Team:Berkeley Wetlab/Passenger: Cellulases

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(→Functional Assay: Azo-CM-Cellulose)
 
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[[Team:Berkeley_Wetlab/Buttons]]
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{{newtemplateBerkeley}}
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__NOTOC__
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==What is it?==
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==Cellulases==
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Cellulases are enzymes that catalyze the degradation of cellulose.  Displaying cellulases on the cell surface allows the enzymes to act on insoluble cellulose present in the media that cannot diffuse into the cytoplasm.  We made four cellulase display systems using cel9a, cel3a, cel5b, and cel6a from Celvibrio Japonicus.  These cellulases contain cellulose binding domains which bind the enzyme to the cellulose substrate and are thought to disrupt the crystal structure of cellulose.  Of the four cellulases, we were able to characterize the activity of the two endoglucanases, cel9a and cel5b. 
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==Applications of Surface Display of scFv==
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==Functional Assay: Azo-CM-Cellulose==
==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'''
'''This assay measures 1,4-Beta-D-endoglucanase activity using a soluble cellulose that has been dyed with Remazolbrilliant Blue'''
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[[Assay Illustration]]<br>
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[[Image:cellassay.jpg|600px|center]]<br>
'''constructs:''' <br>
'''constructs:''' <br>
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#inoculate cells from stock into LB with appropriate antibiotics and grow to saturation overnight (12+ hours)
#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.
#take OD 600 measurements of saturated culture to ensure approximately uniform cell densities.
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#add 20uL of saturated culture to 0.5mL of LB containing 0.01g dissolved Azo-CM and 500uL each of arabinose and the appropriate antibiotics
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#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
#allow cells to grow overnight
====precipitate undegraded cellulose====
====precipitate undegraded cellulose====
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#Add 1mL Precipitation Buffer to each culture.
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#Add 1mL [https://2009.igem.org/Team:Berkeley_Wetlab/Recipes#Precipitation_Buffer 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
#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.   
#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.
#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.
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[https://2009.igem.org/Recipes TBST recipe]
 
==Results==
==Results==
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====Data====
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[[Image:cellulaseData.jpg|900px]]<br>
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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.
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====Displayers + TypeIII Needle scFv====
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====Conclusions====
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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.
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====Negative Controls====
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=====1363 (no surface display)=====
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=====Displayers Only=====
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[[Image:Displayers only.jpg|300px]]<br>
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=====Displayers + different scFv=====
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===10-10-09===
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*Results of the dot blots with the 3rd set of cells.
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[[Image:100909 dot blot scfv - overlay - better version.jpg|150px]]<br>
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unnormalized. with No Pass on the right. 0.5ul of cell lysate. block 45min. 3X washes. antibody incubated 1hr. 1:500 ab:TBST. <br>
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There are no signals on the no pass. there is some background and also the negative controls also have a bit of signal. however, some of the constructs had definite signals that were a lot darker than the other signals. namely, yuaQ and upaG_short. of the three dot blots done, 3 constructs were consistent-ish hits: AIDA, espP, and cpg_l2. YuaQ, azo, and ecpx were hits on 2nd and 3rd blot (they were not present in the first blot). 3 constructs were hits on the first and 3rd blot: hia, hag, and upaG_short.
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===10-8-09===
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*just induced another set of cells for another dot blot: this one will include more negative controls: just LB, no surface display (1363), displayer only, displayer with a different passenger. the inoculated cells were grown for 27hours. The No pass set had huge cell mass settling at the bottom.
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-goals of this experiment: is to verify the results obtained on 10-7-09; get neater data for the Jamboree (i.e. neat rows on nitrocellulose).
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induced cells for 12 hours. There were considerable pellet loss in the no pass plate, except for cl02365, pcryo, CPG_L2, espP, yuaQ, and azo1653. These constructs were also the ones that did not resuspend and needed pipeting.
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dot blotted (#1) unnormalized 0.5ul dots. some of the volumes were not entirely uniform. need to establish neater blots for the presentation.
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===10-6-09===
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*dot blot #2 10-6-09
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some notes: newly purified espA was used<br>
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some modifications: 45min block with BSA, 4ml of 1:200 ab: TBST, 90min wait<br>
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results: will post blot and analyze. the results were hard to interpret since everything was so dark. <br>
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do again: <br>
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use same lysate and blot again on nitrocellulose .5ul instead of 1ul (again unnormalized), since some of the stuff on blot#2 seems to have washed off. (note that lysate was put out on bench top for ~9hours before blotting on nitrocellulose)<br>
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[[Image:100709 dot blot scfv 0.5ul 2nd set of cells.jpg|150px]]<br>
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these results show hits on: ehab, vta, opr, ecpx, yua, azo, cpg, espp, and AIDA. Also note that the negative controls did not have significant signal.
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===10-5-09===
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*analyze results from first dot blot
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[http://openwetware.org/wiki/Image:10-2_needle_dot_blot_assay.xls OD600 measurements for 10-2-09 dot blot experiment]
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[[Image:10-2-09 dot blot unnormalized.JPG|150px]]<br>
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some possible hits: hag, hia, upaG_short,CPG_L2, espP, possibly AIDA. there is also one dot on what should be 1363. Not sure if that is significant.<br>
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there is no significant difference between ODs of the negative controls and the Needle scFv constructs that showed on the dot blot.<br>
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note: the orientation of the blot is not completely sure.<br>
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Latest revision as of 18:03, 21 October 2009

Cellulases

Cellulases are enzymes that catalyze the degradation of cellulose. Displaying cellulases on the cell surface allows the enzymes to act on insoluble cellulose present in the media that cannot diffuse into the cytoplasm. We made four cellulase display systems using cel9a, cel3a, cel5b, and cel6a from Celvibrio Japonicus. These cellulases contain cellulose binding domains which bind the enzyme to the cellulose substrate and are thought to disrupt the crystal structure of cellulose. Of the four cellulases, we were able to characterize the activity of the two endoglucanases, cel9a and cel5b.

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

Cellassay.jpg

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

  1. inoculate cells from stock into LB with appropriate antibiotics and grow to saturation overnight (12+ hours)
  2. take OD 600 measurements of saturated culture to ensure approximately uniform cell densities.
  3. 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
  4. allow cells to grow overnight

precipitate undegraded cellulose

  1. Add 1mL Precipitation Buffer to each culture.
  2. The high molecular weight fragments of cellulose will precipitate out leaving only the digested lower molecular weight fragments in solution
  3. Pellet for 10 minutes at 5400x.
  4. 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

Data

CellulaseData.jpg
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.

Conclusions

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.

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