Team:Berkeley Wetlab/Passenger: TypeIII Needle scFv

From 2009.igem.org

(Difference between revisions)

Revision as of 20:15, 18 October 2009

What is it?

Applications of Surface Display of scFv

Functional Assay: Dot Blot

This assay measures the success of E. coli surface display of typeIII needle scFv and its ability to bind specifically to espA protein, a subunit of the typeIII needle complex in EHEC.

constructs:
Needle scFv (14)
Gliadin neg control (2)
No pass neg control (14)
1363 negative control (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)
dilute culture 1:100 into media with arabinose and induce for 5-12hours
pipet 100ul of cells to Costar V-bottom polystyrene plate and take OD
pellet cells and flick out media

incubation with espA protein

add 5ul of espA protein to 95ul of PBS buffer and resuspend cells in solution
incubate for 1.5 hours
wash 3X with PBS buffer on plate washer
take OD on 3rd wash
after last wash, pellet cells, and resuspend in 50ul lysis buffer
heat for 15-20 minutes to lyse all cells

dot blot and visualization

pipet 5ul of cell lysate onto nitrocellulose paper and allow to dry completely
wet nitrocellulose with 10ml of 1X TBST for 5minutes
block with 2% BSA (.2g BSA in 10ml 1X TBST)for 1 hour
wash 3X with TBST - 5minutes/wash
add 4ml of 1:500 Anti-His HRP: TBST, wait 30minutes
wash 3X with TBST - 5minutes/wash
leave the last wash on for developing
add 3mls of chemiluminescent reagents to the nitrocellulose paper and take pictures

TBST recipe

Results

Displayers + TypeIII Needle scFv

lysis buffer only negative
gliadin scFv negative
No display (1363) negative
The negative controls did not have significant signal.

lysis buffer only negative
gliadin scFv negative
No display (1363) negative
There is some detectable background as denoted by the slight signals in the column with just lysis buffer.
Several of the Needle scFv constructs did have positive signals higher than that of the negative controls. (yuaQ and upaG_short)

Displayers Only (negative)

No detectable signal is present in the set of experiments run with only displays expressed from the cells.

There were three constructs that showed higher signals consistently in all three sets of experiments. They are AIDA, espP, and cpg_L2.

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.

@@ Line 45: / Line 45: @@
 ====Displayers + TypeIII Needle scFv====
 [[Image:Results2.jpg]]<br>
+<font color="turquoise">lysis buffer only negative</font><br>
+<font color="green">gliadin scFv negative</font><br>
+<font color="blue">No display (1363) negative</font><br>
+The negative controls did not have significant signal.
 [[Image:Results3.jpg]]<br>
@@ Line 60: / Line 64: @@
-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]
@@ Line 69: / Line 71: @@
 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>
 there is no significant difference between ODs of the negative controls and the Needle scFv constructs that showed on the dot blot.<br>
-note: the orientation of the blot is not completely sure.<br>