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By Lee McIntosh (Eds.)
General Description of the Volume:
The articles during this Methods in Enzymology quantity replicate the combination of molecular genetics with photosynthesis. They specialise in the genetic engineering of the photosynthesis gear, new mechanisms for genetic number of photosynthetic mutants, biophysical and biochemical research of Photosystem I and Photosystem II Mutations, and Photosynthetic Gene Expression.
General Description of the Series:
The severely acclaimed laboratory regular for greater than 40 years, Methods in Enzymology is among the such a lot hugely revered courses within the box of biochemistry. given that 1955, each one quantity has been eagerly awaited, usually consulted, and praised through researchers and reviewers alike. Now with greater than three hundred volumes (all of them nonetheless in print), the sequence comprises a lot fabric nonetheless suitable today--truly a necessary e-book for researchers in all fields of lifestyles sciences.
Key Features
* Genetic engineering of the photosynthesis gear
* New mechanisms for genetic collection of photosynthetic mutants
* Biophysical and biochemical research of Photosystem I and Photosystem II Mutations
* Photosynthetic Gene Expression
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Example text
P. Wolk, Methods Enzymol. 167, 747 (1988). 45 j. Elhai, A. Vepritskiy, A. M. Muro-Pastor, E, Flores, and C. P. Wolk, J. Bacteriol. 179, 1998 (1997), 46 M. F. D. Thesis, University of California, Davis (1996). 47 T. Thiel and C. P. Wolk, Methods Enzymol. 153, 232 (1987). [1] TRANSPOSON MUTAGENESIS OF HETEROCYST-FORMING CYANOBACTERIA 11 to methylate AvaI, AvaII, and AvaIII sites on the DNA of the transposonbearing cargo plasmid prior to transfer. Transfer of RK2 oriT-containing plasmids to N. punctiforme 6 is enhanced by use of conjugal plasmid pRK2013, as whose copy number is greater than that of RK2.
For mutagenesis, the number of plates is determined by the expected frequency of mutations of interest, and the number of colonies that can reasonably be screened. Forty milliliters of cultured cells are required for each plate. Materials 20% Polyethylene glycol (PEG)-8000 (Sigma P-2139, St. Louis, MO), autoclaved. PEG solutions are somewhat unstable, so they should be prepared fresh every 1-2 months. There is some controversy as to whether PEG is always necessary or helpful; we have found that with cwl5 mutants and cells treated with GLE, PEG substantially increases the number of transformants.
Double mutant strains CC-2986 (arg2nitlmt*), CC-3379 (arg2nitlmt-), CC-3396 (arg7nitlmt-) or CC-3397, CC-3398, or CC-3399 (all arg7nitlmt ÷) are useful for sequential transformation by ARG7 and NIT1. Note that arginine interferes with the selection for nitrate reductase, so ARG7 transformation must be carried out first. 812 (Chlamydomonas Genetics Center P-389) contains the ARG7 gene cloned into pUC19. Plasmid DNA should be purified by CsCI centrifugation or an alternative method to give DNA of comparable purity.