By Nathan P. Kaplan, Nathan P. Colowick, Sidney Fleischer, Becca Fleischer
The significantly acclaimed laboratory common, Methods in Enzymology, is likely one of the so much hugely revered guides within the box of biochemistry. due to the fact 1955, each one quantity has been eagerly awaited, usually consulted, and praised via researchers and reviewers alike. The sequence includes a lot fabric nonetheless correct this day - really a necessary ebook for researchers in all fields of existence sciences
Read or Download Biomembranes Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts) PDF
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Additional info for Biomembranes Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)
The technique we employ is depicted in Fig. 4. The rationale behind this m e t h o d is as follows: I f a mutation known to block expression of the wild-type target gene also blocks expression of the hybrid protein, it can be concluded that the hybrid protein is being expressed from target gene signals. j ~:" Z ', Y ,A' ~J ;. N~: P ompF I Lac- OmpF + FIG. 4. Genetic verification of ompF-lac fusions. (a) An ompF::Tn5 insertion strain is lysogenizedwith a 2 transducing phage isolated from a potential ompF-lacfusion strain.
N. Cohen, J. Bacteriol. 143, 971 (1980). 22 S. Michaelis, L. Guarente, and J. Beckwith, J. BacterioL, in press. _,3A. Sarthy, S. Michaelis, and J. Beckwith, J. Bacteriol. 145, 293 (1981). 22 PROKARYOTIC MEMBRANES  Since such phages do not exist for most other genes, the utility of this approach may be limited. On the other hand, it should be possible, given the appropriate restriction enzyme sites, to clone the gene fusion from the plasmid onto a phage. C h a r a c t e r i z a t i o n of G e n e F u s i o n S t r a i n s The techniques described for fusion construction were designed to minimize the occurrence of abnormal events.
After the agar hardens, drops ofa 2pSG 1 lysate (approximately 108 ~bper milliliter) are spotted onto the surface of the plate. 2pSG 1 is a derivative of2p 1(209) and is identical to the parent phage except that it carries a transposon, Tn9, which confers resistance to chloramphenicol (Cam r) inserted in the lacY gene. ~8 This allows direct selection of lysogens. When the spots are dry, the plates are incubated at 30 ° overnight. From the turbid centers of the lysed areas, cells are picked and streaked onto lactose MacConkey agar containing 25/zg of chloramphenicol per milliliter.
Biomembranes Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts) by Nathan P. Kaplan, Nathan P. Colowick, Sidney Fleischer, Becca Fleischer