Team:Berkeley Wetlab/Passenger: MGFP

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Contents

What is it?

Applications of surface display of mgfp-5

Functional Assay: BCA Protein Quantification Assay

BCA Assay.jpg


This assay tests for the presence of mgfp-5 displayed on the surface of E. coli through its ability to bind to the bottom of polystyrene plates.

Constructs used:

  • mgfp-5 + spacers + displayers (79)
  • mgfp-5 + displayers (14)
  • displayers only (15)

Experiment run with 5x replicate of each condition


Day 1: Grow Cells

  • Pick cells, using pin tool, with appropriate constructs from frozen stock plates into 96-well blocks with 1 ml of LB+AC media
  • Place blocks in 37C shaker overnight to grow to saturation

Day 2: Induce Cells

  • Set up 96-well blocks with 1 ml of LB+AC+arabinose media:
    • Add 1 ul of arabinose at a concentration of (100mg/ml) to one ml of LB+AC media (1: 1000)
  • Take plates with constructs out of the 37C incubator and add 10 ul of each construct to the LB+AC+arabinose media to induce
  • Incubate blocks at 37C in shaker overnight.

Day 3: Incubate in Artificial Seawater

  • Remove blocks from incubator
  • Pellet cells at 5400 rpm for 7 minutes
  • Remove supernatant by flicking block over a sink (swiftly inverting block in one motion)
  • Re-suspend cells in 100 ul of artificial seawater
  • Transfer 50 ul of cell suspensions with multi-pipette to labeled flat bottom 96-well costar plates (polystyrene plates)
  • Take OD 600 readings in TECAN
  • Cover plates with plastic film and let cells settle overnight

Day 4: Run BCA Protein Quantification Assay

  • Uncover plates and flick out the liquid from each plate over a sink.
  • Wash each plate with 100 ul of artificial seawater (using dispense 100 ul program on the washing robot)
  • Flick out liquid again (this should remove the cells that are unbound to the surface of the polystyrene plates)
  • Add 25 ul of Tris buffer + 2% SDS to each well, to lyse cells
  • Set up BCA standards in empty wells on the plates for each condition replicate:
    • 0.25, 0.75 and 1.0 mg of bovine serum albumin(BSA)/ ml of Tris Buffer + 2% SDS
  • Incubate plates at 50C for 15 minutes
  • While plates are incubating, set up another set of flat-bottom polystyrene plates with 190 ul of BCA reagents for each construct: BCA reagents are (1:50 ratio of BCA reagent (A) little blue bottle : (B) big white bottle.
  • Remove plates from incubator and transfer 10 ul of each lysate to the corresponding well in the plates with the BCA reagents.
  • Incubate plates for 40 minutes at 37C
  • Take OD 562 measurements on the TECAN
  • Analyze Data

Results

One-tailed t-test analysis

Mgfp-5 with linkers/spacers and mgfp-5 without spacers:

9-30 t-tests.jpg