The only colonies that are obtained will therefore be ones that comprise cells containing the desired recombinant DNA molecule. Privacy Policy https://doi.org/10.1007/BF00267933, Over 10 million scientific documents at your fingertips, Not logged in tetracycline resistance of this plasmid. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli Sampali Banerjee , 1 Jitendra Kumar , 1 Anjali Apte-Deshpande , … kanamycin agar, on which the only cells able to survice 5. Direct selection for recombinants by supplemented minimal media from polyethylene-glycol (PEG)-induced fusion of protoplasts of polyauxotrophic strains of B. megaterium revealed striking physiological influences on the yield of recombinants. A Direct Selection of the E. coli Recombinant Clones that Produce the Y. Pestis FI Antigen The electrophoretically heterogeneous suspension containing both the transformed and intact cells was studied in model experiments using the E. coli DH1 cells transformed with hybrid plasmids pFSK3 and Fodor, K., Alföldi, L. Polyethylene-glycol induced fusion of bacterial protoplasts. 135, 68–70 (1978), Fodor, K., Hadlaczky, G., Alföldi, L.: Reversion of Bacillus megaterium protoplasts to the bacillary form. Lehrstuhl für Genetik der Universität Bayreuth, D‐8580 Bayreuth, F.R.G. When such recombinants (containing target DNA in tef gene) are grown onto medium containing tetracycline, they will not grow because their tef gene has been inactivated. TOS Subscription will auto renew annually. PUC19). Transformants are plated onto By continuing you agree to the use of cookies. When such recombinants (containing target DNA in tetr gene) are grown into medium containing tetracycline, they will not grow because their tetr gene has been inactivated. If the cloned DNA itself codes for resistance to the antibiotic ampicillin (ampr) the recombinants can be allowed to grow on minimal medium containing ampicillin. The vector or foreign DNA present in recombinant cells express the characters, while the non-recombinants do not express the traits. Another powerful method of screening for the presence of recombinant plasmid is referred to as blue-white selection. Colonies which appear white are both ampicillin resistant and have the insert rDNA and thus are the cells to be used for future experiment. This approach ranges in sophistication, from simple selection for … After transformation of competent mutant cells with a ligation mixture, simultaneous selection for the plasmid-coded drug resistance and for the inactivation of the complementing gene directly yields colonies, which harbor recombinant plasmids. Because ampr gene is present in both the recombinants. Thus, only transformed cells, however few, will be selected for growth and division. It is demonstrated that this cloning system can be used with an efficiency beyond 90%. If the lac-Z gene is inactivated due to the presence of the insert, then the enzymes is not expresses. Summary. 13 EcoRI fragments (a). Sci. ADVERTISEMENTS: carries the gene for kanamycin resistance. Vector sequences are provided. DNA molecule. Disclaimer The simplest example of Direct Selection occurs when – The simplest example of Direct Selection occurs when the desired gene specifies resistance to an antibiotic. - 162.213.252.64. FEMS Microbiology Letters 43 (1987) 201-205 Published by Elsevier FEM 02845 201 Direct selection of recombinant plasmids with chlorate Jochen Reiss and Walter Klingmler Lehrstuhl f Genetik der Uniuersit Bayreuth, D-8580 Bayreuth, ER. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. 121, 390–391 (1975), Hadlaczky, G., Fodor, K., Alföldi, L.: Morphological study of reversion to bacillary form of Bacillus megaterium protoplasts. Copyright © 2020 Elsevier B.V. or its licensors or contributors. After transformation of competent mutant cells with a ligation mixture, simultaneous selection for the plasmid‐coded drug resistance and for the inactivation of the complementing gene directly yields colonies, which harbor recombinant plasmids. This potentially lethal function can be inactivated by the insertion of DNA fragments into a multiple cloning site. G. Received 20 March 1987 Revision received 26 March 1987 Accepted 27 March 1987 Key words: Chlorate resistance; Cloning vector; Positive selection; … the cloned kanamycin resistance gene(c). immaterial for cloning the kanamycin resistance gene All the articles you read in this site are contributed by users like you, with a single vision to liberate knowledge. 2. USA. Corresponding Author. In this approach, one of the genetic traits is disrupted by inserting foreign DNA. Plasmid pBR322 contains two antibiotic resistance genes, one for ampicillin (ampr gene), and the other for tetracycline (tetr gene). PreserveArticles.com is a free service that lets you to preserve your original articles for eternity. The lacZ encodes the enzyme p-galactosidase and contains several recognition sites for restriction enzymes. Because p-galactosidase is not synthesised due to inactivation of lacZ. In the present study, the efficacy of the galE-based positive selection system for direct selection of recombinants in various α-complementation plasmids was examined.

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