Team:Berkeley Wetlab/Passenger: TypeIII Needle scFv

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Contents

Dot Blot

This assay measures the success of 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 neg control (1)

All experiments are done in triplicate

cell growth and induction

  1. grow cells to saturation overnight (12+ hours)
  2. dilute culture 1:100 into media with arabinose and induce for 5-12hours
  3. pipet 100ul of cells to Costar V-bottom polystyrene plate and take OD
  4. pellet cells and flick out media

incubation with espA protein

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

dot blot and visualization

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

TBST recipe

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