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Team:Berkeley Wetlab/Passenger: MGFP
From 2009.igem.org
Contents |
What is it?
Applications of surface display of mgfp-5
Functional Assay: BCA Protein Quantification Assay
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:
