a spore forming type of bacteria.

They are gram-positive and contain rods. Due to the fact that this bacteria is known to survive cooking, it is a common cause of food poisoning and diarrhea. Seratia Marscens a usually anaerobic bacteria which contains gram-negative rods. This bacteria feeds on decaying plant and animal material.

The Essay on Sickle Cell Anemia Disease Blood Cells

BY BETTY J ALLEN OUTLINE Thesis statement: Sickle Cell Disease is an inherited disease that affects the red blood cells. This disease can be treated so a person can live a long health life. 1. Sickle Cell Disease a. What is sickle cell disease? b. Whom does this disease affect? c. What race and geographic region do they belong to? d. c. Causes of sickle cell disease. e. Signs and symptoms and how .

S. are found in water, soil, milk, foods, and certain insects. In spite of the fact that bacteria are harmful to the body, certain measures can be taken in order to inhibit their growth and reproduction. The most common form of bacteria fighting medicines are antibiotics. Antibiotics carry out the action which their Greek origin suggests: anti meaning against, and bios meaning life. In the early parts of the 20 th century, a German chemist, Paul Ehrlich began experimentation using organic compounds to combat harmful organisms without causing damage to the host.

The results of his experimentation began the study and use of antibiotics to fight bacteria. Antibiotics are classified in various ways. They can be arranged according to the specific action it has on the cell. For example, certain antibiotics attack the cell wall, others concentrate on the cell membrane, but most obstruct protein synthesis. Another form of indexing antibiotics is by their actual chemical structure. Practically all antibiotics deal with the obstruction of synthesis of the cell wall, proteins, or nucleic acids.

Some antibacterials interfere with the messenger RNA, consequently mixing up the bacterial genetic code. Penicillins act by inhibiting the formation of a cell wall. Thisantibiotic works most effectively against gram-positive streptococci, staphylococci (e. g. Micrococcus Luteus) as well as certain gram-negative bacteria. Penicillin is usually prescribed to treat syphilis, gonorrhea, meningitis, and anthrax.

Tetracycline inhibits protein synthesis in pathogenic organism. Thisantibiotic is obtained from the culture of Streptomyces. Streptomycin an antibiotic agent which is obtained from Streptomycesgriseus. This antibiotic acts by limiting normal protein synthesis.

Streptomycin is effective against E. Coli, gram-negative bacilli, as well as many cocci. Neomycin an antibiotic derived from a strain of Streptomyces. Neomycin effectively destroys a wide range of bacteria. Kanamycin an antibiotic substance derived from Streptomyces. Its antibacterial action is very similar to that of neomycin.

The Essay on Cause and effect of cell phone

Cell phone is one of great devices that improve the lives of the society. Cell phone provides some services of access to communicate to people in worldwide/in the world. Technology is used by cell phone developer is making rapid advancement. Nowadays, obviously, there will be more and more the people in the society are using cell phone in their lives. In 2012 year, the number of cell phone sold in .

Kanamycin works against many aerobic gram-positive and gram-negative bacteria, especially E. coli. Protracted use may result in auditory as well as other damages. Erythromycin is an antibiotic produced by a strain of Streptomyceserythreaus. This antibiotic works by inhibiting protein synthesis but not nucleic synthesis. Erythromycin has inhibitory effects on gram-negative cocci as well as some gram-positive bacteria.

Chloramphenicol is a clinically useful antibiotic in combating serious infections caused by certain bacteria in place of potentially hazardous means of solving the problem. In lab tests, it has been shown that this medicine stopped bacterial reproduction in a wide range of both gram-positive and gram-negative bacteria. The inhibition of cell reproduction caused by Chloramphenicol takes place through interference with protein synthesis. An experiment was conducted in order to determine which antibiotics are most effective in inhibiting bacterial growth. First, the different bacteria were placed on agar inside petri dishes. Then, antibiotic discs were placed intothe dishes.

Each bacteria was exposed to every one of the antibiotics listed above. The bacteria used in the experiment were: Bacillus Cerus, EscerichiaColi, Seratia Marscens, and Micrococcus Luteus. After a 24 hour incubation period, the results were measured. In order to determine which antibiotic had the most effect their zones of inhibition we rerecorded.

The zone of inhibition refers to the distance from the disc to the outermost section around the disc where no bacterial growth was present. There sults can be seen on the graph and data chart. The following is a table showing the different zones of inhibition of each antibiotic in the bacteria culture: Tetracycline Chloramphenicol Kanamycin Neomycin Penicillin Streptomycin Erythromycin B. Cerus 5. 5 9 56.

6 1 7 13 E. Coli 74. 2 5. 5 4.

5 no effect 4. 6 no effectS. Marscens no effect no effect 4. 5 4 no effect 3 no effect. Luteus 2322 10 11 23. 5 11.

5 19 After analysis of the data obtained it is obvious that each antibiotic had a distinct effect on the growth of the different bacteria. The results of this experiment are very important, since they teach of how each bacteria reacts to different antibiotics. This is very valuable because it is the information which assists physicians in prescribing certain medications to cure diseases caused by bacteria. Bibliography 1) En cart Encyclopedia 1994, CD-ROM.

The Essay on Bacteria Classification By Gram Staining

Bacteria Classification By Gram Staining THE AMERICAN UNIVERSITY IN CAIRO BIOLOGY DEPARTMENT SCIENCE 453: BIOLOGY FOR ENGINEERS REPORT No. 1 Presented By: Karim A. Zak lama 92-1509 Sci. 453-01 24/2/96 Objective: To test a sample of laboratory prepared bacteria and categorise it according to Christians gram positive and gram negative classes and also by viewing it under a high powered microscope .

2) McGraw-Hill Encyclopedia of Science and Technology, 1992. 3) Physicians’ Desk Reference, 1996.

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Purification and Digestion of Plasmid (Vector) DNA

Susan Carson , . D. Scott Witherow , in Molecular Biology Techniques (Third Edition) , 2012

Principles of Gene Expression

In order to understand how expression vectors function, it is important to recall the Central Dogma of Molecular Biology ( Figure 2.1 ). For a gene to be expressed, it must first be transcribed into messenger RNA (mRNA), and then translated into protein. In the simplest example, RNA polymerase binds to the promoter of a gene and then proceeds with transcription, producing mRNA. Transcription ends at the terminator sequence.

Fig. 2.1 . Central Dogma of Molecular Biology. DNA is transcribed into mRNA, which in turn is translated into protein. RNA polymerase binds to the promoter of a gene on DNA and proceeds with transcription, producing a new mRNA. The ribosome and tRNA work together to translate the nascent mRNA into protein.

The ribosome then binds the mRNA at the ribosome binding site (RBS) and the ribosome moves along the mRNA. As the ribosome moves along the mRNA, transfer RNA (tRNA) is responsible for decoding the mRNA and specifically depositing an amino acid residue on the nascent polypeptide chain. The translational start codon, which is usually encoded by AUG (ATG on the DNA), encodes the first amino acid (usually methionine), and the translation stop codon (TAA, TAG or TGA) ends translation.

Expression Vectors

The expression vector you will use for your project is pET-41a ( Figure 2.2 ). This expression vector utilizes a kanamycin resistance gene as a selectable marker and the glutathione-S-transferase gene ( gst) as a fusion tag. The multiple cloning site is downstream (3′) of the gst gene and there is no stop codon or termination signal following the gst gene. Therefore, when our gene of interest (egfp) is cloned into the multiple cloning site it will be expressed as a fusion protein with gst, resulting in expression of the fusion protein, GST::EGFP. You will learn more about how creating this fusion protein will aid in the purification of the EGFP protein later in the semester.

Fig. 2.2 . Salient features of pET-41a.

The expression of the gst gene, and consequently the fusion gene in your future construct, is under the control of the T7 promoter and is inducible using isopropyl-β-D-thiogalactopyranoside (IPTG). In nature, the promoter is induced by lactose, and IPTG mimics lactose with regard to the induction properties, but is not cleaved by the E. coli enzyme β-galactosidase. Inducibility is due to the fact that pET-41a uses two components of the lac operon, the lac operator and the lacI gene, to regulate transcription. In this vector, the lac operator is located adjacent to the T7 promoter. lacI encodes a repressor and is constitutively expressed, so the repressor protein LacI is always present. LacI binds to the lac operator in the absence of inducer and prohibits RNA polymerase from initiating transcription from the T7 promoter. When the inducer molecule IPTG is added, it interacts with LacI in such a way that LacI will no longer bind to the lac operator, and thus transcription by the T7 RNA polymerase proceeds. This process is called derepression of the promoter ( Figure 2.3 ).

Fig. 2.3 . Promoter repression by LacI and derepression by IPTG. (A) The repressed state of the promoter. (B) The derepressed state of the promoter due to the inducer molecule, IPTG.


Watch the video: The Antibiotic Apocalypse Explained (August 2022).