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Team:Berkeley Wetlab/Passenger: Cellulases
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==Results== | ==Results== | ||
| - | After running the assay, activity was observed for all cel5b constructs independent of the display system chosen. As this seemed to be an unusual result when compared to other passenger data, the assay was run again using a control of Cel5b with only the periplasmic targeting prepro sequence and no fused display system. It was found that Cel5b activity required only a prepro sequence and was not dependent upon the attached display system. | + | After running the assay, activity was observed for all cel5b constructs independent of the display system chosen. As this seemed to be an unusual result when compared to other passenger data, the assay was run again using a control of Cel5b with only the periplasmic targeting prepro sequence and no fused display system. It was found that Cel5b activity required only a prepro sequence and was not dependent upon the attached display system. The high standard deviation is hypothesized to be due to unusual cell growth condition due to the dissolved Azo-CM-cellulose. Attempts to run the assay in lower concentrations of dissolved Azo-CM-cellulose (10 times dilution) gave no results for any construct including the positive control. |
====Assay Results from September 14th==== | ====Assay Results from September 14th==== | ||
[[Image:cellulaseData.jpg|900px]]<br> | [[Image:cellulaseData.jpg|900px]]<br> | ||
Revision as of 20:31, 18 October 2009
Contents |
What is it?
Applications of Surface Display of scFv
Functional Assay: Azo-CM-Cellulose
This assay measures 1,4-Beta-D-endoglucanase activity using a soluble cellulose that has been dyed with Remazolbrilliant Blue
constructs:
Cel9a, an endoglucanase from Celvibrio japonicus (16)
Cel5b, an endoglucanase from Celvibrio japonicus (16)
Industrial cellulose degrading strain (14)
1363 negative control (1)
Cel5b, only prepro (1)
All experiments are done in triplicate
cell growth and induction
- inoculate cells from stock into LB with appropriate antibiotics and grow to saturation overnight (12+ hours)
- take OD 600 measurements of saturated culture to ensure approximately uniform cell densities.
- add 20uL of saturated culture to 0.5mL of LB containing 0.01g dissolved Azo-CM and 0.5 uL each of arabinose and the appropriate antibiotics
- allow cells to grow overnight
precipitate undegraded cellulose
- Add 1mL Precipitation Buffer to each culture.
- The high molecular weight fragments of cellulose will precipitate out leaving only the digested lower molecular weight fragments in solution
- Pellet for 10 minutes at 5400x.
- Take OD 590 measurements of 100uL of each sample. The higher the reading, the more blue digested fragments were present in the media and the stronger the endoglucanase activity.
Results
After running the assay, activity was observed for all cel5b constructs independent of the display system chosen. As this seemed to be an unusual result when compared to other passenger data, the assay was run again using a control of Cel5b with only the periplasmic targeting prepro sequence and no fused display system. It was found that Cel5b activity required only a prepro sequence and was not dependent upon the attached display system. The high standard deviation is hypothesized to be due to unusual cell growth condition due to the dissolved Azo-CM-cellulose. Attempts to run the assay in lower concentrations of dissolved Azo-CM-cellulose (10 times dilution) gave no results for any construct including the positive control.
Assay Results from September 14th
