The protocol is simple and uses either miniprep plasmid DNA or cesium-chloride-purified DNA. In addition, the QuikChange site-directed mutagenesis system requires no specialized vectors, unique restriction sites, or multiple transformations. For long (~8 kb) or No evidence was obtained for mutagenesis by undecomposed NTG at pH 5. However, low lipid productivity and costly downstream processing continue to hamper the commercial deployment of oleaginous microorganisms. The primers, one or both with desired mutation(s), are designed so that they anneal back to back to the plasmid (for schematic presentation, see Fig. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). There are many reasons to make specific DNA alterations (insertions, deletions and substitutions), including: To study changes in protein activity that occur as a result of the DNA manipulation. Resistances to rifampicin (100 mg/liter), streptomycin (200 mg/liter), and nalidixic acid (40 mg/liter) are due to base substitutions. Five-ml aliquots were centrifuged and washed with one volume of citrate buffer (100 mM sodium citrate, pH 5.5).
... a NTG-based mutagenesis system was established. Abstract. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals. C. elegans is highly amendable to functional genetics because of its short generation time, ease of use, and wealth of available gene-alteration techniques.
Oleaginous microalgae and yeasts represent promising candidates for large-scale production of lipids, which can be utilized for production of drop-in biofuels, nutraceuticals, pigments, and cosmetics. The NAD(P)H-dependent Pichia stipitis xylose reductase (PsXR) is one of the key enzymes for xylose fermentation, and has been cloned into the commonly used ethanol-producing yeast Saccharomyces cerevisiae.
plasmid-mediated enhancement of mutagenesis. PCR amplificaton of target plasmid with two phosphorylated primers. The protocol is simple (see Figure 1) The mutagenesis protocol comprises four steps: 1. ELSEVIER Mutation Research 310 (1994) 187-209 Fundamental and Molecular Mechanisms of Mutagenesis Comparative mutagenicity of chemicals selected for test in the International Program on Chemical Safety's collaborative study on plant systems for the detection of environmental mutagens William F. Grant a,,, Michael F. Salamone b a Genetics Laboratory, Department of Plant Science, … Assays for Mutagenesis.