Team:Berkeley Wetlab/Passenger: Cellulases

From 2009.igem.org

(Difference between revisions)
(→Functional Assay: Azo-CM-Cellulose)
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'''constructs:''' <br>
'''constructs:''' <br>
-
Needle scFv (14)<br>
+
Cel9a, an endoglucanase from Celvibrio japonicus (16)<br>
-
Gliadin neg control (2)<br>
+
Cel5b, an endoglucanase from Celvibrio japonicus (16)<br>
-
No pass neg control (14)<br>
+
Industrial cellulose degrading strain (14)<br>
1363 negative control (1)<br>
1363 negative control (1)<br>
 +
Cel5b, only prepro (1)<br>
''All experiments are done in triplicate''
''All experiments are done in triplicate''
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====cell growth and induction====
====cell growth and induction====
#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)
-
#dilute culture 1:100 into media with arabinose and induce for 5-12hours
+
#take OD 600 measurements of saturated culture to ensure approximately uniform cell densities.
-
#pipet 100ul of cells to Costar V-bottom polystyrene plate and take OD
+
#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
-
#pellet cells and flick out media
+
#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.
 +
 
 +
 
-
====incubation with espA protein====
 
-
#add 5ul of espA protein to 95ul of PBS buffer and resuspend cells in solution
 
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#incubate for 1.5 hours
 
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#wash 3X with PBS buffer on plate washer
 
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#take OD on 3rd wash
 
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#after last wash, pellet cells, and resuspend in 50ul lysis buffer
 
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#heat for 15-20 minutes to lyse all cells
 
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====dot blot and visualization====
 
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#pipet 5ul of cell lysate onto nitrocellulose paper and allow to dry completely<br>
 
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#wet nitrocellulose with 10ml of 1X TBST for 5minutes<br>
 
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#block with 2% BSA (.2g BSA in 10ml 1X TBST)for 1 hour<br>
 
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#wash 3X with TBST - 5minutes/wash
 
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#add 4ml of 1:500 Anti-His HRP: TBST, wait 30minutes
 
-
#wash 3X with TBST - 5minutes/wash
 
-
#leave the last wash on for developing
 
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#add 3mls of chemiluminescent reagents to the nitrocellulose paper and take pictures
 
[https://2009.igem.org/Recipes TBST recipe]
[https://2009.igem.org/Recipes TBST recipe]

Revision as of 19:12, 18 October 2009

Team:Berkeley_Wetlab/Buttons

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

Assay Illustration

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 500uL 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.



TBST recipe

Results

Displayers + TypeIII Needle scFv

Negative Controls

1363 (no surface display)
Displayers Only

Displayers only.jpg

Displayers + different scFv

10-10-09

  • Results of the dot blots with the 3rd set of cells.

150px
unnormalized. with No Pass on the right. 0.5ul of cell lysate. block 45min. 3X washes. antibody incubated 1hr. 1:500 ab:TBST.
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.

10-8-09

  • 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.

-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).

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.

dot blotted (#1) unnormalized 0.5ul dots. some of the volumes were not entirely uniform. need to establish neater blots for the presentation.


10-6-09

  • dot blot #2 10-6-09

some notes: newly purified espA was used
some modifications: 45min block with BSA, 4ml of 1:200 ab: TBST, 90min wait
results: will post blot and analyze. the results were hard to interpret since everything was so dark.

do again:
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)

150px
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.

10-5-09

  • analyze results from first dot blot

[http://openwetware.org/wiki/Image:10-2_needle_dot_blot_assay.xls OD600 measurements for 10-2-09 dot blot experiment]

150px
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.
there is no significant difference between ODs of the negative controls and the Needle scFv constructs that showed on the dot blot.
note: the orientation of the blot is not completely sure.

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