"

 

From 2009.igem.org

Contents 1 Mussel Adhesive Protein: MGFP-5 2 Functional Assay: BCA Protein Quantification Assay 2.1 Day 1: Grow Cells 2.2 Day 2: Induce Cells 2.3 Day 3: Incubate in Artificial Seawater 2.4 Day 4: Run BCA Protein Quantification Assay 3 Results 3.1 MGFp-5 + Linkers data 3.1.1 INP-Repeats 3.1.2 Beta Roll 3.1.3 Beta Helix 3.1.4 Gly-Ser Repeats 3.1.5 GFP-LVA 3.2 MGFP - no linkers data 3.3 Displayers only data 3.4 One-tailed t-test analysis

Mussel Adhesive Protein: 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:

1. mgfp-5 + spacers + displayers (79)
2. mgfp-5 + displayers (14)
3. displayers only (15)

Experiment run with 5x replicate of each condition

Day 1: Grow Cells

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

Day 2: Induce Cells

1. 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)
2. Take plates with constructs out of the 37C incubator and add 10 ul of each construct to the LB+AC+arabinose media to induce
3. Incubate blocks at 37C in shaker overnight.

Day 3: Incubate in Artificial Seawater

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

Day 4: Run BCA Protein Quantification Assay

1. Uncover plates and flick out the liquid from each plate over a sink.
2. Wash each plate with 100 ul of artificial seawater (using dispense 100 ul program on the washing robot)
3. Flick out liquid again (this should remove the cells that are unbound to the surface of the polystyrene plates)
4. Add 25 ul of Tris buffer + 2% SDS to each well, to lyse cells
5. 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
6. Incubate plates at 50C for 15 minutes
7. 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.
8. Remove plates from incubator and transfer 10 ul of each lysate to the corresponding well in the plates with the BCA reagents.
9. Incubate plates for 40 minutes at 37C
10. Take OD 562 measurements on the TECAN
11. Analyze Data

Results

MGFp-5 + Linkers data

INP-Repeats

Graph of the mean OD562 signals normalized to OD600 data from September 30th:

Beta Roll

Beta Helix

Gly-Ser Repeats

GFP-LVA

MGFP - no linkers data

Displayers only data

One-tailed t-test analysis

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

• Recent changes
• What links here
• Related changes
• Special pages
• My preferences

• Printable version
• Permanent link
• Privacy policy
• Disclaimers