Quantification of BC Production
Updated 10/13/2019 03:32 AM
Introduction
Materials
- Equipment
- Incubator
- High sensitivity scale
- Baking paper
- Vacuum desiccator
- Reagents
- HS medium
- K. rhaeticus
- distilled water
- 25mL 0.1M NaOH
Procedure
- Add 50mL of HS medium (or other medium of choice) to 250mL conical flask
- Grow K. rhaeticus in HS medium for 7 days standing, at 30C. Don’t seal the flasks using foam buns in order to allow diffusion of oxygen (don't seal hermetically)
- After 7 days of growth, extract and wash the cellulose twice with distilled water
- While you are washing the cellulose, turn on your waterbath to 65o C. Make sure there is enough water to submerge the cellulose sample that is in its respective container/tube.
- Add 25mL* of 0.1M NaOH to cellulose, incubate at 65o C for 4 hours in a waterbath.
*Depending on the thickness and size of the BC, there should just be enough NaOH to submerge the sheets
- Wash the cellulose twice using distilled water
- Place the formed cellulose pellicle on baking paper and dry the pellicle at 30 degrees for 4 hours or overnight at room temperature. Before drying, cut out and measure the weight of a piece of baking paper, and dry the pellicle together with the paper. This is because the pellicle will invariably stick to the surface, and removal of it results in loss of cellulose.
- Place the pellicle and paper into a vacuum desiccator for 2 hours
- Weigh the pellicle and paper using a high-sensitivity scale. Subtract the weight of the paper to determine the weight of cellulose.
This procedure aims to quantify the mass of dried cellulose produced. This was adapted from 2014 iGEM Imperial's procedures.