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Team:Berkeley Wetlab/Passenger: TypeIII Needle scFv
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Contents |
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
Results
==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=== *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<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> 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] [[Image:10-2-09 dot blot unnormalized.JPG|150px]]<br> 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> *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<br> Also transform and plate more espA *dot blot without normalization - block nitrocellulose and visualize. <br> *preparation/materials for dot blot with normalization (should happen on Tues): cells = grow sunday<br> protein = purify, should have by Tues<br> PBS = should have 10X big bottle<br> .05% and .1% SDS = need to make (ask Terry Sunday) <br> benzonase nuclease = need to get (from LSA?)<br> negative controls = restreak 1363, set of no pass grow as well <br> ==September== ===9-1-09=== *cloning espA into histag vector: PCR espA analytical gel - <br> [[Image:8-28 espA pcr analytical.jpg|150px]]<br> expected size is 580bp gel purified vector - <br> [[Image:8-29 T4lig and T7RNAP vector purification.jpg|150px]]<br> expected sizes: T7RNAP = 2383/4326bp (NcoI/EcoRI) <br> restriction map of espA in his tag vector - <br> [[Image:8-31 restriction map of espA in histag vector.jpg|150px]]<br> expected sizes: 4326/580bp (NcoI/EcoRI)<br> 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. <br> ===8-29-09=== cloned espA into his tag vector. pick colonies tomorrow.
