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Team:Michigan/Project
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<b>Discussion</b> | <b>Discussion</b> | ||
| - | <p>In the graph above, CY3 with Gli1 DNA binding domain recognition sequence was named 1, and 6-FAM labeled Zif268 recognition sequence was named 2. CY3's excitation wavelength is 550 nm, and emission is 564 nm; 6-FAM excitation is 495 nm and emission is 520 nm. We conclude that the set of data collected from the UV/Vis spec is similar to our results from the fluorescence anisotropy assay. A possible error may arise from the high fluorescence:buffer control ratio. </p> | + | <p>In the graph above, CY3 with Gli1 DNA binding domain recognition sequence was named "1", and 6-FAM labeled Zif268 recognition sequence was named "2". CY3's excitation wavelength is 550 nm, and emission is 564 nm; 6-FAM excitation is 495 nm and emission is 520 nm. Measurements across multiple wavelengths were conducted in part to find peak emission intensity as CY3 has a generally lower emission intensity. We conclude that the set of data collected from the UV/Vis spec is similar to our results from the fluorescence anisotropy assay. A possible error may arise from the high fluorescence:buffer control ratio. </p> |
Revision as of 04:17, 29 September 2011
Cell Patterning
This year’s project explores developing a cell patterning platform based on oligonucleotide-directed cell binding to substrate surfaces. Our approach entails engineering cells to selectively bind to certain nucleotide sequences (via surface display of DNA binding proteins, such as zinc fingers), allowing for guided assembly of defined cell patterns on surfaces patterned with oligonucleotides.
Abstract
The ability of zinc finger domains to selectively bind specific double stranded DNA sequences have largely been applied intracellularly, such as in engineered zinc finger nucleases for genomic manipulations. Proteins containing zinc finger domains can also be used extracellularly to precisely adhere objects to surfaces containing bound oligonucleotides. This project aims to utilize the specificity of zinc finger protein to direct binding of ''Escherichia coli'' to oligonucleotides bound on surfaces. The fusion protein engineered to contain a fragment of the OmpA membrane domain and a zinc finger domain allows the protein to be expressed on the outside of the cell while remaining bound to the host cell. Possible applications of this project include creating patterns with fluorescently labeled cells or studying cell-cell interactions.
Research Modules
Surface Display
The goal of the SD team is to identify, construct, and test various surface display systems to support the team's ultimate aim of displaying a Zn-Finger protein on the surface of ''E. coli''. Specifically, we have chosen to focus on systems utilizing OmpA, INP, and AIDA-1, allowing for the use of both established and unestablished BioBrick parts. Currently we are trying to build plasmids to test display by combining promoters, RBS’s, carrier’s, and test display proteins (e.g. GFP). Combining these parts we will be able to build biobricks and test our display systems so that we are ready to attempt surface display for a zinc finger.
DNA-Binding Proteins
The objective of the DNA binding team is to find and characterize DNA binding proteins in order to identify one with 1) the strongest affinity to DNA and 2) a structure that is predicted to tolerate the linker attachment in order to surface display it on ''Escherichia coli''. Our preliminary approach is to use zinc finger motifs as it has been well-known for its strong ability to bind to DNA and there exists a plethora of literature documenting its mechanism and combinations for optimizing binding specificity and affinity. We currently have three zinc finger protein candidates, two freely available from the iGEM Parts Registry and the third is a designer zinc finger described in Jantz et al. 2010. Binding of fluorescently labeled ''E. coli'' expressing the fusion protein will be confirmed under fluorescence microscope, and binding affinity assayed with fluorescence anisotropy.
DNA-Printing (Microarrays)
The purpose of the DNA printing team is to determine the best way to print a specific pattern of oligonucleotides onto a glass slide. After looking at several methods, we concluded that there is a huge barrier to entry for synthesizing our own glass slides and that the best way to proceed would be to order the slides pre-synthesized. This still leaves several aspects to the design of the slide that we need to work on. These include oligonucleotide density, sequences and linker sizes. We are currently conducting assays to determine the optimal density. The sequences we work with are determined by the zinc fingers that we will use. They consist of repeats of the zinc finger recognition sites, sometimes with filler DNA in between.
Results
Assaying Binding Affinity with Fluorimeter We assayed the binding affinity of the cells to the fluorescence-labeled oligonucleotides, or "oligos", by measuring the intensity of fluorescence after polarized excitation through an emission polarizer. We have two oligos: 6-FAM and CY3. 6-FAM is bound to the recognition sequence corresponding to Zif268, while CY3 is bound to the recongnition sequence of Gli-1. This method is a derivative of fluorescence anisotropy, an assay that measures the tumbling frequency of any molecule tagged with a fluorophore. It requires a polarized excitation of the fluorophore to produce a partially oriented population, and to measure emission with a polarizer parallel or perpendicular to the orientation of the excitation polarization. Factors that affect the tumbling frequency depends on the mass and shape of the tagged molecule as while as the viscosity of the solvent. When another, significantly larger, untagged molecule is tightly bound to the tagged molecule, the tumbling frequency decreases due to the change in inertia and shape of the complex.
We carried out an experiment that measured the binding affinity of cells expressing the zinc finger DNA binding domain on the outer membrane to the fluorescently-labelled oligos. We had two sets of negative controls: one with uninduced BL21 and the other is BL21 expressing the other zinc finger domain. The negative control with the uninduced BL21 is to test nonspecific binding of oligo to the outer membrane of ''E. coli'', and the second control of BL21 expressing a zinc finger domain with a different recognition sequence (e.g. Cy3 mixing with Zif268) is to test for nonspecific binding of zinc finger domain to oligo. We carried out our experiment in FluoroMax-2 machine with autopolarizer accessory.
Discussion
Based on the data from our experiment, we conclude that there was not a significant difference in the tumbling frequency of the oligos whether it was in the presence of uninduced BL21 or zinc finger domain expressing cells. Unfortunately, the software controlling the fluorimeter was not fully compatible with the anisotropy accessory, and we were unable to alter emission polarization orientation. Errors may be introduced by a lack of data from a emission polarizer readout that is perpendicular to the polarization from the current set of readings.
Binding Assay with UV/Vis Spectrophotometer
A second attempt to assay the binding affinity of the expressed cells with the fluorescence oligos was carried out with a UV/Vis Spectrophotometer. Instead of measuring the tumbling frequencey, UV/Vis spec solely looks at the emission frequency from the molecules. Ideally, the more binding there is, larger the emission value. We normalized the fluorescence values to the cell density by dividing fluorescence intensity by OD. In this experiment, we used the same samples from the previous fluorescence anisotropy assay and the fluorescence oligos.
Discussion
In the graph above, CY3 with Gli1 DNA binding domain recognition sequence was named "1", and 6-FAM labeled Zif268 recognition sequence was named "2". CY3's excitation wavelength is 550 nm, and emission is 564 nm; 6-FAM excitation is 495 nm and emission is 520 nm. Measurements across multiple wavelengths were conducted in part to find peak emission intensity as CY3 has a generally lower emission intensity. We conclude that the set of data collected from the UV/Vis spec is similar to our results from the fluorescence anisotropy assay. A possible error may arise from the high fluorescence:buffer control ratio.
