Molecular cloning of recombinant DNA : proceedings of the Miami winter symposia, January 1977 / edited by W.A. Scott, R. Werner ; sponsored by the Department of Biochemistry, University of Miami, School of Medicine, Miami, Florida.

Date:
1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Molecular cloning of recombinant DNA : proceedings of the Miami winter symposia, January 1977 / edited by W.A. Scott, R. Werner ; sponsored by the Department of Biochemistry, University of Miami, School of Medicine, Miami, Florida. Source: Wellcome Collection.

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    USE OF SAFER BACTERIAL HOST STRAINS 101 survive passage through the intestinal tract of rats. These same three mutations confer upon extreme sensitivity to detergents, antibiotics, drugs and chemicals and thus cause it to be less likely to survive in envir¬ onments in which these compounds might be encountered, such as in sewers. Due to the Д40[gal-uvrB] mutation, Xl776 is unable to carry out either dark or photo repair of UV-induced damage and is thus several orders of mag¬ nitude more sensitive to sunlight than wild-type strains of coli. xLllb is resistant to many transducing phages and is conjugation deficient for inheritance of many different types of conjugative plasmids due to the presence of the tonA53, Д29[bioH-asd], Д40[gal-uvrB], rfb-2, oms-1 and oms-2 mutations (7). Although conjugational trans¬ mission of cloning vector DNA in triparental matings is detectable under optimal permissive laboratory conditions, neither transductional nor conjugational transmission of cloning vector DNA is detectable under a variety of non- permissive conditions that might be analogous to those encountered in nature (7). xl776 is resistant to nalidixic acid (nalA25), cycloserine (cycAl, cycB2) and trimethoprim (thyA57*). These drugs can therefore be added to media to permit monitoring of the strain and to preclude trans¬ formation of contaminants during transformation. Since x1776 has a rather fragile cell wall and exhibits extreme sensitivity to detergents, it is essential that all glassware and plasticware used in the cultivation of this strain be extremely clean and well rinsed so as to be free from all residual detergents. xl776 grows with a generation time of about 50 min in either L broth or Pen- assay broth when supplemented with 100 yg DAP/ml and 4 yg thymidine/ml and reaches final densities of 8-10 x 10^ cells/ml at stationary phase. xl776 is particularly sensitive to mechanical shear due to its fragile cell wall and somewhat elongated cell shape. It is thus essential to avoid use of vortex mixers when suspending cells pel¬ leted by centrifugation. We find that gentle agitation by swirling and shaking back and forth for a period of 1-2 min adequately suspends cells with maintenance of 100% viability. To illustrate, if a xl776 culture is sedi- mented and then the pellet suspended by vortex mixing for 60 sec, between 90 to 99.9% of the cells die with many of them lysing. We therefore recommend to those individuals who have had difficulty in the growth and maintenance of Xl776 to exhibit a little more tender loving care. xl776 will reward them for their efforts.
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