Team:Michigan/Project

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.

Results

To test whether the fusion proteins are present on the cell surface in active form, we assayed the binding affinity of the cells to the fluorescence-labeled oligonucleotides (oligos) by measuring the intensity of fluorescence after polarized excitation through an emission polarizer. We labeled our recognition oligos with 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. CY3's excitation wavelength is 550 nm, and emission is 564 nm; 6-FAM excitation is 495 nm and emission is 520 nm. 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.

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. As a result, we are only able to compare between negative control and sample values, but no meaningful anisotropy or polarization measurements can be extracted from the data.

A second attempt to assay the binding affinity of the expressed cells with the fluorescence oligos was carried out with a UV/Vis Spectrophotometer. A binding assay was performed with fluorescently-labeled oligos. Three types of cells were grown: BL21 with no fusion construct, BL21 carrying an uninduced fusion construct (LOG or LOZ), and BL21 carrying an induced fusion product. The latter two were then incubated with double-stranded, fluorescently-labeled oligos corresponding to the Gli-1 or Zif268 recognition sequence. After incubation, cells were pelleted, washed, and resupended in 20μM ZnSO4, 20mM Tris, pH 8.0 buffer. Emission data were recorded at wavelengths corresponding to the fluorophores used.

Left: Samples with Gli-1 expression under an excitation frequency of 550 and emission freq of 564 (bottom plate read). Right: Samples with Znf268 expression under excitation of 495 and emission of 520 (bottom plate read). Dup1, 2 and Rep1 and 2 represent the two different colonies selected from the same plate. Samples on both graphs from left to right: lac-ompA-Gli (induced), lac-ompA-Gli (uninduced), lac-ompA-Znf268 (induced), LOZ (uninduced), buffer alone, buffer and CY3, buffer and 6FAM.

Unfortunately, none of the samples showed fluorescence higher than BL21 alone, which is consistent with the fluorescent polarization assay. We cannot say whether this is due to failure to establish proper conditions for binding, inefficient transport to the membrane, improper folding or instability of the fusion protein.