why do plasmids contain an antibiotic resistance gene
What are antibiotics? Antibiotics are generally defined as agents that kill bacteria, or inhibit their growth. Although originally sourced from natural products, many common antibiotics used in labs today are semi-synthetic or fully synthetic compounds. Antibiotics can be categorized based on whether they directly kill bacteria (bactericidal) or slow growth/prevent cell division (bacteriostatic); however, the distinction between the two categories may be a bit of a gray area as some bacteriostatic reagents can kill bacteria when used at high concentrations (and vice versa). Looking around the lab, you'll likely find many of the antibiotics listed in the table below. Note, in this post we'll focus primarily on antibiotics against
Gram negative bacteria. In f uture posts, we'll detail selection in non-bacterial cells such as or. *In prokaryotes. **Dissolve in dH O and sterile filter unless otherwise specified. The above table lists some antibiotics commonly found in the lab, their mechanism for killing bacteria, and general working concentrations. For instructions on how to prepare antibiotic stocks, see. How else can antibiotics be used in the lab? Historically, antibiotics have also been used to disrupt genes at the chromosomal level. Scienists introduce an antibiotic resistance cassette within the coding region of the gene they are trying to disrupt or delete, which both inactivates the gene and acts as a marker for the mutation. When designing these types of experiments it is best practice not to use the same resistance cassette for the mutation and for plasmid selection.
Additionally, scientists can use the loss of resistance as a marker for successful cloning. In these instances, the cloning vector typically has two separate resistance cassettes and your gene of interest is cloned into/inactivates or completely removes (in the case of ) one cassette. Counterselection allows the scientist to select bacteria that are only resistant to the antibiotic that remains intact. Use fresh stocks. Most antibiotics are stable in powder form, but quickly breakdown in solution. Storing aliquots at -20 C and avoiding repeated freeze/thaw cycles will keep most antibiotics viable for at least 6 months. Ampicillin breaks down especially fast and plates should be used witin 1 month for optimal efficiency. Beware of satellite colonies! Carbenicillin is more stable than Ampicillin and can be used in place of Ampicillin in most applications. C and store plates/stocks wrapped in foil if a light-sensitive antibiotic like Tetracycline is used. Keep in mind that some E. coli strains have natural antibiotic resistances, so make sure your plasmid and E. coli strain are compatible! Check out this E. coli genotypes and their natural resistances. Additional Resources on the Addgene Blog Read other Learn How to Use See Where You Can Find Antibiotic Resistance on Resources on Addgene. org Browse Our View our Find Your Sinkovics, J; Harvath J; Horak A. (1998). "The Origin and evolution of viruses (a review)". Acta Microbiologica et Immunologica Hungarica. 45 (34): 34990.
P. Smillie, Chris; Garcilln-Barcia, M. Pilar; Francia, M. Victoria; Rocha, Eduardo P. C. ; Cruz, Fernando de la (1 September 2010). Microbiology and Molecular Biology Reviews. 74 (3): 434452. :. P. Thomas, Christopher M; Summers, David (2008). "Bacterial Plasmids". Encyclopedia of Life Sciences. :. P. Wolfgang Schumann (2008). "Chapter 1 - Escherichia coli Cloning and Expression Vectors". In Georg Lipps. Plasmids: Current Research and Future Trends. Caister Academic Press. pp. P12. P. CS1 maint: Uses editors parameter ( Lederberg J (1952). "Cell genetics and hereditary symbiosis". Physiol. Rev. 32 (4): 403430. P. ^ Finbarr Hayes (2003). "Chapter 1 - The Function and Organization of Plasmids". In Nicola Casali, Andrew Presto. Methods in Molecular Biology. 235. Humana Press. pp. P15. P. CS1 maint: Uses editors parameter ( Stanley Falkow. Microbiology Society. ^ T. A. Brown (2010). "Chapter 2 - Vectors for Gene Cloning: Plasmids and Bacteriophages". (6th ed. ). Wiley-Blackwell. P. David Summers (1996). "Chapter 1 - The Function and Organization of Plasmids". Wiley-Blackwell; First Edition. pp. P2122. P. David P. Clark; Nanette Jean Pazdernik (2012). (2nd ed. ). Academic Cell. p. P795. P. Margaret C. M. Smith and R. Elizabeth Sockett, eds. (1999). Methods in Microbiology, vol. 29. Academic Press. pp. P7577. P. CS1 maint: Uses editors parameter ( ^ Russell, David W. ; Sambrook, Joseph (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, N. Y: Cold Spring Harbor Laboratory. Uldis N. Streips, Ronald E. Yasbin, eds. (2002). (2nd ed. ).
Wiley-Blackwell. p. P248. P. CS1 maint: Uses editors parameter ( Andrew Preston (2003). "Chapter 2 - Choosing a Cloning Vector". In Nicola Casali, Andrew Preston. Methods in Molecular Biology, Vol. 235. Humana Press. pp. P1926. P. CS1 maint: Uses editors parameter ( Kandavelou K, Chandrasegaran S (2008). "Plasmids for Gene Therapy". Plasmids: Current Research and Future Trends. Caister Academic Press. P. Morange M (2009). Journal of Biosciences. 34 (6): 8458. :. P. Jacob F Wollman EL (1958), "Les pisomes, elements gntiques ajouts", Comptes Rendus de l'Acadmie des Sciences de Paris, 247 (1): 154156, P Hayes, W (1969). "What are episomes and plasmids? ". In Gordon E. W. Wolstenholme; Maeve O'Connor. pp. P48. P. Gordon E. W. Wolstenholme; Maeve O'Connor, eds. (1969). pp. P244245. P. T. A. Brown (2011). Garland Science. p. P238. P. Kathleen Van Craenenbroeck, Peter Vanhoenacker and Guy Haegeman (2000). Eur. J. Biochem. 267 (18): 56655678. :. P. CS1 maint: Uses authors parameter ( Colosimo A1, Goncz KK, Holmes AR, Kunzelmann K, Novelli G, Malone RW, Bennett MJ, Gruenert DC. (2000). Biotechniques. 29 (2): 3148, 3202, 324 passim. P. Archived from on 24 July 2011. CS1 maint: Multiple names: authors list ( Gerdes K, Rasmussen PB, Molin S (1986). Proc. Natl. Acad. Sci. U. S. A. 83 (10): 311620. :. :. P. Kroll J, Klinter S, Schneider C, Vo I, Steinbchel A (2010). Microb. Biotechnol. 3 (6): 634657. :. P. Gunge, N; Murata, K; Sakaguchi, K (July 1982). Journal of Bacteriology. 151 (1): 4624. P. The DNA Lab.
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