Difference between revisions of "Team:UESTC-China/Demonstrate"

Fig. 1 Double restriction enzyme digestion of three constructed vectors analyzed by using agarose gel electrophoresis.

(a) piGEM2018-Module001 digested by Eco32Ⅰ+NcoⅠ(lane 1), piGEM2018-Module001 digested by NcoⅠ+XhoⅠ(lane 2);

(b) piGEM2018-Module002 digested by PstⅠ+KpnⅠ(lane 1), piGEM2018-Module002 digested by HindⅢ+KpnⅠ(lane 2);

(c) piGEM2018-Module003 digested by EcoRⅠ+NcoⅠ(lane 1), piGEM2018-Module003 digested by BamHⅠ+BglⅡ(lane 2).

Fig. 2 Enzyme activity detection using crude extract fraction of strain with or without. Module001：All sample were collected after fermentation for 24 h.
(a) The activity of xylanase. U/mL definition: 1 ml of enzyme solution (at 40 °C pH = 6.0) 1 h decomposition of xylan to produce 1 mg xylose, regarded as an enzyme unit [1];
(b) The activity of ferulic acid esterase. U/mL definition: The amount of enzyme (at 40 °C pH = 6.4) required to degrade 1 μmol of 4-nitrophenyl trans-ferulate per minute is one Unit of ferulic acid esterase activity [2];
(c) The activity of total cellulase. U/mL definition: 1 ml of enzyme solution (at 40 °C pH = 7.0) hydrolyzed the filter paper per minute to produce 1.0 μmol of glucose, which is one Unit of total cellulase activity [3];
(d) The activity of endoglucanase. U/mL definition: 1 ml of enzyme solution (at 40 ° C pH = 7.0) hydrolyzed CMC-Na per minute to produce 1.0 μmol of glucose, which is one Unit of endoglucanase activity [4].

Fig. 3 The production of ferulic acid determined using GC. The supernatants of fermented liquid of E. coli BL21(DE3) with or without piGEM2018-Module001were used for the determination of ferulic acid, respectively. Samples were shaking with corn straw and taken from reaction mixtureat 0h, 24h.

Fig. 4 Mass spectrums of ferulic acid derivative. Sample was the supernatants of E. coli BL21(DE3) carrying piGEM2018-Module001 after fermentation for 24h.

Fig. 5 Activity determination of cellulase using Congo Red assay. (a) CMC agar plate before staining with Congo Red. (b)CMC agar plate after staining with Congo Red. Module001: BL21(DE3) carrying piGEM2018-Module001（OD600: 1, 3, 5 from left to right); Positive Control: commercial cellulase (Enzyme concentration: 0.2, 0.3, 0.4 mg/ml from left to right); Negative Control: BL21 (DE3) carrying empty vector (OD600: 1, 3, 5 from left to right)

Fig. 6 The production of butanol determined using GC. The supernatants of fermented liquid of E. coli BL21(DE3) with or without piGEM2018-Module002 were used for the determination of butanol, respectively. For anaerobic growth, precultures were adjusted to OD600 10 with 2 mL of fresh medium with 50mg/mL kanamycin. The culture was transferred to a sealed 10ml vacuum tube. Cultures were shaken (200 rpm) at 37 °C for 24 h. Then samples were taken from reaction mixture at 0h, 24h.

Fig. 7 The butanol production curve at optimized conditions (temperature=34℃, initial OD=13, initial pH=8)

Fig. 8 Overall setup of the hydrogen collection device.

Video 1. The video of gas combustion test. Samples were collected from the engineered E. coli DH5α carrying the plasmid piGEM2018-Module003 and wild type plasmid after shaking 48h in TB medium.

Fig. 9 A diagram of the volume of hydrogen generated over time in a gas collection device. Recombinant strain and original strain were fermented in 200 ml 1% (W/V) glucose M9 medium.

Fig. 10 Comparing our hydrogen production with relevant literatures.

Fig. 11 After adding 0.3ml butanol in 30ml LB medium, the measurement of optical density (OD600) deference between DH5α with GroESL and Negative Control in 24 hour.

Fig. 12 E. coli self-metabolic pathway