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E.Cargo

• Overview
• Background
• Rationale/Evolution
• Experimental Design
• Results/future

Rationale and Project Evolution

Our Tat-PTD project was originally developed as a way of delivering various fluorescent single-chain variable fragments (scFvs) extracted from an antibody library to mammalian cells in order to provide a staining mechanism that would allow visualization of cell structures in vitro without requiring fixation of the cells.

In the first month of summer, we reached a point where we were ready to express Tat-PTD fusion proteins, but were stalled waiting for a response from our university's Environmental Health and Safety committee as to BSL2 precautions and lab space. During that time, we focused our efforts into the numerous other projects that we were pursuing at that time. The Light-pattern-control project was designed in that period, and as it developed into a flagstone project, we struggled to manage so many projects at once. We soon came up with the idea to integrate E.Cargo with the Light-Pattern project, both to ease the workload of multiple projects with different goals, and to promote our project's philosophy of dealing with ever-increasing complexity.

Our idea was to use Tat-PTD fused to various bacterial transcription factors involved in the Light-Pattern circuitry in order to transiently introduce transcription factors to cells that only contained some of the circuit's components. This way, we could test constructs of the circuit individually without creating a massive amount of similar bricks and reporters for testing, and without having to worry about transforming cells with many plasmids at a time. We decided to test whether we could fuse Tat to transcription factors, and use these "Tat-TFs" to "remotely" induce gene expression.

After construction of Tat-fused parts and reporter constructs had been underway for quite some time, we discovered in our literature research that E.cargo may not be able to traverse the bacterial cell wall of E.coli. We will continue with our construction and testing of the Tat-linker and reporters in E.coli (just in case) while searching for an alternate bacterial chassis that is compatible with our constructs.

In the meantime, we offer the modular Tat-PTD_Linker construct in protein fusion assembly (RFC25) to the iGEM community as a means of introducing proteins to eukaryotic cells. A researcher needs simply to perform a BioBrick ligation of a his-tagged protein with our E.Cargo system, and transform the resulting product into E.coli. From there, it is a simple matter of Ni-NTA purification to isolate the Tat fusion protein for direct application to cell cultures. By fusing transcription factors to E.Cargo, one can easily test small parts of a complex circuit without need for another transformation. By fusing fluorescently-tagged proteins with specific targets to E.Cargo, it becomes a simple matter to visualize localization of target proteins or cellular structures. We hope that E.Cargo will be a valuable tool to future iGEM teams in many ways.