Cellulose degradation

The complete cellulose hydrolysis reaction require Endo5a cellulase, B-Glucosidase, exo-β-1,4-glucanase and Endo-1,4-beta-glucanase.

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/

Acetate production

AatA is an ABC transporter found in Acetobacter acetii which moves acetate out of the cell. It is known that this gives resistance to high levels of acetic acid in the cellular environment and drives the acetate production. PoxB,Pyruvate dehydrogenase, is a peripheral cell membrane enzyme that catalyzes the oxidative decarboxylation of pyruvate to form acetate and CO2. In this project, poxB is regulated by lac promoter and aatA is constitutively expressed. Cultivate transformed E. coli in medium supplemented with IPTG and measure OD600 and acetic acid concentration every hour.

Procedures

1. Gibson assembly

   Forming the plasmid contains aatA and poxB.

2. Transformation

   Please refer to "cellulose degradation - Transformation"

3. Culturing

   Pick colonies and culture them in LB-cam containing ampicillin overnight.

4. IPTG induction

   Add 30μl of culture fluid to 3 ml of LB-medium containing ampicillin and 0-1.5μmol of IPTG. Cultured for 4-5h at 37℃ in culture tubes in a shaking water bath at 168 min^-1

5. Measure the cocentration of acetic acid every hour

   • According to the protocol offered by assay kit's manifacture, make the standard curve. Since LB medium might contains acetate and absorbrance, acetate samples used in calibration was made by dliting acetate Standard with LB mediums instead of water.
     The protocols is here; https://www.bioassaysys.com/datasheet/EOAC.pdf
   • Sampleing culture medium and mix with the working reagent of assay kit.
   • Mesure absorbrance of 455-630nm by spectrometer (USIO Picoscpe) every hour to determine the concentration of acetic acid based on the standard curve.
   • OD600 was measured too by spectometer.

Measuring the pH of the culture

To prove the production and transportation of acetate ion, pH tests were performed on cultures containing E. coli transformed with AatA and PoxB.

Procedures

1. Culture the acetobacteria

   Propagate the E.Coli transformed in "Acetate production". Add the 30μl of culture fluid and ampicillin to 3ml of LB medium. Concurrently, IPTG induction is coducted; each tube contain 0-1.5μM of IPTG. Under the circumstance, the E.Coli have been cultured overnight in culture tubes in shaking water bath at 168min^-1, 37℃.

2. Measure pH of the culture fluid

   After culturing overnight, pH tests are conducted based on pH test paper.