Team:Washington/Project/Display

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=Background= Our system hinged on finding a protein which could bind other proteins to the outside of the cell, but whose interaction with these proteins was weak enough to be disrupted by another small molecule. streptavidin presented itself as a logical choice. Several protein tags (such as the Nano-Tag1) have been developed to bind streptavidin at the biotin binding site, but can be released when biotin is added to the system and binds to streptavidin. The ability for peptides to bind streptavidin and be released upon the addition of biotin is a technology currently used on a daily basis world-wide by labs purifying proteins. The major difference between the traditional system and our system is that streptavidin is attached to beads which are used in a column format in the traditional protein purification method. Our system will have streptavidin attached to the surface of the cell. Combining the display system with our target and secretion vector our vision is complete. A single cell can then produce, secrete, bind, and release any protein of interest.

Harvard 2006 Surface Display System: Legacy Parts
When we checked the Parts Registry to see if someone had already built this or sometime similar that we could use as a starting part for our Idealized Protein Purification Display System, we found the Lpp-OmpA-streptavidin construct submitted by the 2006 Harvard iGEM team7. In fact, Harvard had submitted four biobrick parts which were variations of the same function. All four are fusion proteins which have a LPP signal peptide, either one or five trans-membrane ompA, and either monomeric or a single-chain dimeric streptavidin. All the variations and the resulting biobricks are shown below.

Harvard 2006 Surface Display System: Documented Data
Data regarding the activity of the Harvard iGEM 2006 Lpp-OmpA-Streptavidin construct has been published in IET Synthetic Biology in 20077. The author showed, by Western blot against the His tag fused to this construct, that their protein was being expressed within the cell. In order to demonstrate binding of their cell-surface-anchored streptavidin to biotin, the author incubated cells expressing Lpp-OmpA-streptavidin with an aptamer that linked biotin to a fluorophore using an oligonucleotide linker (see figure below). Any cells that express streptavidin on the surface of the cell should fluoresce in their assay - concordantly, when cells were incubated with this biotin-oligo-fluorescein aptamer, the author saw fluorescence on cells expressing Lpp-OmpA-Streptavidin, but not on Lpp-OmpA negative control. However, cells that expressed streptavidin on the surface also bound to the negative aptamer control (oligo-fluorescein) that lacked biotin, indicating that the oligo aptamer itself (and not biotin) may have been responsible for the association of the fluorophore to the cell surface. Thus, the Harvard 2006 iGEM team was never able to definitively show specific affinity of their Lpp-OmpA-streptavidin construct.

Based on these experiments, we decided to try using this display system for our project. But since binding of the system to streptavidin had not be definitively shown, we wanted to verify binding of the Lpp-OmpA-streptavidin construct to biotin for ourselves, and characterize this interaction.

=Experiments=

Harvard's Legacy 2006 Cell Surface Display Parts: Do They Work As Predicted?
In order to determine whether the Harvard 2006 iGEM streptavidin cell surface display parts were, first, expressing properly, and, second, binding biotin on the surface of the cell, we decided to do the following tests:


 * 1) Western blot to verify expression of Harvard 2006 surface display parts
 * 2) Assess biotin binding to cell surface by fluorescence microscopy
 * 3) Assess biotin binding to cell surface by flow cytometry

Western Blot Demonstrates that Harvard 2006 iGEM Parts Are Expressed
The goal of this experiment was to make sure that the proteins were being expressed in our cell strains, and also to make sure that they were the correct length. This was crucial to ascertain before we moved on and began to test the parts. Even though we had the individual parts sequenced confirmed we needed to make sure that the proteins were being expressed correctly in the cell. Since all the Harvard parts are His tagged, we used a Western blot reagent (horseradish peroxidase) that was conjugated to nickel-NTA which binds His tags.8.

Data


The expected sizes of each of these proteins are shown in the table below:

Based on the table above, we were able to verify expression of the Harvard 2006 iGEM surface display parts. The varying intensity of the bands does not indicate the strength of expression because the protein amount was not normalized before it was inserted into the gel. Next we wanted to determine whether functional, biotin-binding streptavidin was displayed on the cell surface in cells expressing these parts. To test this, we used two different methods: visualization by fluorescence microscopy and by flow cytometer.

Streptavidin-Biotin Binding Is Not Visualized By Fluorescence Microscopy
The goal of this experiment was to confirm the display of streptavidin on the surface of the cell. To do this, we incubated cells expressing the Harvard 2006 iGEM streptavidin surface display parts with a biotinylated fluorophore9. If cells are expressing functional streptavidin on the surface of the cell, they should bind the biotinylated fluorophore, and this binding should be detectable in our florescence microscope as a halo of fluorescence surrounding each individual cell. As a positive control for streptavidin-biotin binding we incubated the biotinylated fluorophore with streptavidin-coated beads that were roughly the same size (with respect to volume) as our cells (except spherical) (see Notebook page for protocols).

Streptavidin-Biotin Binding Is Not Visualized By Flow Cytometry
The goal of this experiment was to visualize biotin binding to streptavidin over a whole population of cells. Whereas the Microscope experiment looked at a few localized cells, we set the cytometer to look 50,000 streptavidin-expressing cells after incubation (and washing) with the biotinylated fluorophore and read the resulting fluorescence (see Notebook page for protocols). Like in the microscopy assay, we used streptavidin-coated beads as a positive control. The cytometer reads the fluorescence of each individual particle (cell or bead) passing through it, which allows for accurate readings, and monitoring of a much larger sample size than we visualized under the microscope. The results of this experiment are described below.

Data
In the flow cytometer, our positive control, streptavidin-coated beads, showed a clear distinction between beads pre-incubated with biotinylated fluorophore and the beads without fluorophore, indicating that this assay is capable of visualizing streptavidin binding to the biotinylated fluorophore. Like in the microscopy assay, we did not observe appreciable binding occurring with any of the ompA-streptavidin parts to biotin at the population level. This data only shows part number 48. However this data is applicable to all of the parts we observed. All streptavidin-expressing cells demonstrated low levels of fluorescence comparable to those of beads not pre-incubated with fluorophore.

Conclusion
In conclusion, we demonstrated expression of the Harvard 2006 streptavidin surface display parts via Western Blot. We were unable, however, to visualize binding between a biotinylated fluorophore and the cells expressing these proteins. This indicates that the streptavidin display system is likely not binding biotin correctly. We hypothesize that streptavidin might not having enough room on the cell surface to form tetramers (which is its native state), and thus may not be binding to biotin with high efficiency. In order to generate a construct that demonstrates tight binding to to biotin, we propose to design a generalized cell surface display system, and to computationally design a biotin-binding streptavidin monomer using software developed at the University of Washington.

These solutions are described in the future directions section.