Corynebacterium glutamicum genes encoding phosphoenolpyruvate: sugar phosphotransferase system proteins
a technology of sugar phosphotransferase and corynebacterium glutamicum, which is applied in the direction of transferases, peptides, bacteria based processes, etc., can solve the problems of time-consuming and difficult process of selecting strains for the production of a particular molecule, and achieve the optimization of activity, increase the quantity of glucose uptake or the rate at which glucose is translocated into the cell, and improve the yield
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example 1
Preparation of Total Genomic DNA of Corynebacterium glutamicum ATCC 13032
[0126] A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30° C. with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded and the cells were resuspended in 5 ml buffer-I (5% of the original volume of the culture—all indicated volumes have been calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g / l sucrose, 2.46 g / l MgSO4×7H2O, 10 ml / l KH2PO4 solution (100 g / l, adjusted to pH 6.7 with KOH), 50 ml / l M12 concentrate (10 g / l (NH4)2SO4, 1 g / l NaCl, 2 g / l MgSO4×7H2O, 0.2 g / l CaCl2, 0.5 g / l yeast extract (Difco), 10 ml / l trace-elements-mix (200 mg / l FeSO4×H2O, 10 mg / l ZnSO4×7 H2O, 3 mg / l MnCl2×4 H2O, 30 mg / l H3BO3 20 mg / l CoCl2×6 H2O, 1 mg / l NiCl2×6 H2O, 3 mg / l Na2MoO4×2 H2O, 500 mg / l complexing agent (EDTA or critic acid), 100 ml / l vitamins-mix (0.2 mg / l biotin, 0.2 mg / l folic acid, 20 mg / l p-amino benzoic a...
example 2
Construction of Genomic Libraries in Escherichia coli of Corynebacterium glutamicum ATCC13032.
[0127] Using DNA prepared as described in Example 1, cosmid and plasmid libraries were constructed according to known and well established methods (see e.g., Sambrook, J. et al. (1 989) “Molecular Cloning : A Laboratory Manual”, Cold Spring Harbor Laboratory Press, or Ausubel, F. M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley & Sons.)
[0128] Any plasmid or cosmid could be used. Of particular use were the plasmids pBR322 (Sutcliffe, J. G. (1979) Proc. Natl. Acad. Sci. USA, 75:3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol 134:1141-1156), plasmids of the pBS series (pBSSK+, pBSSK− and others; Stratagene, LaJolla, USA), or cosmids as SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T. J., Rosenthal A. and Waterson, R. H. (1987) Gene 53:283-286. Gene libraries specifically for use in C. glutamicum may be constructed using plasmid pSL109 (Lee, H.-S. and ...
example 3
DNA Sequencing and Computational Functional Analysis
[0129] Genomic libraries as described in Example 2 were used for DNA sequencing according to standard methods, in particular by the chain termination method using ABI377 sequencing machines (see e.g., Fleischman, R. D. et al. (1995) “Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496-512). Sequencing primers with the following nucleotide sequences were used:
5′-GGAAACAGTATGACCATG-3′or5′-GTAAAACGACGGCCAGT-3′.
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