Coding

Part:BBa_K5238003

Designed by: Yishi Jia   Group: iGEM24_NJMU-CHINA   (2024-07-10)


Produce inosine for the treatment of depression

Inosine Inosine, a purine nucleoside, has shown potential therapeutic effects in the treatment of Alzheimer's disease (AD) and depression. It acts as a neuroprotective agent by promoting axonal regeneration and enhancing neuronal survival. Studies have indicated that inosine can improve cognitive functions and alleviate depressive symptoms by modulating neurotransmitter levels and reducing oxidative stress. Additionally, inosine has been found to enhance synaptic plasticity and neurogenesis, which are critical for cognitive and emotional health[1][2].

In our research, we have demonstrated through molecular docking simulations and molecular interaction experiments that inosine can bind to the neuronal nitric oxide synthase (nNOS) receptor. Current antidepressant drugs on the market suffer from insufficient targeting, significant side effects, and delayed onset of action. Research has found that in the dorsal raphe nucleus (DRN), serotonin transporter (SERT) is highly colocalized with nNOS, but this colocalization is largely absent in the postsynaptic region[2]. After chronic stress, SERT-nNOS coupling in the DRN increases, leading to reduced membrane localization of SERT, increased intercellular serotonin concentration, activation of serotonin autoreceptors, and enhanced negative feedback inhibition of serotonergic neuron firing, resulting in reduced serotonin concentration in the postsynaptic synaptic cleft and inducing depression. By uncoupling SERT from nNOS, increasing DRN membrane SERT, reducing intercellular serotonin, and lowering negative feedback, serotonergic neuron firing is stimulated, leading to a rapid increase in serotonin concentration in the postsynaptic synaptic cleft, thereby exerting a rapid antidepressant effect independent of serotonin autoreceptor desensitization. Therefore, we aim to target the nNOS receptor by using gut microbiota-produced small molecules, which can reach the brain via the gut-brain axis, bind to depression-related nNOS receptors, uncouple SERT from nNOS, and alleviate depression symptoms[3].

References: 1. Chen, P., Goldberg, D. E., Kolb, B., Lanser, M., & Benowitz, L. I. (2002). Inosine induces axonal rewiring and improves behavioral outcome after stroke. *Proceedings of the National Academy of Sciences*, 99(13), 9031-9036. 2. Walker, T. L., & Kempermann, G. (2014). One mouse, two cultures: isolating adult hippocampal neurogenesis in vitro. *Frontiers in Neuroscience*, 8, 384. 3. Sun, N., Qin, Y. J., Xu, C., et al. (2022). Design of fast-onset antidepressant by dissociating SERT from nNOS in the DRN. *Science*, 378(6618), 390-398.


Gsk

We successfully transformed the plasmid into Lactobacillus plantarum L168, and validation through PCR and agarose gel electrophoresis confirmed the success of this step. Following this, we cultured Lactobacillus plantarum L168 and extracted RNA using bacteriophage, with the supernatant being used for metabolite measurement. Next, we conducted reverse transcription and quantitative polymerase chain reaction (qPCR) experiments to quantify the gene expression levels in Lactobacillus plantarum L168, both with and without the pSIP403-P9-gsk plasmid. The qPCR results, as shown in figure 3, indicated our designed plasmid effectively and significantly upregulated the gene expression of inosine in Lactobacillus plantarum L168 (Fig. 3).

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Figure 3. Relative Expression Analysis of gene gsk by qPCR.


Liquid Chromatograph Mass Spectrometer (LC-MS) was also utilized to examine the production of inosine in the supernatant of Lactobacillus plantarum L168. It is evident that the level of inosine rose significantly in Lactobacillus plantarum L168 containing the pSIP403-P9-gsk plasmid compared to the control group, revealing that, as anticipated, the gsk gene was effectively expressed in Lactobacillus plantarum L168 and led to inosine production (Fig. 4).

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Figure 4. Relative levels of inosine between two groups by LC-MS.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 943
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 943
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 943
    Illegal BglII site found at 834
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 943
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 943
    Illegal AgeI site found at 663
  • 1000
    COMPATIBLE WITH RFC[1000]


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