Experiment notes TypeIII Needle scFv

From 2009.igem.org

(Difference between revisions)
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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>
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.
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.
-
 
-
*to dos:
 
-
-with same lysate, add DNAseI and run unnormalized set of dot blots with same lysate (from 10-9-09) to see if could get better reading.
 
-
-with same lysate, add DNAseI, do a BSA, quantify the protein, add the appropriate amts of SDS. dot blot that onto nitrocellulose.
 
-
-visualize both of those on Monday
 
===10-8-09===
===10-8-09===
Line 32: Line 27:
[[Image:100709 dot blot scfv 0.5ul 2nd set of cells.jpg|150px]]<br>
[[Image:100709 dot blot scfv 0.5ul 2nd set of cells.jpg|150px]]<br>
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.
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.
-
 
-
play with: DNaseI concentration to get optimal. BCA assay and total protein normalization. use smaller set of cells.
 
-
 
-
grow more cells. scfv, no pass, 1363, other scfv.
 
===10-5-09===
===10-5-09===
-
[[protocol for the dot blot 10-5-09]]
 
-
 
-
to dos:
 
-
*do a coomassie test of purified protein in PBS buffer to test for presence of protein (protein is present)
 
-
*try a test run of .5ul DNAseI and write out the protein quantification protocol for the dot blot
 
*analyze results from first dot blot  
*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]
[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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*run the second dot blot tmr
*run the second dot blot tmr
modification: added .5-1ul DNaseI to the cell lysate. [DNaseI] unknown. did not effectively get rid of genomic DNA. DNaseI more in the first rows. took 1ul for dot blot. froze the rest of the ~49ul.  
modification: added .5-1ul DNaseI to the cell lysate. [DNaseI] unknown. did not effectively get rid of genomic DNA. DNaseI more in the first rows. took 1ul for dot blot. froze the rest of the ~49ul.  
-
 
-
===10-4-09===
 
-
to dos:<br>
 
-
*DNaseI is active in 2% SDS. for a 50ul suspension, can add 1ul of DnaseI. Will try to see if will work for .5ul of DNaseI.
 
-
*purify espA - put through column and dialyze overnight
 
-
*run the dot blot
 
-
*induce cells for tues
 
===10-3-09===
===10-3-09===
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benzonase nuclease = need to get (from LSA?)<br>
benzonase nuclease = need to get (from LSA?)<br>
negative controls = restreak 1363, set of no pass grow as well <br>  
negative controls = restreak 1363, set of no pass grow as well <br>  
-
 
-
===10-2-09===
 
-
*Run Needle experiment (dot blot) again.
 
-
-used PBS, not TRIS, for washes and incubation steps<br>
 
-
-for the first row of triplicates, I pipeted up and down to resuspend in espA. The second row, I shook it on the shaker. could the cells have been sheared or otherwise damaged by pipetting up and down?<br>
 
-
-it seems that the first gliadin scFv (used as a negative control), has this clumping phenotype, and when resuspended in espA, is just a clump with surrounding solution being clear.<br>
 
-
 
-
===10-1-09===
 
-
*ran a test BCA assay with inoculated cells. Initial OD was taken. Cells were spun down and the LB flicked out. Resuspended in 2% SDS and incubated at 50C for 30minutes. put in various amounts of cell lysate: 20ul to 200ul of reagents (10x dil), and 2ul to 198ul of reagents (100X dil). Also ran standard with BSA diluted to 1ml in 2%SDS and TRIS. Standard concentrations were 0, .25, and .50 mg/ml of BSA. Froze lysate.
 
-
 
-
results: There was purple. The controls showed that putting in too much of the protein can reduce signal. 20ul set showed purple, the 2ul set didn't. after incubating for 30minutes.
 
-
yay it works.<br>
 
-
[http://openwetware.org/wiki/Image:10_1_test_bca_562nm.xls 562nm BCA test assay results]<br> A very rough ball park of protein concentration of the whole cell is ~.1mg/ml (i think)
 
==September==
==September==
-
===9-22-09===
 
-
[[DotBlot Assay]]
 
-
 
===9-19-09===
===9-19-09===
*outgrowth assay #4 with modified protocol:
*outgrowth assay #4 with modified protocol:
Line 95: Line 58:
*The results for the 9-19-09 outgrowth assay is here: [http://openwetware.org/wiki/Image:9-19-09_outgrowth_assay.xls results for outgrowth assay]<br>
*The results for the 9-19-09 outgrowth assay is here: [http://openwetware.org/wiki/Image:9-19-09_outgrowth_assay.xls results for outgrowth assay]<br>
It seems that the no pass constructs grow much faster than the needle constructs, and the OD measurements do not show significant different between the needle and no pass constructs. Although, when compared to the gliadin negative controls, the needle constructs did have a significantly higher signal.  
It seems that the no pass constructs grow much faster than the needle constructs, and the OD measurements do not show significant different between the needle and no pass constructs. Although, when compared to the gliadin negative controls, the needle constructs did have a significantly higher signal.  
-
 
-
 
-
to dos: 1. calculate protein conc. based on bsa standard curve 2. analyze data from 9-19-09 outgrowth assay(make sure write down exp conditions 3. write protocol for dot blot assay by Sunday
 
-
 
-
===9-18-09===
 
-
[[new and improved outgrowth assay]] (includes BCA assay protocol)
 
-
 
-
*grew up cells for outgrowth assay tomorrow. Needle scFv (pin tool and cell issue)
 
===9-16-09===
===9-16-09===
Line 113: Line 68:
Results: [http://openwetware.org/wiki/Image:9-16-09_outgrowth_assay.xls 9-16-09 outgrowth data] <br>
Results: [http://openwetware.org/wiki/Image:9-16-09_outgrowth_assay.xls 9-16-09 outgrowth data] <br>
somethings to do next time: take OD right after adding media, use nonFITC conjugated espA in case the FITC has a negative effect on binding.<br>
somethings to do next time: take OD right after adding media, use nonFITC conjugated espA in case the FITC has a negative effect on binding.<br>
-
 
-
to do: gather materials and make up protocol for dot blot assay.<br>
 
there is espA protein, purified, in the fridge
there is espA protein, purified, in the fridge
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looked at after 7hours: growth, cl02365, ehab, azo <br>
looked at after 7hours: growth, cl02365, ehab, azo <br>
looked at after 8hours: growth, cl02365, ehab, upaG short, azo<br>
looked at after 8hours: growth, cl02365, ehab, upaG short, azo<br>
-
 
-
Will repeat experiment on 9-15 to verify results<br>
 
===9-12-09===
===9-12-09===
-
*Outgrowth assay: [[protocol for 9-12-09 experiment]]
+
*Outgrowth assay:  
results: [http://openwetware.org/wiki/Image:9-12-09_outgrowth_assay_1.xls 9-12-09 outgrowth assay]
results: [http://openwetware.org/wiki/Image:9-12-09_outgrowth_assay_1.xls 9-12-09 outgrowth assay]
-
 
-
*[[protocol for western blot needle assay]]
 
===9-11-09===
===9-11-09===
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===9-08-09===
===9-08-09===
-
*centrifuge speeds for the experiment: slowly ramp up to prevent shear of protein from scFv (actually Josh said that it's not a problem)
 
-
 
[http://openwetware.org/wiki/Image:9-8-09_pull_down_needle_assay.xls 9-8-09 pull down assay 1st run data]<br> This is the first run of the pull down assay. This assay assumes that the cells will bind to a certain amount of FITC-labeled espA and when centrifuged, will be pulled down, leaving the supernatant with less fluorophore. <br>
[http://openwetware.org/wiki/Image:9-8-09_pull_down_needle_assay.xls 9-8-09 pull down assay 1st run data]<br> This is the first run of the pull down assay. This assay assumes that the cells will bind to a certain amount of FITC-labeled espA and when centrifuged, will be pulled down, leaving the supernatant with less fluorophore. <br>
The results are not very telling. And there are some things that need to be improved.  <br>
The results are not very telling. And there are some things that need to be improved.  <br>
Conditions for this experiment include: overnight inoculation to saturation. 5hr induction of 1:10 dilution of cells. incubation of cells with espA-FITC (1000X diluted) for 1.5hours. A titration curve was generated. <br>
Conditions for this experiment include: overnight inoculation to saturation. 5hr induction of 1:10 dilution of cells. incubation of cells with espA-FITC (1000X diluted) for 1.5hours. A titration curve was generated. <br>
Improvements: need better aspirating technique for taking up supernatant. need to establish centrifuge speed and time. need a better titration curve. <br>
Improvements: need better aspirating technique for taking up supernatant. need to establish centrifuge speed and time. need a better titration curve. <br>
-
 
-
*Things to do/try tomorrow: 1. Tecan the 16hr incubation pull down experiment (include a FITC blank) <br> 2. Look at cells under the microscope <br> 3. purify protein for the growth assay.<br> 4. continue to read literature about espA and Needle scFv to understand the system more and also other ways that people assayed the system.<br>
 
===9-07-09===
===9-07-09===
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*also grew up 10ml starter culture for more espA protein. Will start protein purification again tomorrow. This is to prepare protein for binding-growth experiment
*also grew up 10ml starter culture for more espA protein. Will start protein purification again tomorrow. This is to prepare protein for binding-growth experiment
-
*should look up the binding affinity of needle scFv. in general, read more about the needle scFv and the cell surface display of scFvs to come up with better conditions and experiments.
 
-
 
-
*looked up induction time for cell surface expression: grow until OD600 = 0.6 (midlog) and then induce for 6hours [source: Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface]
 
-
 
-
<pre>
 
-
results for assay: crappy
 
-
- should maybe test the lysate. histag scFv.
 
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- know for sure that binding of espA and scFv is good? according to a paper
 
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- are you making protein period? tag with GFP. or functional assay (fail so far). increase volume to a larger scale system. run stuff on a gel preinduction and postinduction. or even do a western.
 
-
- functionality period? test if in cytosol. lyse cells, do soluble binding assay.
 
-
- if is functional, why is it not going on surface?? other ways to display.
 
-
- what is it is functional but not functional on surface? add linker. test different linkers. other than linkers what other schemes can you do? is it N or C terminally fused.
 
-
- buffers, salts, need something else to be active, sterics (like cells binding to eachother?), protein could have non specific binder but not likely. Tecan reading? What about the cell junk?
 
-
</pre>
 
[http://openwetware.org/wiki/Image:9-7-09_8hr_incubation_1to100dil_sol_binding.xls 9-7-09 data]<br>
[http://openwetware.org/wiki/Image:9-7-09_8hr_incubation_1to100dil_sol_binding.xls 9-7-09 data]<br>
-
changes made from last time: incubation is 4hours and Tris buffer is used instead of PBS. A 1:100dilution and growth for 8hours is done in addition to 5hr induction
+
changes made from last time: incubation is 4hours and Tris buffer is used instead of PBS. A 1:100dilution and growth for 8hours is done in addition to 5hr induction<br>
 +
results are still inconclusive.
-
===9-05-09===
+
===9-05-09==
-
[[protocol for Needle Assay]]<br>
+
[http://openwetware.org/wiki/Image:9-5-09_needle_binding_assay_2.xls results for the assay]<br>
[http://openwetware.org/wiki/Image:9-5-09_needle_binding_assay_2.xls results for the assay]<br>
-
There was no significant readings for the needle scFv.<br>
+
There was no significant readings for the needle scFv. It could be that fluorescence is too low to be detected.<br>
-
could it be: the induction (induce longer), amount of protein (add 10ul instead), the buffer (10XPBS), the affinity of the scFv to the protein (maybe too weak, so 3 washes washed all out), experimental design (the design does not give meaningful results in this assay), ''incubation time could also be too short (next time incubate for 4hours)'', ''a different buffer can be used (Tris instead of PBS, and pH it to 7.5)'', there might be an optimal pH for scFv activity<br>
+
-
 
+
-
the pH of the 10X pbs used is 7.12. <br>  
+
===9-4-09===
===9-4-09===
-
* [http://openwetware.org/wiki/Image:9-3-09_cellulase_assay_2.xls updated version of data with graphs of cellulase assay (9-3-09)]
 
-
 
* FITC conjugated the proteins: We did a spot test after pipetting the proteins out of the dialysis bag. There was protein. The protein was at exactly the pH in the protocol (which can be found in this notebook). Weighed out 3.8mgs of FITC. Added 300ul of the FITC solution into ~3ml of protein solution. Allowed incubation for 1hr at room temp. Put in dialysis bag, and will change solution tomorrow.  
* FITC conjugated the proteins: We did a spot test after pipetting the proteins out of the dialysis bag. There was protein. The protein was at exactly the pH in the protocol (which can be found in this notebook). Weighed out 3.8mgs of FITC. Added 300ul of the FITC solution into ~3ml of protein solution. Allowed incubation for 1hr at room temp. Put in dialysis bag, and will change solution tomorrow.  
-
*Also grew up cells for the assay tomorrow. All 14 constructs and negative control is Gliadin scFv with cl02365 AtD. They were grown in 1ml of LB with AC antibiotics. They will be induced into triplicated tomorrow morning and allowed to grow for 5hrs before the assay.
+
*Grew cells for the 1st soluble espA assay. All 14 constructs and negative control is Gliadin scFv with cl02365 AtD. They were grown in 1ml of LB with AC antibiotics. They will be induced into triplicate tomorrow morning for 5hours
===9-3-09===
===9-3-09===

Revision as of 15:57, 13 October 2009

Contents

October

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.

  • run the second dot blot tmr

modification: added .5-1ul DNaseI to the cell lysate. [DNaseI] unknown. did not effectively get rid of genomic DNA. DNaseI more in the first rows. took 1ul for dot blot. froze the rest of the ~49ul.

10-3-09

  • purify more espA - need to innoculate and then go through process
    Also transform and plate more espA
  • dot blot without normalization - block nitrocellulose and visualize.
  • preparation/materials for dot blot with normalization (should happen on Tues):

cells = grow sunday
protein = purify, should have by Tues
PBS = should have 10X big bottle
.05% and .1% SDS = need to make (ask Terry Sunday)
benzonase nuclease = need to get (from LSA?)
negative controls = restreak 1363, set of no pass grow as well

September

9-19-09

  • outgrowth assay #4 with modified protocol:

modifications: 3 plates with negative controls on each plate, added 5ul of espA protein in 90ul of TRIS, incubated for 5hours, took out induced cells and put them in fridge for 6.5hours

  • The results for the 9-19-09 outgrowth assay is here: [http://openwetware.org/wiki/Image:9-19-09_outgrowth_assay.xls results for outgrowth assay]

It seems that the no pass constructs grow much faster than the needle constructs, and the OD measurements do not show significant different between the needle and no pass constructs. Although, when compared to the gliadin negative controls, the needle constructs did have a significantly higher signal.

9-16-09

  • Outgrowth assay attempt #3. repeating.

changes made from the previous protocol:
1. incubated in 100ul total of espA (5ul espA protein and 95ul TRIS ph7.5)
2. incubate for ~1.75hrs
3. (washed 2X) blocked with BSA for 1.25hrs
concern: the film that covered the plate had pores, wasnt sure if when smoothing the film with hand w/out gloves caused any contamination.

Results: [http://openwetware.org/wiki/Image:9-16-09_outgrowth_assay.xls 9-16-09 outgrowth data]
somethings to do next time: take OD right after adding media, use nonFITC conjugated espA in case the FITC has a negative effect on binding.

there is espA protein, purified, in the fridge

9-14-09

  • Outgrowth assay attempt #2. looking kinda good.

Refer to the previous protocol, but with the following changes:
1. induced cells for 12hours
2. incubate maxisorp plate with espA for 1hour
3. added 100ul of 1mg/ml BSA in TRIS and incubated for 30min
4. added 5ul of cells with 95ul of 1mg/ml BSA in TRIS and incubated for 2hours
5. aspirated out cells and washed plate 3X

results:
looked at after 2.5hours, no growth
looked at after 6hours, growth, more than neg control: cl02365, ehab, oprF, hia [http://openwetware.org/wiki/Image:9-14-09_outgrowth_assay.xls Tecan data for 9-14-09]
looked at after 7hours: growth, cl02365, ehab, azo
looked at after 8hours: growth, cl02365, ehab, upaG short, azo

9-12-09

  • Outgrowth assay:

results: [http://openwetware.org/wiki/Image:9-12-09_outgrowth_assay_1.xls 9-12-09 outgrowth assay]

9-11-09

  • Ran the pull down experiment after inducing for 13hrs and incubating in espA-FITC for 8hrs.

The results are [http://openwetware.org/wiki/Image:9-9-09_fluor_Needle_assay.xls here] from the experiment done on 9-09-09.
The results are not helpful because the wells with no cells had lower readings (so maybe the proteins stuck to the bottom of the plate?).

9-08-09

[http://openwetware.org/wiki/Image:9-8-09_pull_down_needle_assay.xls 9-8-09 pull down assay 1st run data]
This is the first run of the pull down assay. This assay assumes that the cells will bind to a certain amount of FITC-labeled espA and when centrifuged, will be pulled down, leaving the supernatant with less fluorophore.
The results are not very telling. And there are some things that need to be improved.
Conditions for this experiment include: overnight inoculation to saturation. 5hr induction of 1:10 dilution of cells. incubation of cells with espA-FITC (1000X diluted) for 1.5hours. A titration curve was generated.
Improvements: need better aspirating technique for taking up supernatant. need to establish centrifuge speed and time. need a better titration curve.

9-07-09

  • cells were inoculated in LB to saturation. Experiment will be repeated, but with Tris Buffer instead of PBS and incubation will be 4hours instead of 30minutes.

-will do another set that was diluted 1:100 and induced for 8hours

  • grew up cells for trying the supernatant/subtraction experiment
  • also grew up 10ml starter culture for more espA protein. Will start protein purification again tomorrow. This is to prepare protein for binding-growth experiment

[http://openwetware.org/wiki/Image:9-7-09_8hr_incubation_1to100dil_sol_binding.xls 9-7-09 data]
changes made from last time: incubation is 4hours and Tris buffer is used instead of PBS. A 1:100dilution and growth for 8hours is done in addition to 5hr induction
results are still inconclusive.

=9-05-09

[http://openwetware.org/wiki/Image:9-5-09_needle_binding_assay_2.xls results for the assay]
There was no significant readings for the needle scFv. It could be that fluorescence is too low to be detected.

9-4-09

  • FITC conjugated the proteins: We did a spot test after pipetting the proteins out of the dialysis bag. There was protein. The protein was at exactly the pH in the protocol (which can be found in this notebook). Weighed out 3.8mgs of FITC. Added 300ul of the FITC solution into ~3ml of protein solution. Allowed incubation for 1hr at room temp. Put in dialysis bag, and will change solution tomorrow.
  • Grew cells for the 1st soluble espA assay. All 14 constructs and negative control is Gliadin scFv with cl02365 AtD. They were grown in 1ml of LB with AC antibiotics. They will be induced into triplicate tomorrow morning for 5hours

9-3-09

  • progress on needle assay: We purified the protein today, and it is dialyzing overnight. Tomorrow, we will FITC conjugate and grow cells in preparation for the soluble binding assay on Saturday.

9-1-09

  • cloning espA into histag vector:

PCR espA analytical gel -
150px
expected size is 580bp

gel purified vector -
150px
expected sizes: T7RNAP = 2383/4326bp (NcoI/EcoRI)

restriction map of espA in his tag vector -
150px
expected sizes: 4326/580bp (NcoI/EcoRI)

sequencing - used ca56 primer. looked good.

August

8-30-09

plates of espA clones look contaminated since there were large and small colonies. picked 3 clones from each plate. restriction map tomorrow.

8-29-09

cloned espA into his tag vector. pick colonies tomorrow.

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