Template:SBB- Streptavidin TECAN Assay

Streptavidin Display TECAN Assay
 Materials   cells expressing a displayer protein harboring a streptavidin binding peptide  R-phycoerythrin conjugated streptavidin  1X PBS  96-well Costar plate (black wall, clear-bottom)

 Procedure  Day 1 1. Make stock media for the experimental and control samples, containing the appropriate antibiotics and/or inducers. E.g., in the case of pBAD on an AMP-resistant plasmid, make up X mL of LB-AMP media + 1/100X dilution of 10mg/mL arabinose. 2. Using a multichannel p1000 pipet and a sterile media trough, transfer 500uL media to an autoclaved, deep well (2mL), 96-well plate. Inoculate 10uL of saturated culture to this plate using a p20 multichannel pipet. Inoculate 1/500X dilution into media containing the appropriate antibiotics and/or inducers. In the case of pBAD, inoculate i 3.  Allow to grow to saturation (~ 5 hours), then centrifuge the plate for 5 min at 5000 rpm. 4. Remove the entire supernatant (500uL) using a multichannel pipet, by aspirating from the CORNER of the wells. 5. Resuspend in 200uL 1X PBS. Add an appropriate volume of fluorescent streptavidin; incubate while shaking for 30min at 37deg. 6. Centrifuge the plate for 5 min at 5000 rpm. Remove the entire supernatant (500uL) using a multichannel pipet, by aspirating from the CORNER of the wells. Repeat 2 more times. 7. Transfer 100uL the resupended solution a black wall, clear-bottom, 96-well Costar plate. 8. Turn on the Tecan Safire2 plate reader. Then, on the computer, start the Xfluor4Safire2 software by clicking on the desktop icon. Enable macros. Go the Xfluor4SafireII menu and click "connect". Open the "Load measurement parameters" from the same menu. Choose "iGEM2009_streptavidin_FluorParameters" file. Finally, click "start measurement" from the menu.

Solutions

1X PBS (scale up or down as necessary) - Dissolve in 800 ml of distilled H2O: - 8 g of NaCl - 0.2 g of KCl - 1.44 g of Na2HPO4 - 0.24 g of KH2PO4 - Adjust the pH to 7.4 with HCl or NaOH - Add H2O to 1 liter.

Positive Control: pBca9495-M10220 induced in arabinose Negative controls: pBca9495-1363 induced in arabinose, pBca9495-1363 uninduced, and pBca9495-M10220 uninduced.

1. Measure cell concentration by measuring OD600.

2a. Centrifuge the R-phycoerythrin suspension at 10,000g for 10 min at 4°C. 2b. Discard the supernatant and resuspend the pellet (R-phycoerythrin ) into the desired buffer. 2c. Desalt the sample using either Sephadex G-25 or dialysis. ?

2. Dilute streptavidin in PBS: 8 uL of streptavidin-R-phycoerythrin to 72 uL PBS. 3. Setup in a 96 well plate the following: Four columns (1-4), one for each control; and 9 rows (A-I) of 3 varying strept concentrations in triplicate (A-C conc. 1, D-F conc. 2, G-I conc. 3) For .5 uL concentration add 5 uL of streptavidin in PBS to 95 uL of cells (total of 100 uL) to wells A-C. For .25 uL concentration add 2.5 uL of streptavidin in PBS to 97.5 uL of cells to each wells D-F. For 1.0 uL concentration add 10 uL of streptavidin in PBS to 90 uL of cells to wells G-I. 4. Incubate for __ at 37 C. 5. Spin cells at 5500 rpm for 5 min. 6. Read phycoerythrin fluorescence of each well at __, __, __ z-positions. 7. Remove supernatant from each well and move to another plate or empty wells. 8. Read phycoerythrin fluorescence of supernatant at TECAN-optimized z-position. 9. Wash cell pellets in PBS. 10a. Resuspend cells in PBS. 10b. Lyse cells in __. 11. Read phycoerythrin fluorescence of cell lysate at TECAN-optimized z-position as well as OD600 to determine remaining cell concentration.

Alternative:

Positive control is pBca9145-Bca9494 {AraC-Pbad}{rbs.cpx}

Negative control is pBca9495CA-Bca1363 {Pbad.rbs.prepro.StrepTag}

Wells containing (a) 140 ul of cell media and 20 uL PBS and (b) 140 ul of cell media and 20 uL streptavidin in PBS should be included to measure background fluorescence.

1. Grow cells to saturation and induce with arabinose. Typically, inoculate overnight, add arabinose in the morning, and assay in the afternoon. Before assaying, measure OD600. 2. Add 140 uL of cells to wells in a 96-well PCR plate (max volume: 200 microliters/well). 3. For each sample, add 20 uL of streptavidin (0.04 mg/mL) in PBS to each well (for a final concentration of 0.005 mg/mL). Incubate at 37C without shaking for 30 minutes. 4. Pellet cells by spinning down plate at 5000 RPM for 5 minutes. 5. Read phycoerythrin fluorescence of cell lysate at the TECAN-optimized z-position, at 5,100um, and 10,100um. 6. Pellet cells by spinning down plate at 5000 RPM for 5 minutes. 7. Move supernatant to empty plate wells and resuspend cells in 140 uL of PBS. 8. Read phycoerythrin fluorescence of cell lysate at the TECAN-optimized z-position, at 5,100um, and 10,100um. 9. Repeat 6-8 (wash steps) until the number of washes is adequate to produce clean results (or the cell loss becomes noticeable). 10. Lyse cells before last TECAN read?