designed by Melike Sabry

Engineering

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Overview

Overview of our engineering cycle.
Figure 1: Overview of our engineering cycle.

Reporter System

Our loop system design is based on three general components (Figure 2A):

  • Secreted ligand
  • Receptor recognizing the ligand, releasing a transcription factor
  • Inducible gene activated by the transcription factor and expressing the ligand

concept drawing of quorum sensing loop
Figure 2: First Concepts of our Quorum Sensing Loop. (A) In the full loop system the ligand 2xmCherry is secreted from the cells and binds to its corresponding receptor. The receptor binding releases a transcription factor further increasing ligand production. BFP can be linked to the transcription factor to check its expression (B) Our first tests employ EYFP as an inducible reporter for the transcription factor. The ligand 2xmCherry is added manually to the cells.

Before testing the full loop experimentally, we wanted to start by adding the ligand manually to test whether it could bind to the receptor and activate it. To test the activation of the transcription factor, we used an inducible reporter different from the loop ligand (Figure 2B).

To investigate the individual processes, we developed a fluorescence reporter setup that enables measuring several parameters parallely. We initially decided on the following fluorescence signals to investigate our MESA system:

  • Reporter: EYFP (Ex513/Em527)
  • Loop Ligand: 2xmCherry (Ex587/Em610)
  • Transcription Factor: BFP (Ex381/Em445)

These three fluorescent proteins are well distinguishable and allowed us to cover the whole system:
Using EYFP we were able to read out our system activation caused by the release of the transcription factor. 2xmCherry allowed us to track expression of the surface receptors as well as ligand binding to the receptor. Finally, BFP was used to correct for variations in expression levels of the receptors that might influence reporter activation.

As our system works through the transcription factor tTA, we ordered a tTA induceable plasmid expressing EYFP from Addgene (Plasmid #58855) [1].

depiction of the three possible receptor ligand dimers
Figure 3: Possible Receptor-Ligand Dimers of a Homodimeric Ligand. Besides the active dimer created from a receptor dimer with both the TEV protease and the transcription factor, combinations with two proteases or transcription factors are also possible. These combinations are not active, potentially affecting the efficiency of the receptor system.

Initially, the EYFP reporter worked well for our loop setup. We were able to investigate the reporter induction by the homodimeric ligand 2xmCherry binding to our MESA system (Figure 2). This setup, however, only allowed us to use a homodimeric ligand (2xmCherry). The receptor activation is dependent on the dimerization of the transcription factor (TF) and Protease (TEV) bearing complementing receptor halves. Using a homodimer as ligand leads to four different receptor dimer configurations (Figure 3), of which some are not functional [2]. To alleviate this problem we also used a heterodimeric ligand (GFP-mCherry). This enabled us to split the receptor halves to recognize a unique antigen, in our case either GFP or mCherry.

This, however, led to the problem of fluorescence signal separation. As mEGFP (Ex488/Em507) and EYFP (Ex513/Em527) have a big overlap in excitation and emission, we could no longer separate ligand binding and reporter activation of our system (Figure 4, Figure 5B).

two graphs of flurescence spectra of mEGFP & EYFP
Figure 4: Fluorescence Spectra of mEGFP (A) and EYFP (B) [3]. Separation of mEGFP and EYFP fluorescence signals is not possible due to their overlap in both emission and excitation wavelengths.

To address this limitation, we decided to change the reporter protein from EYFP to miRFP680 (Ex661/Em680) [4], which allowed for differentiation from the other three used fluorescent proteins. We assembled the new reporter using Gibson assembly and it worked as hoped (Figure 5). Using the engineered miRFP reporter system, the overlapping FITC signal is eliminated (Figure 5A-C). In its place, reporter activity is now determined through APC signal intensity (Figure 5D). We incorporated it into all our measurements for better comparison of our different MESA system setups (See Results).

four flow cytometry histograms
Figure 5: Flow Cytometry Histograms comparing the usage of EYFP and miRFP in our loop system. (A): Gating for FITC positive cells expressing either the miRFP or EYFP reporter. Both cell samples were also transfected with the heterodimeric MESA system G1TA & M1TEV. 500 ng/mL GFP-mCherry were added to the samples. (B) Gated FITC Histogram of the EYFP reporter sample. It is not possible to differentiate the GFP-mCherry receptor binding from the induced reporter activation. (C) Gated FITC Histogram of the miRFP reporter sample. GFP binding is visible by the signal shift of the right wing of the curve. (D) Gated APC-A Histogram of the miRFP reporter sample. The reporter signal is visible in the APC-A channel separated from the FITC signal.

References

[1] Daringer, N. M., Dudek, R. M., Schwarz, K. A., & Leonard, J. N. (2014). Modular Extracellular Sensor Architecture for Engineering Mammalian Cell-based Devices.
https://doi.org/10.1021/sb400128g

[2] Schwarz, K. A., Daringer, N. M., Dolberg, T. B., & Leonard, J. N. (2017). Rewiring human cellular input-output using modular extracellular sensors. Nature Chemical Biology, 13(2), 202–209.
https://doi.org/10.1038/nchembio.2253

[3] Lambert, TJ (2019) FPbase: a community-editable fluorescent protein database. Nature Methods. 16, 277–278.
https://doi.org/10.1038/s41592-019-0352-8

[4] Matlashov, M. E., Shcherbakova, D. M., Alvelid, J., Baloban, M., Pennacchietti, F., Shemetov, A. A., Testa, I., & Verkhusha, V. v. (2020). A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales. Nature Communications, 11(1), 1–12.
https://doi.org/10.1038/s41467-019-13897-6

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