Team:Berkeley Wetlab/Passenger: Streptavidin

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(Functional Assay: Fluorescent Plate Reader)
 
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{{newtemplateBerkeley}}
{{newtemplateBerkeley}}
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__NOTOC__
==Strepavidin Binding Peptide==
==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.
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==
==Functional Assay: Fluorescent Plate Reader==
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'''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)'''<br>
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'''This assay tests for the presence of a functional strep binding peptide on the E. coli cell surface via its ability to bind to streptavidin, R-phycoerythrin conjugate (SAPE)'''<br>
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[[Image:IGem2009Berkeley_strep_cartoon.JPG]]<br>
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[[Image:IGem2009Berkeley_strep_cartoon.JPG|center]]<br>
'''Constructs:''' <br>
'''Constructs:''' <br>
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====incubate with SAPE and Assay====
====incubate with SAPE and Assay====
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1. Add 100 uL of cells to a polystyrene (PS) V-bottomed 96-well plate. Centrifuge and pellet the cells. <br>
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2. Flick away LB media and resuspend pellets in 100 uL PBS with 5.0 ug/mL of Streptavidin-PE.<br>
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3. Seal well plate with a film and incubate at 37C without shaking for 30
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minutes.<br>
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4. Pellet the cells, remove the seal, and flick away PBS/Streptavidin-PE, keeping the cell pellets.<br>
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5. Pellet cells again and flick away the PBS wash.<br>
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6. Resuspend the pellets in 100 uL PBS, then pellet the cells and flick away
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the PBS wash.<br>
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7. Resuspend one last time in 100 uL PBS, taking care that the pellet has been completely resuspended.<br>
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8. Take an OD measurement after 3 washes, to ensure that we have not lost too much of the cells. <br>
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9. Take a Fluorescence measurement at Excitation wavelength of 488nm and emission wavelength 575 nm<br>
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==Results==
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===Data===
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<center>[[image:IGem2009Berkeley_strep_linkers.JPG|300px]]<br> '''UV image of strepavidin binding peptides''' <br>
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'''incubated with strepavidin and washed.'''<br></center>
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[[Image:IGem2009Berkeley_linkers_comparison.JPG|900px]]<br>
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Fluorescence of strepavidin binding protein with GGSG linker and INP repeats. The INP repeats greatly improved display.
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===Conclusion===
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While at first it seems trivial to display a small peptide such as the strepavidin binding protein, from the results, it seems that this is not the case. Many of the displayed strepavidin binding proteins did not work. There seems to be a strong context effect between the linker sequence used and the function of the displayed protein. The use of a larger linkers such as an INP repeat seems to help the cell surface display.
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When pelleting the cells, use the centrifuge at 5000 RPM for 5 minutes.<br><br>
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==References==
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'''Experiment:'''<br>
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Jeffrey J. Rice and Patrick S. Daugherty. Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides. Protein Engineering, Design and Selection. July 2008; 21(7): 435–442. Available online at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2427320/
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0. Grow cells to saturation (overnight)<br><br>
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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>
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2. Add 100 uL of cells to wells in a polystyrene (PS) V-bottomed 96-well plate.
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Seal plate with a film and pellet the cells.<br><br>
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3. Remove seal and flick away LB media, keeping the cell pellets. Always
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visually inspect the plate to confirm that no pellets were lost.<br><br>
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4. Resuspend pellets in 100 uL PBS with 5.0 ug/mL of Streptavidin-PE.<br><br>
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5. Seal well plate with a film and incubate at 37C without shaking for 30
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minutes.<br><br>
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6. Pellet the cells, remove the seal, and flick away PBS/Streptavidin-PE, keeping the cell pellets.<br><br>
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7. Resuspend pellets in 100 uL PBS.<br><br>
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8. Pellet cells again and flick away the PBS wash.<br><br>
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9. Invert plate very gently and visualize dry pellets using an epi UV lamp and an
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EtBr filter. Photograph the plate (TIF or RAW preferably).<br><br>
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Hint: the camera focus may be off if it's been used to photograph gels. If so,
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move the camera focus switch on the side of the barrel to AF (auto focus) and
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take a picture of the plate with no UV and the door open. A piece of paper
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towel on top of the plate can also help. Once you have the camera focused on
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the top of the plate, switch the camera back to MF (manual focus) to preserve
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your focus setting.<br><br>
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10. Resuspend the pellets in 100 uL PBS, then pellet the cells and flick away
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the PBS wash.<br><br>
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11. Resuspend one last time in 100 uL PBS, taking care that the pellet has been completely resuspended.<br><br>
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12. Take an OD measurement after 3 washes, to ensure that we have not lost too much of the cells. <br><br>
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13. TECAN the plate using the following settings:<br><br>
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Measurement mode: Fluorescence<br>
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Excitation wavelength: 488 nm<br>
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Emission wavelength: 575 nm<br>
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Excitation bandwidth: 20 nm<br>
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Emission bandwidth: 20 nm<br>
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Gain (Manual): 50 <br>
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Number of reads: 20 <br>
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FlashMode: High sensitivity<br>
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Integration time: 40 µs<br>
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Lag time: 0 µs<br>
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Z-Position (Manual): 5100 µm<br>
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Shaking: Medium, linear<br><br><br>
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'''Image analysis (to be performed only when visual observation doesn't
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K. Terpe. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology. January 2003; 60(5): 523-533. Available online at: http://www.springerlink.com/content/euj72wc9a8l23dtu/
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match up with the TECAN analysis):'''<br>
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1. Open the image in ImageJ. Go to Image > Type > RGB Stack. This will
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separate the image into three grayscale images representing the brightness in
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the red, green, and blue channels. Analyze the red channel image.<br><br>
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2. Go to Analyze > Set Measurements and make sure that "Integrated Density". is checked.<br><br>
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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.<br><br>
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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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==Results==
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===Data===
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[[image:IGem2009Berkeley_strep_linkers.JPG|300px]]<br>
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===Conclusions===
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Latest revision as of 18:02, 21 October 2009

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 via its ability to bind to streptavidin, R-phycoerythrin conjugate (SAPE)

IGem2009Berkeley strep cartoon.JPG

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. Before assaying, measure OD600.

incubate with SAPE and Assay

1. Add 100 uL of cells to a polystyrene (PS) V-bottomed 96-well plate. Centrifuge and pellet the cells.
2. Flick away LB media and resuspend pellets in 100 uL PBS with 5.0 ug/mL of Streptavidin-PE.
3. Seal well plate with a film and incubate at 37C without shaking for 30 minutes.
4. Pellet the cells, remove the seal, and flick away PBS/Streptavidin-PE, keeping the cell pellets.
5. Pellet cells again and flick away the PBS wash.
6. Resuspend the pellets in 100 uL PBS, then pellet the cells and flick away the PBS wash.
7. Resuspend one last time in 100 uL PBS, taking care that the pellet has been completely resuspended.
8. Take an OD measurement after 3 washes, to ensure that we have not lost too much of the cells.
9. Take a Fluorescence measurement at Excitation wavelength of 488nm and emission wavelength 575 nm

Results

Data

IGem2009Berkeley strep linkers.JPG
UV image of strepavidin binding peptides
incubated with strepavidin and washed.

IGem2009Berkeley linkers comparison.JPG
Fluorescence of strepavidin binding protein with GGSG linker and INP repeats. The INP repeats greatly improved display.

Conclusion

While at first it seems trivial to display a small peptide such as the strepavidin binding protein, from the results, it seems that this is not the case. Many of the displayed strepavidin binding proteins did not work. There seems to be a strong context effect between the linker sequence used and the function of the displayed protein. The use of a larger linkers such as an INP repeat seems to help the cell surface display.

References

Jeffrey J. Rice and Patrick S. Daugherty. Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides. Protein Engineering, Design and Selection. July 2008; 21(7): 435–442. Available online at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2427320/

K. Terpe. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology. January 2003; 60(5): 523-533. Available online at: http://www.springerlink.com/content/euj72wc9a8l23dtu/