Team:Berkeley Wetlab/Passenger: TypeIII Needle scFv

From 2009.igem.org

(Difference between revisions)

Latest revision as of 18:03, 21 October 2009

TypeIII Needle scFv

The TypeIII secretion system (TTSS) is present in many pathogenic bacteria such as E. Coli O157:H7 and is a mechanism of their virulence. Specifically, EspA, a subunit of the TypeIII secretion complex, is a virulence factor expressed by both Enteropathogenic and Enterohaemorrhagic (EHEC)bacteria. It has a filament structure that aids in delivery of effector proteins to the host cell. Conditions such as haemorrhagic colitis are caused by EHEC. The surface display of an scFv (single-chain variable fragment) that can bind to espA can disrupt the function of the TTSS, which may have probiotic as well as pathogen detection applications.

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.

basic theory behind experiment The incubation of cells expressing the anti-espA scFv with espA protein allows for specific binding. The washes following incubation separates the bound and unbound espA proteins, such that when the cells are lysed, only the bound espA protein is released into solution and subsequently blotted onto the nitrocellulose. An anti-his antibody conjugated to HRP is used to probe the blot for the his-tagged espA. Signals are read via chemiluminescence.

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

Most of the constructs have signals larger than the negative control, but two of them gave higher signals than the rest: hia and upaG_short.

These dot blots have not been normalized to protein concentration. However, this does not make a significant difference in the results since the starting ODs (indicating growth) of the bacteria for the constructs and negative controls do not significantly differ.

The negative controls did not have significant signal.

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.

Quantitaive Image J Analysis of Dot Blots

This data represents the averaged fold increase of three sets of data normalized to the negative control. The data indicates preliminarily the functional display of scFv with displayers Hia, upaG_short, yuaQ, CPG_L2, and espP(beta).

References

La Ragione, R et al. Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro. Microbes and Infection. September 2005; 8: 426–433. Available Online: http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6VPN-4H4T215-1-7&_cdi=6211&_user=4420&_orig=search&_coverDate=02%2F28%2F2006&_sk=999919997&view=c&wchp=dGLbVtz-zSkzS&md5=f35699db5dcd202f4cd1c1d8739d89b0&ie=/sdarticle.pdf (Accessed: 20 October 2009).

Retrieved from "http://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_TypeIII_Needle_scFv"

@@ Line 1: / Line 1: @@
-==What is it?==
+{{newtemplateBerkeley}}
+__NOTOC__
+==TypeIII Needle scFv==
+The TypeIII secretion system (TTSS) is present in many pathogenic bacteria such as E. Coli O157:H7 and is a mechanism of their virulence. Specifically, EspA, a subunit of the TypeIII secretion complex, is a virulence factor expressed by both Enteropathogenic and Enterohaemorrhagic (EHEC)bacteria. It has a filament structure that aids in delivery of effector proteins to the host cell. Conditions such as haemorrhagic colitis are caused by EHEC. The surface display of an scFv (single-chain variable fragment) that can bind to espA can disrupt the function of the TTSS, which may have probiotic as well as pathogen detection applications.
-==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.'''
-[[Assay Illustration]]<br>
+[[Image:Scfvassay1 2.jpg|center]]
+'''basic theory behind experiment''' The incubation of cells expressing the anti-espA scFv with espA protein allows for specific binding. The washes following incubation separates the bound and unbound espA proteins, such that when the cells are lysed, only the bound espA protein is released into solution and subsequently blotted onto the nitrocellulose. An anti-his antibody conjugated to HRP is used to probe the blot for the his-tagged espA. Signals are read via chemiluminescence.
 '''constructs:''' <br>
@@ Line 43: / Line 47: @@
 ==Results==
 ====Displayers + TypeIII Needle scFv====
+[[Image:Results1.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><br>
+[[Image:Dotblot set1.jpg]]<br>
+Most of the constructs have signals larger than the negative control, but two of them gave higher signals than the rest: hia and upaG_short.<br>
-====Negative Controls====
+These dot blots have not been normalized to protein concentration. However, this does not make a significant difference in the results since the starting ODs (indicating growth) of the bacteria for the constructs and negative controls do not significantly differ.<br>
+[[Image:10-2 OD600.jpg]]<br>
-=====1363 (no surface display)=====
-=====Displayers Only=====
-=====Displayers + different scFv=====
-==October==
-===10-10-09===
-*Results of the dot blots with the 3rd set of cells.
-[[Image:100909 dot blot scfv - overlay - better version.jpg|150px]]<br>
-unnormalized. with No Pass on the right. 0.5ul of cell lysate. block 45min. 3X washes. antibody incubated 1hr. 1:500 ab:TBST. <br>
-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===
+[[Image:Results2.jpg]]<br><br>
-*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.
+[[Image:Dotblot set 2.jpg|900px]]<br>
--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).
+The negative controls did not have significant signal.
-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.
+[[Image:Results3.jpg]]<br><br>
+[[Image:Dotblot set 3.jpg|900px]]<br>
+There is some detectable background as denoted by the slight signals in the column with just lysis buffer.<br>
+Several of the Needle scFv constructs did have positive signals higher than that of the negative controls. (yuaQ and upaG_short) <br>
+====Displayers Only (negative)====
+[[Image:Displayers numbered.jpg]]<br>
+No detectable signal is present in the set of experiments run with only displays expressed from the cells.
-===10-6-09===
+There were three constructs that showed higher signals consistently in all three sets of experiments. They are AIDA, espP, and cpg_L2.
-*dot blot #2 10-6-09
-some notes: newly purified espA was used<br>
-some modifications: 45min block with BSA, 4ml of 1:200  ab: TBST, 90min wait<br>
-results: will post blot and analyze. the results were hard to interpret since everything was so dark. <br>
-do again: <br>
-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>
-[[Image:100709 dot blot scfv 0.5ul 2nd set of cells.jpg|150px]]<br>
+====Quantitaive Image J Analysis of Dot Blots====
-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===
+[[Image:Average chart.jpg|900px]]<br>
-*analyze results from first dot blot
+[[Image:ScFv fold increase.jpg|600px]]<br>
-[http://openwetware.org/wiki/Image:10-2_needle_dot_blot_assay.xls OD600 measurements for 10-2-09 dot blot experiment]
+This data represents the averaged fold increase of three sets of data normalized to the negative control. The data indicates preliminarily the functional display of scFv with displayers Hia, upaG_short, yuaQ, CPG_L2, and espP(beta).
-[[Image:10-2-09 dot blot unnormalized.JPG|150px]]<br>
+===References===
-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>
+La Ragione, R et al. Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro. Microbes and Infection. September 2005; 8: 426–433. Available Online: http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6VPN-4H4T215-1-7&_cdi=6211&_user=4420&_orig=search&_coverDate=02%2F28%2F2006&_sk=999919997&view=c&wchp=dGLbVtz-zSkzS&md5=f35699db5dcd202f4cd1c1d8739d89b0&ie=/sdarticle.pdf (Accessed: 20 October 2009).
-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>