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Team:Berkeley Wetlab/Passenger: Streptavidin
From 2009.igem.org
(→Functional Assay: Fluorescent Plate Reader) |
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====cell growth and induction==== | ====cell growth and induction==== | ||
| + | 1. Grow a cells in a liquid culture overnight to saturation. | ||
| + | 2. Make a 1:10 dilution the following day of the saturated cultures and innoculate with arabinose (100 μg/mL final concentration) and incubate for 5-6 hours. | ||
| + | 3. | ||
| + | |||
| + | 1. inoculate cells from stock into LB with appropriate antibiotics and grow to saturation overnight (12+ hours) | ||
| + | 2. take OD 600 measurements of saturated culture to ensure approximately uniform cell densities. | ||
| + | 3. add 20uL of saturated culture to 0.5mL of LB containing 0.01g dissolved Azo-CM and 0.5 uL each of arabinose and the appropriate antibiotics | ||
| + | 4. allow cells to grow overnight | ||
When pelleting the cells, use the centrifuge at 5000 RPM for 5 minutes.<br><br> | When pelleting the cells, use the centrifuge at 5000 RPM for 5 minutes.<br><br> | ||
'''Experiment:'''<br> | '''Experiment:'''<br> | ||
0. Grow cells to saturation (overnight)<br><br> | 0. Grow cells to saturation (overnight)<br><br> | ||
| - | 1. Induce with arabinose: for 500uL total volume in blocks - dilute culture 1:10 in media. Typically, inoculate 1:1000 add arabinose | + | 1. Induce with arabinose: for 500uL total volume in blocks - dilute culture 1:10 in media. Typically, inoculate 1:1000 add arabinose Before assaying, measure OD600.<br><br> |
2. Add 100 uL of cells to wells in a polystyrene (PS) V-bottomed 96-well plate. | 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.<br><br> | Seal plate with a film and pellet the cells.<br><br> | ||
Revision as of 02:36, 21 October 2009
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Contents |
Strepavidin Binding Peptide
With the display of numerous of on the surface of a cell, there is a need determine the viability of the proteins. One such method is to tag the displayed proteins. By displaying a strepavidin binding protein, we can effectively tag displayers as well as other proteins of interest. The strepavidin binding protein is a short peptide sequence (SAECHPQGPPCIEGRK) that binds onto strepavidin. Using a streptavidin, R-phycoerythrin conjugate (SAPE), we can fluorescently tag proteins and displayers for high through-put, automated analysis.
Functional Assay: Fluorescent Plate Reader
This assay tests for the presence of a functional strep binding peptide on the E. coli cell surface, and its ability to bind to streptavidin, R-phycoerythrin conjugate (SAPE)
Constructs:
Strepavidin binding protein with GGSG linker (15)
Strepavidin binding protein with INP repeat linker (14)
1363 Negative Control (1) (Strepavidin binding protein targeted to periplasm)
9494 Positive Control (1) (Displayed circularly permuted OmpX)
All experiments are done in triplicate
cell growth and induction
1. Grow a cells in a liquid culture overnight to saturation. 2. Make a 1:10 dilution the following day of the saturated cultures and innoculate with arabinose (100 μg/mL final concentration) and incubate for 5-6 hours. 3.
1. inoculate cells from stock into LB with appropriate antibiotics and grow to saturation overnight (12+ hours) 2. take OD 600 measurements of saturated culture to ensure approximately uniform cell densities. 3. add 20uL of saturated culture to 0.5mL of LB containing 0.01g dissolved Azo-CM and 0.5 uL each of arabinose and the appropriate antibiotics 4. allow cells to grow overnight
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 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.
Results
Data
