Template:SBB- Streptavidin Pellet Assay

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Positive Controls: (Amp resistant)

  • pBca9145-Bca9494 in TG1 cell-line{AraC-Pbad}{rbs.cpx} (this also contains a strep tag and has been tested for display)
  • pBca9145-Bca9494 in EC100D cell-line
  • __________-Bca9494 in EC100D cell-line (?)

Negative controls:(CA resistant)

  • pBca9495CA-Bca1363 in EC100D

When pelleting the cells, use the centrifuge at 5000 RPM for 5 minutes.

Experiment:
0. Grow cells to saturation (overnight)

1. Induce with arabinose: for 500uL total volume in blocks - dilute culture 1:10 in media. Typically, inoculate 1:1000 add arabinose (100 μg/mL final concentration) and incubate for 5-6 hours. Before assaying, measure OD600.

2. Add 100 uL of cells to wells in a polystyrene (PS) V-bottomed 96-well plate. Seal plate with a film and pellet the cells.

3. Remove seal and flick away LB media, keeping the cell pellets. Always visually inspect the plate to confirm that no pellets were lost.

4. Resuspend pellets in 100 uL PBS with 5.0 ug/mL of Streptavidin-PE.

5. Seal well plate with a film and incubate at 37C without shaking for 30 minutes.

6. Pellet the cells, remove the seal, and flick away PBS/Streptavidin-PE, keeping the cell pellets.

7. Resuspend pellets in 100 uL PBS.

8. Pellet cells again and flick away the PBS wash.

9. Invert plate very gently and visualize dry pellets using an epi UV lamp and an EtBr filter. Photograph the plate (TIF or RAW preferably).

Hint: the camera focus may be off if it's been used to photograph gels. If so, move the camera focus switch on the side of the barrel to AF (auto focus) and take a picture of the plate with no UV and the door open. A piece of paper towel on top of the plate can also help. Once you have the camera focused on the top of the plate, switch the camera back to MF (manual focus) to preserve your focus setting.

10. Resuspend the pellets in 100 uL PBS, then pellet the cells and flick away the PBS wash.

11. Resuspend one last time in 100 uL PBS, taking care that the pellet has been completely resuspended.

12. Take an OD measurement after 3 washes, to ensure that we have not lost too much of the cells.

13. TECAN the plate using the following settings:

Measurement mode: Fluorescence
Excitation wavelength: 488 nm
Emission wavelength: 575 nm
Excitation bandwidth: 20 nm
Emission bandwidth: 20 nm
Gain (Manual): 50
Number of reads: 20
FlashMode: High sensitivity
Integration time: 40 µs
Lag time: 0 µs
Z-Position (Manual): 5100 µm
Shaking: Medium, linear


Image analysis (to be performed only when visual observation doesn't match up with the TECAN analysis):
1. Open the image in ImageJ. Go to Image > Type > RGB Stack. This will separate the image into three grayscale images representing the brightness in the red, green, and blue channels. Analyze the red channel image.

2. Go to Analyze > Set Measurements and make sure that "Integrated Density". is checked.

3. Use the Ellipse tool to draw a circle around one of your pellets. If your circle is too large it will pick up a lot of noise from the plastic well. Click Control-M to measure the integrated density of your selection - a spreadsheet will open containing your data.

4. Drag the ellipse to the next pellet. (Do not redraw the ellipse - that will give you messy data.) Click Control-M. Repeat until you've measured every pellet.

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