Team:Washington/Project/Display

From 2009.igem.org

(Difference between revisions)
(Streptavidin-Biotin Binding Is Not Visualized By Fluorescence Microscopy)
 
(266 intermediate revisions not shown)
Line 2: Line 2:
{{Template:Team:Washington/Templates/Header}}
{{Template:Team:Washington/Templates/Header}}
-
=Display System=
+
<html><style>
-
[[Image:Titlepic.jpg]]
+
#uw_himg {
 +
  display:none;
 +
}
 +
</style></html>
 +
<div>
 +
<div style="float:left;">'''[https://2009.igem.org/Team:Washington/Project &lt; Project Description]'''</div>
 +
<div style="float:right;">'''[https://2009.igem.org/Team:Washington/Project/Release Release &gt;]'''</div>
 +
</div>
-
==Background==
+
[[Image:Main_graphic3_display_banner.png|center]]
-
Our system hinged on finding a protein which could bind other protines to the outside of the cell, but whose interaction with these proteins was weak enough to be disrupted by another small molecule. Streptavadin presented itself as a logical choice because it can bind biotin (a small molecule) as well as a nanotag sequence which can be added easily to any protein sequence  [1]. The strepatavadin biotin complex is shown below. <br>
+
-
{|
+
='''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-Tag<sup>1</sup>) 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.
 +
{| cellpadding="1" cellspacing="8"
|-
|-
-
|[[Image:Biotin-strept.gif | 300px]]<br>[11]  
+
|[[Image:Strept-biotin.png | 175px]]
-
| Enter info on strepatavadin and biotin binding here to fill up the formatted space on the right side of this image
+
|The Nano-Tag binds streptavidin with 4nM binding affinity and has been used to purify multiple proteins. The ability of biotin to compete off the Nano-Tag in biding assays indicates that the binding occurs at the same site of streptavidin. Of great importance for incorporating streptavidin into our display system is that streptavidin is a common protein used in bio-chemical assays. Its ability to bind biotin is one of the strongest non-covalent interactions known. This allows for the utilization of biotin and streptavidin to act as molecular connectors in protein systems. Furthermore the availability of biotinylated and streptavidin linked fluorophores allows for an easy assay of their presence, using fluorescence microscopy<sup>2</sup>.
|-
|-
|}
|}
-
Overview of streptavadin biotin binding [2].
+
{| cellpadding="1" cellspacing="8"
 +
|-
 +
|Next we needed a way to anchor the streptavidin to the outer membrane. The obvious choice was to use the LPP signal peptide and Outer Membrane Protein A (OmpA) because this system has been extensively characterized as an expression system to display proteins on the cell surface. This LPP-OmpA system has been used to display a diverse group of proteins, including Green Fluorescent Protein (GFP)<sup>3</sup>, Organophosphorus Hydrolase (OPH)<sup>4</sup>, Cyclodextrin Glucanotransferase (CGTase)<sup>5</sup>,  Methyl Parathion Hydrolase (MPH)<sup>6</sup>, and Enhanced Green Fluorescent Protein (EGFP)<sup>6</sup>.  Thus, an appropriate display construct for our system would be a Lpp-OmpA-streptavidin fusion protein, which would be anchored in the outer membrane and display streptavidin on the surface of the cell. Streptavidin would bind the nanotag on our target protein in an interaction that could be disrupted by adding biotin.
 +
|[[Image:Display image.jpg|400px|right]]
 +
|-
 +
|}<br>
 +
==== Harvard 2006 Surface Display System: Legacy Parts ====
-
Next we needed a way to anchor the streptavadin to the outer membrane. The obvious choice was to use the LPP signal peptide and Outer Membrane A (ompA) because it has been extensively studied. This LPP-ompA system has been used to display the following proteins: <br>
 
-
# Green Fluorescent Protein (GFP) [3]
+
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 team<sup>7</sup>. 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.
-
# Organophosphorus Hydrolase (OPH) [4]
+
-
# Cyclodextrin Glucanotransferase (CGTase)  [5]
+
-
# Methyl Parathion Hydrolase (MPH)  [6]
+
-
# Enhanced Green Fluorescent Protein (EGFP)  [6] <br>
+
-
 
+
{|cellpadding="3" cellspacing="1" border="2" width="75%" align="center"
-
Now we knew what we wanted. The next step was to check the parts registry to see if someone had already built this part, or anything similar that we could use as a starting part for our Idealized Protein Purification system. Low and behold this system was already in the parts registry, and it was submitted by Harvard in 2006 [7]. In fact Harvard had submitted four biobrick parts which where of interest to us. They used a  They submitted four biobricks which where all variations on the same theme. All four are fusion proteins which have a LPP signal peptide, either one or five trans-membrane ompA, and either monomeric or dimeric streptavidin. All the variations and the resulting biobricks are shown below.
+
-
 
+
-
{|style="color:#B5A642;background-color:#702963;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%"
+
! Bio Brick
! Bio Brick
! OmpA trans-membrane domains
! OmpA trans-membrane domains
-
! Type of Streptavadin
+
! Type of Streptavidin
-
|-
+
|- align="center"
|J36848
|J36848
|1
|1
|monomeric
|monomeric
-
|-
+
|- align="center"
|J36849
|J36849
|1
|1
|dimeric
|dimeric
-
|-
+
|- align="center"
|J36850
|J36850
|5
|5
|monomeric
|monomeric
-
|-
+
|- align="center"
|J36851
|J36851
|5
|5
Line 50: Line 56:
|-
|-
|}
|}
 +
<br>
 +
==== Harvard 2006 Surface Display System: Documented Data ====
-
A basic graphic of the structure of this display system is shown below:
+
Data regarding the activity of the Harvard iGEM 2006 Lpp-OmpA-Streptavidin construct has been published in IET Synthetic Biology in 2007<sup>7</sup>.  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.
-
[[Image:Display image.jpg|700px]] <br>
+
{|
 +
|-
 +
|[[Image:HarvardIETSYNTH.jpg|400px|center]]
 +
|''
 +
L01W: Lpp-OmpA-Streptavidin monomer, no His Tag.<br>
 +
L01H: Lpp-OmpA-Streptavidin monomer with His tag.<br>
 +
L01S: Lpp-OmpA-Streptavidin dimer.<br>
 +
L01: Lpp-OmpA alone).<br>
 +
A: no aptamer added.<br>
 +
B: With only ‘F’ 50 -fluorescently-tagged oligonucleotide.<br>
 +
C: With fluorescently tagged streptavidin aptamer.<br>
 +
D: With ‘B-F’ hybrid of 50 -biotinylated oligonucleotide annealed with 50-fluorescently-tagged oligonucleotide<sup>7</sup>.''
 +
|-
 +
|}
 +
-
Now we had the parts that we wanted. After looking at the work Harvard did on these parts, we decided to do the following set of tests.  
+
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.
-
# Western against the his tags on all of these proteins
+
='''Experiments'''=
-
# Micrscope Assay
+
== Harvard's Legacy 2006 Cell Surface Display Parts: Do They Work As Predicted? ==
-
# Flow Cytometry Assay
+
 +
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:
-
==Experiments==
+
# Western blot to verify expression of Harvard 2006 surface display parts
 +
# Assess biotin binding to cell surface by fluorescence microscopy
 +
# Assess biotin binding to cell surface by flow cytometry
-
=== Western ===
+
=== 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 lines, 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 our the Harvard parts are conveniently his tagged this means that we could use a his tag antibody to detect the presence of these proteins in our western [8].
+
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.<sup>8</sup>.
-
==== Results ====
+
==== Data ====
-
[[Image:Western.jpg]]
+
[[Image:HarvardExpressionWestern.jpg|700px|center]]
-
''Waiting for image from Ingrid''
+
<br>
-
The expected values of each of these proteins are shown in the table below:
+
The expected sizes of each of these proteins are shown in the table below:
-
{|
+
{|cellpadding="3" cellspacing="1" border="2" width="50%"
! Bio Brick
! Bio Brick
-
! Length (in kD)
+
! Length (in Da)
-
|-
+
|- align="center"
|J36848
|J36848
|21478.7
|21478.7
-
|-
+
|- align="center"
|J36849
|J36849
|34600.9
|34600.9
-
|-
+
|- align="center"
|J36850
|J36850
|31215.4
|31215.4
-
|-
+
|- align="center"
|J36851
|J36851
|44972.3
|44972.3
Line 93: Line 117:
|}
|}
-
This data showed that indeed our parts did come out at the expected lengths. The varying brightness of the bands does not indicate the strength of expression because the protein amount was not normalized before it was inserted into the gel. This was an important conclusion, which proved to us that these proteins were indeed being expressed by the cell. Now we could move on to check to see if the proteins were being expressed on the surface, and if they were functional. To do this we did a microscope assay.
+
<br>
 +
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. <br>
 +
=== Streptavidin-Biotin Binding Is Not Visualized By Fluorescence Microscopy ===
-
=== Microscope Assay ===
+
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 fluorophore<sup>9</sup>. 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 [https://2009.igem.org/Team:Washington/Notebook/Microscope Notebook] page for protocols).
-
The goal of this experiment was to see if we could confirm the presence of streptavadin on the outside of our cell. We wanted to do this using a biotyntalated flourophore. We expected that this flourophore would bind to the streptavadin on the outside of the cell in such a high level that we would be able to detect it under a high powered florescence microscope. The resulting concentration of florescence if high enough, would result in a halo of florescence around the cells. As a positive control we used streptavadin coated beads which were roughly the same size (with respect to volume) as our cells (except spherical).
+
==== Data ====
==== Data ====
-
[[Image:Beads.jpg | 800px]]
 
 +
<table>
 +
<tr>
 +
<td><p style="font-size:18px"> Positive Control </p> </td>
 +
<td><p style="font-size:18px"> Negative Control </p> </td>
 +
<td rowspan="4">These images were analyzed using imageJ<sup>10</sup>. The image on the left shows an image of the beads with fluorophore added to them. These beads were diluted and spun diluted and spun down until the background level of fluorescence was low enough to get an accurate reading. We then used imageJ to analyze the intensity of a line going through the bead, which is demonstrated by the above schematic. On the positive control the edges of the streptavidin-coated beads show spikes in fluorescence, indicating binding of the biotinylated fluorophore to the beads. The negative control (beads without fluorophore) showed no such increase. This meant that our biotinylated fluorophore binds to streptavidin in a detectable manner.  When our cells were examined in the same manner, no difference could be seen in biotinylated fluorophore binding between cells that had induced expression of surface streptavidin (left) and uninduced cells which should not express the surface display protein (right).  This was evidence that the streptavidin surface display part binding to biotin was very low / nonexistent.  In order to verify this, we measured the binding of the biotinylated fluorophore to entire populations of streptavidin-expressing cells by flow cytometry.</td>
 +
</tr>
 +
<tr>
 +
<td>[[Image:M_beads1.png| 210px]]</td>
 +
<td>[[Image:M_beads2.png| 210px]]</td>
 +
</tr>
 +
<td><p style="font-size:18px"> Induced Cells </p></td>
 +
<td><p style="font-size:18px"> Uninduced Cells </p></td>
 +
<tr>
 +
<td>[[Image:M_cells1.png| 210px]]</td>
 +
<td>[[Image:M_cells2.png| 210px]]</td>
 +
</tr>
 +
</table>
-
These images were analyzed using imageJ [9]. The image on the right shows an image of the beads with flourophore added to them. These beads were diluted and spun diluted and spun down until the background level of florescence was low enough to get an accurate reading. We then used imageJ to analyze the intensity of a line going through the bead. On the positive control the edges of the beads show spikes in florescence, which is what we expected. The negative control (beads without florophore) showed no such increase. This meant that our biotyntalated flouophore was working. Unfortunate when our cells were measured in the same manner, no difference could be seen between the induced (left) and the uninduced (right).  However, this assay only looked at single cells and their binding properties. Before we could dismiss this part as not working, we need to take a look at a while population of cells. To do this was used flow cytomtery.
+
=== 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 ([https://2009.igem.org/Team:Washington/Notebook/Flow_Cytometry 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.
-
 
+
-
 
+
-
=== Flow Cytometry ===
+
-
The goal of this experiment was to see how the biotin bonded to streptavadin over a whole population of cells. Where as the Microscope experiment looked at a few localized cells, we set the cytometer to look 50,000 cells and read the resulting florescence. Our positive control was again the strepatavadin coated beads. The cytomtery would be able to read the flouresence of each individual particle passing through it, which would allow us to get much more accurate reading, and look at a much larger sample size than we could under the microscope. The results of this experiment are described below.
+
==== Data ====
==== Data ====
-
[[http://soslab.ee.washington.edu/igem/2009/images/f/f9/Hists.png]]
+
<gallery heights=300px widths=350>
-
[[Image:2009-08-07_51s.png]]
+
Image:48.png|'''BBa_J36848''' ''This image shows both the induced and uninduced cells for part 48 in varying levels of  fluorophore (0nM to 100nM). The cells were induced at 1mM IPTG. This data shows that there is no appreciable difference between the induced and uninduced cells at any given level of fluorophore. All curves appear to have the same amount of fluorescence. We found similar results using a microscopy assay.''
 +
Image:StreptBead cyto.png|'''+ Control''' ''We used the streptavidin coated beads<sup>11</sup> to show us what the magnitude of fluorescence increase we should see with increased fluorophore levels. We were able to see that as the level of fluorophore was increased we could see increased retention between the beads and the fluorophore. The black is beads with no fluorophore, the red is with 10 nM, and the purple is 100 nM. These showed a clear difference between the beads without fluorophore, and the beads with fluorophore. The cells shown at right, matched the readings of the beads when they had no fluorophore added.''
 +
</gallery>
-
Our positive control showed nice separation between the beads with flourophore and the beads without flourophore. This confirmed what we saw under the microscope, and also indicated that our assay was working. However this data showed us that we did not have any appreciable binding occurring with any of the ompA-sterptavadin parts to biotin at the global level. All our the cells have low levels of florescence which are comparable to those of the beads.
+
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 ===
=== Conclusion ===
-
In conclusion we were able to shown that the proteins were being expressed in the cell by a western blot. We were unable however to see any sort of binding between the biotyntalated flourophore and the cells expressing these proteins. This meant that our display system was likely not binding biotin correctly. We hypothesized this could have been do to streptavadin not having enough room to form tetrameters on the surface (which is its native state)[10]. We came up with the following solutions to this problem.
 
-
 
-
=== Solutions ===
 
-
==== Fold It ====
 
-
 
-
==== New Display Constructs ====
 
 +
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.<br>
 +
'''These solutions are described in the [https://2009.igem.org/wiki/index.php?title=Team:Washington/Future future directions section].'''
-
=== References ===
+
----
-
# TBA
+
-
# TBA
+
-
# Display of green fluorescent protein on Escherichia coli cell surface. Shi H, Wen Su W. Department of Biological & Agricultural Engineering, University of Missouri-Columbia, 65211, Columbia, MO, USAEnzyme Microb Technol. 2001 Jan 2;28(1):25-34.
+
-
# Cell surface display of organophosphorus hydrolase using ice nucleation protein. Shimazu M, Mulchandani A, Chen W. Biotechnol Prog. 2001 Jan-Feb;17(1):76-80. Department of Chemical and Environmental Engineering, and Environmental Toxicology Program, University of California, Riverside, CA 92521, USA.
+
-
# Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli. Wan HM, Chang BY, Lin SC. Biotechnol Bioeng. 2002 Aug 20;79(4):457-64. Department of Chemical Engineering, National Chung Hsing University, Taichung, 402, Taiwan.
+
-
# Cell surface display of functional macromolecule fusions on Escherichia coli for development of an autofluorescent whole-cell biocatalyst. Yang C, Zhao Q, Liu Z, Li Q, Qiao C, Mulchandani A, Chen W. Environ Sci Technol. 2008 Aug 15;42(16):6105-10. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
+
-
# Reference to Harvard's website from 2006
+
-
# http://www.kpl.com/catalog/productdetail.cfm?catalog_ID=17&Category_ID=448&Product_ID=1085
+
-
# http://rsbweb.nih.gov/ij/docs/index.html
+
-
# TBA
+
-
# Fritz Lipmann Institute (http://www.imb-jena.de/image_library/PROTEINS/proteins/1stp/)
+
 +
=== Citations ===
 +
#The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins [http://www.ncbi.nlm.nih.gov/pubmed/14680960 doi:10.1016/j.physletb.2003.10.071]
 +
#Structural origins of high-affinity biotin binding to streptavidin. PC Weber, DH Ohlendorf, JJ Wendoloski, and FR Salemme (6 January 1989). Science 243 (4887), 85. [http://www.ncbi.nlm.nih.gov/pubmed/2911722 DOI: 10.1126/science.2911722]
 +
#Display of green fluorescent protein on ''Escherichia coli'' cell surface. Shi H, Wen Su W. Department of Biological & Agricultural Engineering, University of Missouri-Columbia, 65211, Columbia, MO, USA [http://www.ncbi.nlm.nih.gov/pubmed/11118595 Enzyme Microb Technol. 2001 Jan 2;28(1):25-34]
 +
#Cell surface display of organophosphorus hydrolase using ice nucleation protein. Shimazu M, Mulchandani A, Chen W.Department of Chemical and Environmental Engineering, and Environmental Toxicology Program, University of California, Riverside, CA 92521, USA. [http://www.ncbi.nlm.nih.gov/pubmed/11170483 Biotechnol Prog. 2001 Jan-Feb;17(1):76-80]
 +
#Anchorage of cyclodextrin glucanotransferase on the outer membrane of ''Escherichia coli''. Wan HM, Chang BY, Lin SC. Department of Chemical Engineering, National Chung Hsing University, Taichung, 402, Taiwan. [http://www.ncbi.nlm.nih.gov/pubmed/12115409 Biotechnol Bioeng. 2002 Aug 20;79(4):457-64]
 +
#Cell surface display of functional macromolecule fusions on ''Escherichia coli'' for development of an autofluorescent whole-cell biocatalyst. Yang C, Zhao Q, Liu Z, Li Q, Qiao C, Mulchandani A, Chen W. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. [http://www.ncbi.nlm.nih.gov/pubmed/18767673 Environ Sci Technol. 2008 Aug 15;42(16):6105-10]
 +
#Cell surface streptavidin. Tsai P. [http://dx.doi.org/10.1049/iet-stb:20070002 IET Synth Biol. 2007;1(1.2):32]
 +
#[http://www.kpl.com/catalog/productdetail.cfm?catalog_ID=17&Category_ID=448&Product_ID=1085 HisDetector Nickel-HRP]
 +
#Biotin-5-fluorescein, [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=53608|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Sigma Aldrich]
 +
#[http://rsbweb.nih.gov/ij/docs/index.html ImageJ]
 +
#SVP-15-5 streptavidin coated polstyerene spheres (1.5-1.9 &micro;m), [http://www.spherotech.com/coa_pol_par.htm Spherotech]
{{Template:Team:Washington/Templates/Footer}}
{{Template:Team:Washington/Templates/Footer}}

Latest revision as of 02:46, 22 October 2009

Uw title logo.png

Main graphic3 display banner.png

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.

Strept-biotin.png The Nano-Tag binds streptavidin with 4nM binding affinity and has been used to purify multiple proteins. The ability of biotin to compete off the Nano-Tag in biding assays indicates that the binding occurs at the same site of streptavidin. Of great importance for incorporating streptavidin into our display system is that streptavidin is a common protein used in bio-chemical assays. Its ability to bind biotin is one of the strongest non-covalent interactions known. This allows for the utilization of biotin and streptavidin to act as molecular connectors in protein systems. Furthermore the availability of biotinylated and streptavidin linked fluorophores allows for an easy assay of their presence, using fluorescence microscopy2.
Next we needed a way to anchor the streptavidin to the outer membrane. The obvious choice was to use the LPP signal peptide and Outer Membrane Protein A (OmpA) because this system has been extensively characterized as an expression system to display proteins on the cell surface. This LPP-OmpA system has been used to display a diverse group of proteins, including Green Fluorescent Protein (GFP)3, Organophosphorus Hydrolase (OPH)4, Cyclodextrin Glucanotransferase (CGTase)5, Methyl Parathion Hydrolase (MPH)6, and Enhanced Green Fluorescent Protein (EGFP)6. Thus, an appropriate display construct for our system would be a Lpp-OmpA-streptavidin fusion protein, which would be anchored in the outer membrane and display streptavidin on the surface of the cell. Streptavidin would bind the nanotag on our target protein in an interaction that could be disrupted by adding biotin.
Display image.jpg

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.

Bio Brick OmpA trans-membrane domains Type of Streptavidin
J36848 1 monomeric
J36849 1 dimeric
J36850 5 monomeric
J36851 5 dimeric


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.

HarvardIETSYNTH.jpg

L01W: Lpp-OmpA-Streptavidin monomer, no His Tag.
L01H: Lpp-OmpA-Streptavidin monomer with His tag.
L01S: Lpp-OmpA-Streptavidin dimer.
L01: Lpp-OmpA alone).
A: no aptamer added.
B: With only ‘F’ 50 -fluorescently-tagged oligonucleotide.
C: With fluorescently tagged streptavidin aptamer.
D: With ‘B-F’ hybrid of 50 -biotinylated oligonucleotide annealed with 50-fluorescently-tagged oligonucleotide7.


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

HarvardExpressionWestern.jpg


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

Bio Brick Length (in Da)
J36848 21478.7
J36849 34600.9
J36850 31215.4
J36851 44972.3


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).


Data

Positive Control

Negative Control

These images were analyzed using imageJ10. The image on the left shows an image of the beads with fluorophore added to them. These beads were diluted and spun diluted and spun down until the background level of fluorescence was low enough to get an accurate reading. We then used imageJ to analyze the intensity of a line going through the bead, which is demonstrated by the above schematic. On the positive control the edges of the streptavidin-coated beads show spikes in fluorescence, indicating binding of the biotinylated fluorophore to the beads. The negative control (beads without fluorophore) showed no such increase. This meant that our biotinylated fluorophore binds to streptavidin in a detectable manner. When our cells were examined in the same manner, no difference could be seen in biotinylated fluorophore binding between cells that had induced expression of surface streptavidin (left) and uninduced cells which should not express the surface display protein (right). This was evidence that the streptavidin surface display part binding to biotin was very low / nonexistent. In order to verify this, we measured the binding of the biotinylated fluorophore to entire populations of streptavidin-expressing cells by flow cytometry.
M beads1.png M beads2.png

Induced Cells

Uninduced Cells

M cells1.png M cells2.png

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.


Citations

  1. The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins [http://www.ncbi.nlm.nih.gov/pubmed/14680960 doi:10.1016/j.physletb.2003.10.071]
  2. Structural origins of high-affinity biotin binding to streptavidin. PC Weber, DH Ohlendorf, JJ Wendoloski, and FR Salemme (6 January 1989). Science 243 (4887), 85. [http://www.ncbi.nlm.nih.gov/pubmed/2911722 DOI: 10.1126/science.2911722]
  3. Display of green fluorescent protein on Escherichia coli cell surface. Shi H, Wen Su W. Department of Biological & Agricultural Engineering, University of Missouri-Columbia, 65211, Columbia, MO, USA [http://www.ncbi.nlm.nih.gov/pubmed/11118595 Enzyme Microb Technol. 2001 Jan 2;28(1):25-34]
  4. Cell surface display of organophosphorus hydrolase using ice nucleation protein. Shimazu M, Mulchandani A, Chen W.Department of Chemical and Environmental Engineering, and Environmental Toxicology Program, University of California, Riverside, CA 92521, USA. [http://www.ncbi.nlm.nih.gov/pubmed/11170483 Biotechnol Prog. 2001 Jan-Feb;17(1):76-80]
  5. Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli. Wan HM, Chang BY, Lin SC. Department of Chemical Engineering, National Chung Hsing University, Taichung, 402, Taiwan. [http://www.ncbi.nlm.nih.gov/pubmed/12115409 Biotechnol Bioeng. 2002 Aug 20;79(4):457-64]
  6. Cell surface display of functional macromolecule fusions on Escherichia coli for development of an autofluorescent whole-cell biocatalyst. Yang C, Zhao Q, Liu Z, Li Q, Qiao C, Mulchandani A, Chen W. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. [http://www.ncbi.nlm.nih.gov/pubmed/18767673 Environ Sci Technol. 2008 Aug 15;42(16):6105-10]
  7. Cell surface streptavidin. Tsai P. [http://dx.doi.org/10.1049/iet-stb:20070002 IET Synth Biol. 2007;1(1.2):32]
  8. [http://www.kpl.com/catalog/productdetail.cfm?catalog_ID=17&Category_ID=448&Product_ID=1085 HisDetector Nickel-HRP]
  9. Biotin-5-fluorescein, [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=53608|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Sigma Aldrich]
  10. [http://rsbweb.nih.gov/ij/docs/index.html ImageJ]
  11. SVP-15-5 streptavidin coated polstyerene spheres (1.5-1.9 µm), [http://www.spherotech.com/coa_pol_par.htm Spherotech]