Team:TUST China/Experiments
Experience And Protocols
G. xylinus
| G. xylinus Broth Preparation(g/L) | G. xylinus Agar Preparation (g/L) | |
|---|---|---|
| Glucose | 25 | 25 |
| yeast extract powder | 7.5 | 7.5 |
| peptone | 10 | 10 |
| Na2HPO4 | 20 | |
| Agar | <20/td> | |
| pH | 6 | |
| Sterilization | Autoclave 121℃ for 20min |
- Inoculum activation
Harvest a loop of bacteria from Agar Slant using inoculation loop, inoculating it onto 50ml seed culture media (containing 4%（V/V） cellulolytic enzyme) in 250ml triangular flask. Incubate at 180 r/min，30℃, until its OD600 reach to 0.4~0.6, and save it as seed solution for later use. - Preparation for seed solution
Inoculate the seed solution into 200mL seed culture media (containing 4%（V/V） cellulolytic enzyme)in 500mL triangular flask. Incubate at 30℃，180r/min in shaker for 24 hours until the OD600 reach to 0.5. - Flask-shaking Fermentation
Following 4.5%(V/V) inoculum size, innoculate the seeds into the 200mL broth culture medium in 500mL triangular flask. Incubate at 30℃，180r/min in shaker for 120 hours. - Stationary culture
Following 4.5%(V/V) inoculum size, innoculate the seeds into the 200mL broth culture medium in 500mL triangular flask. Incubate at 30℃ in incubator.
Preparation of electro-competent G,xylinus cells(Imperial)
If the goal of transformation is to produce cellulose-producing transformed G.xylinus, use regular HS media for culturing. If cellulose production after transformation is not primary and speed is required, HS-cellulase medium can be used. Usage of HS-cellulase medium during growth results in the formation of a higher number of cellulose negative mutants, but results in much higher transformation efficiencies, and requires less time. Even with HS-cellulose medium, cellulose producing colonies can be identified on the plate after transformation, as cellulose-producing colonies differ in morphology from cellulose non-producing colonies (see section Gluconacetobacter for images of colony morphology).
Transformation of G.xylinus using electroporation
under oscillating condition.
Inoculate a loop of overexpression strain, parent strain, and empty vector strain(control group) onto seed culture media respectively.Incubate them until OD600＝0.5. Following 4.5%（v/v）inoculum size, inoculate the strains onto fermentation media(containing 4%（v/v） cellulolytic enzyme) . Incubate at 30℃180rpm for 120h. Sampling and measure OD600, PH, residual sugar and gluconic acid for periodically.
under still condition.
Inoculate a loop of overexpression strain, parent strain, and empty vector strain(control group) onto seed culture media respectively.Incubate them until OD600＝0.5. Following 4.5%（v/v）inoculum size, inoculate the strains onto fermentation media(containing 4%（v/v） cellulolytic enzyme) Incubate at 30℃ ,let stew for 16 days. Sampling and measure OD600, PH, residual sugar and gluconic acid for periodically.
Methods of measuring these parameters.
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Measuring yield of cellulose
Using 0.1M NaOH to remove the cells and residual media in cellulose, soaking it in water until pH reach to neutral. Dry to constant weight and weigh its mass.
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Measuring optical density of bacteria solution.
Take 3mL fermenting liquid from each sampling points, use empty control group as zero setting. Utilize ultraviolet-visible spectrophotometer under 600nm to measure its OD.
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Measuring pH of fermentation solution.
Take 3mL fermenting liquid from each sampling points, utilize pH meter to measure its pH and make sure it has been adequately shaken.
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Measuring residual sugar
Take 3mL fermenting liquid from each sampling points, centrifuge 5000 r/min for 5 min. fetch the supernatant liquid. Dilute and measure the concentration of glucose in the liquid using SBA-40C biosensor.
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Measuring gluconic acid
Preparation of chromatography: Aminex HPX-87H Strong acid cation exchange resin column, mobile phases 1Mm HClO4, flow velocity 0.5ml/min, column temperature 25℃,UV detector, wavelength 210nm, Injection amount 20μL , ESTD method.
