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Module 1:

The objective of Module 1 is to investigate the basal expression level of the pLasI promoter. Basal or leaky expression is observed in inducible promoters, wherein the regulated gene is expressed without the inducer molecule. This issue is pervasive in synthetic biology and significantly impacts the efficiency of genetic circuits. To address this concern, we intend to express the mCherry fluorescent protein under the control of the pLasI promoter. The strategy involves cloning the mCherry gene and the pLasI promoter into the pJUMP 28-1A plasmid. Subsequently, we will assess the fluorescence intensity of mCherry in the E. coli BL21 strain without any inducer, shedding light on the inherent expression levels mediated by the pLasI promoter under basal conditions. This investigation is vital for understanding the behavior of the pLasI promoter and will contribute valuable insights to further experiments in our project.

Fig. 1

Module 2:

This module is structured to accomplish two specific objectives:

  1. Evaluation of Bacterial Serotonin Transporter (SERT) Protein Expression: We aim to assess the expression of CUW_0748, the bacterial analog of the Human Serotonin Transporter, in E. coli. This will be achieved by expressing SERT under the control of a constitutive promoter. Additionally, the expression of SERT will be monitored using the TagBFP Fluorescent Reporter. A T2A linker, a self-cleaving peptide sequence, is placed between SERT and TagBFP to separate the two proteins during translation within the same operon. This approach was chosen over a SERT+TagBFP fusion protein to ensure the protein’s functionality, as fusion proteins may interfere with protein function.
  1. Verification of SERT's Impact on pLasI Activation: It is imperative to confirm that SERT does not bind to and influence the functionality of the pLasI promoter. SERT could interact with pLasI, potentially affecting downstream gene expression. To assess this, we intend to investigate the impact of SERT on pLasI's functionality by measuring fluorescence intensity. This evaluation is crucial to understanding any potential interactions between SERT and pLasI, ensuring the integrity of our experimental system.

Both of these cassettes will be expressed together in the pJUMP 28-1A plasmid. This module is instrumental in elucidating potential interactions between the Serotonin Transporter (SERT) and the pLasI promoter.

Fig. 2

Module 3:

This module is designed to address two specific objectives:

  1. Evaluation of LasR Expression: Our main objective is to evaluate the expression of the LasR protein using a constitutive promoter. TagBFP will facilitate this assessment. Similar to Module 2, a T2A linker sequence will be incorporated, ensuring separation between LasR and TagBFP. This design choice prevents potential interference with LasR's functionality that might occur with a LasR+TagBFP fusion.
  1. Analysis of Basal Expression of pLasI in the Presence of LasR: We aim to investigate the capability of the LasR protein to independently activate the pLasI promoter, leading to the expression of the downstream mCherry gene. We will employ the same genetic cassette utilized in Module 1, which will be cloned into the pJUMP 28-1A plasmid to achieve this. Through this process, we intend to examine the basal expression levels of pLasI in response to the presence of LasR.

Both of these cassettes will be expressed together in the pJUMP 28-1A plasmid. This module holds the key to determining the expression of the LasR protein and its ability to autonomously induce the pLasI promoter, thereby advancing our understanding of these regulatory mechanisms.

Fig. 3

Module 4:

The primary objectives of this module encompass the following:

  1. Verification of SERT and LasR Protein Functionality
    2.
  1. Evaluation of pLasI Activation in the Presence of Serotonin+LasR Complex
    2.

This Module examines the activation of the pLasI promoter when exposed to the Serotonin+LasR complex. Initially, the SERT membrane protein facilitates the entry of external serotonin into the cell. Subsequently, LasR binds to the internalized serotonin, forming the Serotonin+LasR complex, which activates the pLasI promoter through binding. This activation process will be assessed by measuring the fluorescence intensity of the mCherry reporter gene. An increased fluorescence intensity of mCherry in the presence of the Serotonin+LasR complex, as compared to the uninduced state of pLasI, would indicate the functional capability of the Serotonin+LasR complex to activate the pLasI promoter.

Fig. 4

Module 5:

The primary objectives of this module are as follows:

  1. Evaluation of SERT, SNAT, and COMT Functionality: The goal is to assess the functionality of Serotonin-N-Acetyl Transferase (SNAT) and Caffeic acid-o-methyl Transferase (COMT), along with Serotonin Transporter (SERT). These three proteins will be expressed under the regulation of a T7 IPTG-induced promoter. A single cassette will be created using two T2A linkers to streamline the process. One T2A linker will be placed between SERT and SNAT, and the other between SNAT and COMT. This design eliminates the need for assembling three separate proteins in different cassettes.
  1. Detection and Quantification of Melatonin via High-Performance Liquid Chromatography (HPLC):
    2.

    SNAT will catalyze the conversion of Serotonin to N-acetyl serotonin (NAS), an intermediate compound. Subsequently, COMT will further convert NAS into Melatonin. The synthesized Melatonin will be detected and quantified using High-Performance Liquid Chromatography (HPLC). This method allows for precise measurement, enabling the accurate assessment of Melatonin production.

Fig. 5

Module 6:

This module is focused on assessing the expression of SNAT and COMT proteins utilizing fluorescent reporter proteins. Specifically, a fusion construct of SNAT with TagBFP and COMT with mCherry is expressed under the control of a T7 IPTG-induced promoter. Incorporating a T2A linker between these fusion proteins eliminates the need for using separate cassettes to express SNAT+TagBFP and COMT+mCherry individually, making the utilization of two distinct cassettes redundant.

Fig. 6

Module 7:

This module is focused on the comprehensive conversion of serotonin to melatonin under the control of a LasR regulatory system. The module comprises two distinct cassettes:

  1. The first cassette involves the expression of Serotonin Transporter (SERT) and LasR under a constitutive promoter. A T2A linker is incorporated between these sequences to ensure the production of separate SERT and LasR proteins.
  1. The second cassette includes Serotonin-N-Acetyl Transferase (SNAT) and Caffeic Acid-O-Methyl Transferase (COMT) under the pLasI inducible promoter. Similar to the first cassette, a T2A linker is also used here to generate SNAT and COMT proteins independently.

In this system, the expressed SERT facilitates the entry of external serotonin into the cell. Subsequently, serotonin binds to the LasR protein, forming the serotonin+LasR complex, which then interacts with the pLasI promoter, activating it. Upon activation, the pLasI promoter expresses SNAT and COMT. These enzymes work sequentially on serotonin, ultimately producing melatonin. The melatonin produced is then detected and quantified using High-Performance Liquid Chromatography (HPLC). This module provides a comprehensive overview of the serotonin-to-melatonin conversion process regulated by the LasR system.

Fig. 7

Assembly Strategy

The Golden Gate assembly strategy has been used to assemble all our biobricks. Golden Gate assembly, a prominent method in synthetic biology, utilizes Type IIS endonucleases to merge numerous DNA components in a single reaction. The resulting assembled DNA can be directly integrated into a host organism for selection and propagation. This technique accommodates both linear and circular DNA molecules, facilitating the generation of standardized collections of assembly-ready parts in storage plasmids.

Fig 8: Illustration of Golden Gate Assembly.

Experimental Workflow

Experimental workflow

Fig. 9

References

  1. Bird, J. E., Marles-Wright, J., & Giachino, A. (2022). A user’s guide to golden gate cloning methods and standards. ACS Synthetic Biology, 11(11), 3551-3563. https://doi.org/10.1021/acssynbio.2c00355
  2. https://www.snapgene.com/guides/golden-gate-assembly

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