Overview

Our project primarily focuses on the development of nitrate and ammonia sensors. To accomplish it, we aimed to develop a cell-free biosensor based on in vitro transcription.

Whole-cell biosensors can have precise functions, but considering regulations such as the Cartagena Protocol, social implementation is challenging. Therefore, we decided to use a cell-free sensor. The reason for using a sensor based solely on transcription without utilizing a translation system is that cell-free translation systems are costly, complex, and potentially difficult to control.

As an in vitro-based biosensor, we referred to the ROSALIND system[1]. In the ROSALIND system, an input is made using a DNA-binding protein that typically binds to DNA but dissociates from it depending on the ligand concentration, and an output is generated using a fluorescent aptamer called Broccoli.

We propose an extended system of the ROSALIND system called MITSUNARI (Modular in vitro transcription-based sensing platform utilizing notable outputs and protein-molecular interaction).

Figure1

Logo of MITSUNARI Platform

Figure2

Concept of MITSUNARI Platform

The MITSUNARI system has two main concepts. The first is to convert any interaction between proteins and ligands into T7 RNAP transcription. The second is to freely transform the RNA produced through transcription into various outputs. We were able to show proof of concept on the two-hybrid system, luminescence, and color change within this project. Although we couldn't demonstrate a proof of concept in this project, we aim to use Split T7 RNAP[2]to convert ligand-dependent interactions between proteins and RNA, as well as protein structural changes, into transcription[3][4].

In the ROSALIND system, it is not possible to implement a mechanism that activates transcription in the presence of a ligand using a protein that binds to DNA when the ligand is present. However, in the two-hybrid system, as shown in Figure 3, by fusing a Leucine Zipper to the DNA-binding protein and also to T7RNAP, and using a promoter with low affinity, it is possible to create a mechanism where transcription is activated only when the DNA-binding protein is bound to the DNA.

Figure3

T7-two hybrid system utilizing Leucine-zipper

Furthermore, while the ROSALIND system used fluorescence as the output, we expanded the output to include luminescence and color change. For luminescence, we utilized a mechanism where Split NanoLuc is reconstituted on RNA. For color changes, we designed a system where csx30 is cleaved by the Cas7-11 system in an RNA concentration-dependent manner, causing the color to emerge in the solution. (We only showed proof of concept on this system, so color change has not fully accomplished)

Figure4

Color change system by Cas7-11

Main Parts of MITSUNARI

Input

BBa_K5411020 Leucine-zipperAN3.5

Leucine-zipperAN3.5 is a peptide sequence which specifically interacts with Leucie-zipperBN3.5 (BBa_K5411021). It can be used for two-hybrid systems by fusing it to DNA binding protein or T7 RNAP.

BBa_K5411021 Leucine-zipperBN3.5

Leucine-zipperBN3.5 is a peptide sequence which specifically interacts with Leucie-zipperAN3.5 (BBa_K5411020). It can be used for two-hybrid systems by fusing it to DNA binding protein or T7 RNAP.

Transcription

BBa_K5411028 T7promoter d1

Sequence containing T7 promoter with the first 5 bases removed from the 5' end, in order to reduce the affinity for T7RNAP and prevent transcription from occurring when T7RNAP is not in close proximity to the DNA.

Output

BBa_K5411053 MS2 coat protein-SmBit

A fused protein consists of MS2 coat protein and SmBiT, which binds specifically to a target RNA and undergoes self-assembly. By using the part with PP7-LgBiT (BBa_K5411054) we can reconstitute Luciferase on RNA and cause luminescence.

BBa_K5411054 PP7 coat protein-LgBit

A fused protein consists of PP7 coat protein and LgBiT, which binds specifically to a target RNA and undergoes self-assembly. By using the part with MS2-SmBiT (BBa_K5411053) we can reconstitute Luciferase on RNA and cause luminescence.

BBa_K5411057 spycatcher003-csx30-CBM9.2

A complex consisting of SpyCatcher003, which forms a covalent bond with proteins containing SpyTag003, csx30, which is cleaved by Cas7-11, and CBM9.2, which binds to cellulose. (This system allows for the release of a dye-protein tagged with SpyTag003 from the solid phase by RNA-dependent cleavage of csx30 via the Cas7-11 system.)

Registered Parts

Although we were unable to fully characterize some parts, we will share our findings for the benefit of future teams.

Parts for nitrogen sensing

Parts for transcription

Parts for output

References

[1]Jung JK, Alam KK, Verosloff MS, et al. Cell-free biosensors for rapid detection of water contaminants. Nature Biotechnology. 2020;38(12):1451-1459. doi:https://doi.org/10.1038/s41587-020-0571-7

[2]Pu J, Zinkus-Boltz J, Dickinson BC. Evolution of a split RNA polymerase as a versatile biosensor platform. Nature Chemical Biology. 2017;13(4):432-438. doi:https://doi.org/10.1038/nchembio.2299

[3]Hussey BJ, McMillen DR. Programmable T7-based synthetic transcription factors. Nucleic Acids Research. 2018;46(18):9842-9854. doi:https://doi.org/10.1093/nar/gky785

[4]Thomas F, Boyle AL, Burton AJ, Woolfson DN. A Set of de Novo Designed Parallel Heterodimeric Coiled Coils with Quantified Dissociation Constants in the Micromolar to Sub-nanomolar Regime. Journal of the American Chemical Society. 2013;135(13):5161-5166. doi:https://doi.org/10.1021/ja312310g