By Qiong Cheng
Metabolic engineering is the perform of genetically optimizing metabolic and regulatory networks inside of cells to extend construction and/or restoration of sure substance from cells. In Microbial Metabolic Engineering: equipment and Protocols professional researchers within the box element a number of the tools that are now established to review metabolic engineering. those contain tools and methods to engineer genes and pathways, use of recent biotechnology instruments in microbial metabolic engineering, and examples of metabolic engineering for genuine international purposes similar to entire telephone biosensors and acetate keep watch over in huge scale fermentation. Written within the hugely profitable tools in Molecular Biology™ sequence layout, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, without problems reproducible laboratory protocols, and key pointers on troubleshooting and averting identified pitfalls. Authoritative and functional, Microbial Metabolic Engineering: equipment and Protocols seeks to supply researchers with an outline of key issues on microbial metabolic engineering.
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Extra resources for Microbial Metabolic Engineering: Methods and Protocols (Methods in Molecular Biology Vol 834)
Sample text
Cultivation conditions for high-throughput expression: when growing small culture volumes at 37°C, prevent uneven evaporation of cultures across the plates by increasing the shaker’s humidity to 80%. 13. 5 mM IPTG worked very well for the expression of wild-type IlvC and its variants. However, the IPTG concentration should be adjusted in cases where inclusion bodies are observed. 14. Freezing the 96-well plates containing the cell pellets is essential for a successful lysis step. The plates should be frozen for a minimum of 2 h, preferably overnight.
3. Digestion and Quantification of Recovered Plasmids 1. Digest 5 mL of eluted plasmid from each miniprep (see Note 10), using an appropriate restriction enzyme and buffer in a 20-mL volume reaction, following the manufacturer’s specifications (see Note 11). 2. 8% agarose gel using TAE buffer. 44 S. Million-Weaver et al. 3. Load the gel with an appropriate DNA ladder. 4. Mix each digest with an appropriate amount of gel loading buffer. Load the entire digest into each well of the gel. 5. Run the gel at 120 V, 400 mA for 60–90 min.
1. Transformation of ColE1 Plasmid by Heat-Shock (Used for Our pGFPuv Experiments; see Note 1–4) 1. Prepare a 4-mL overnight culture in LB media of the cell line of interest. Include selective antibiotic in the media for the desired cell line. 2. Expand this overnight culture into a sterile 2-L Erlenmeyer flask containing 400 mL of LB media. 3. 6). 4. Chill the flask containing cells on wet ice for 15 min. 5. Transfer the liquid cultures to plastic centrifuge bottles, and centrifuge for 15 min at 4°C and 4,000 rpm.