Part:BBa_K4206002:Experience
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Applications of BBa_K4206002
Procedures
1. Gibson assembly
Forming the plasmid contains cell-glu1and cell-glu2.
2. Transformation
Following the Single Tube Transformation Protocol provided by iGEM, except for the process of heat shock.
We conducted heat-shock for 30sec at 42°C on a heat block, according to Mr. Tashiro, working in the synbio lab at Kyushu Univ, who offers us cells.
3. Culturing
Picking the colonies and cultured them in LB mediums with ampicillin. The shaking culture was conducted in a shaking water bath at 168 min^-1 overnight.
4. Dissolving cellulose
Culturing like "3. Cultureing" with cellulose. Added 0.03-0.06mg of cellulose powder (Wako 28μm) to the working mixture, containing 3μl of culture medium, ampicillin and 3ml of LB medium.
5. Assay consentration of glucose
According to the protocol offered by assay kit's manifacture, make the standard curve. Since LB medium contains glucose and absorbrance, glucose samples used in calibration was made by dliting 10mmol/L Glucose Standard with LB mediums instead of water.
Sampleing culture medium containing cellulose and mix with the glucose assay kit.
Mesuring absorbrance of 400-540nm by spectrometer (USIO Picoscpe).
The protocol is here;https://www.dojindo.co.jp/products/G264/
Result
In table1, there is a trend toward increased glucose levels up to 6 hours, while there is no significant trend in CONTROL
After one night of incubation, glucose levels in both cases have decreased significantly.
This is assumed that the rate of cellulose degradation exceeded the rate of glucose consumption, as a result of E. coli growth and more glucose being used as a nutrient source, while cellulose was precipitated and was not degraded as much.
User Reviews
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