iGEM 2024 — Team njmu-china

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ENGINEERING

![](images/back.png)

This section we will introduce our engineering success in our four systems
from four
parts:Design,Build,Test and Learn.For
Design,we will explain why we chose this small molecule as the therapeutic target. For
Build, we will explain how we
constructed plasmids. Test and Learn, we performed different experiments for each system and
successfully verify their
function and provided conclusions of our successes, failures, and future plans for each
system.

* [SYSTEM1: tetrahydrofolate-folKE](#section1)
* [SYSTEM2: theanine(glnA)](#section2)
* [SYSTEM3: inosine(Gsk)](#section3)
* [SYSTEM4: niacin(pncA)](#section4)

# **SYSTEM 1:tetrahydrofolate--folKE**

**Design:**

![](https://static.igem.wiki/teams/5238/engine-success/1.png)

Tetrahydrofolate (THF) plays a crucial role as a coenzyme in one-carbon metabolism, which is
essential for the proper
functioning of the nervous system. Studies have indicated that tetrahydrofolate has
potential benefits in treating
Alzheimer's disease (AD) and depression. It aids in the synthesis of neurotransmitters and
DNA methylation, thereby
improving cognitive function and mood regulation. Furthermore, tetrahydrofolate can lower
homocysteine levels, which
helps reduce oxidative stress and neuroinflammation, providing therapeutic benefits for
individuals with AD and
depression[1,2].

In our research, we have demonstrated through molecular docking simulations and molecular
interaction experiments that
tetrahydrofolate can effectively target the latest LilrB3 receptor, which is closely
associated with APOE4 in AD and
represents a pivotal target. By competitively inhibiting APOE4-LilrB3 receptor binding, this
strategy addresses the root
cause of amyloid protein generation and deposition, significantly advancing AD treatment and
prevention and opening up
new avenues for engineered bacterial therapy in AD.

We choose *Lactobacillus plantarum* L168 as the chassis and pSIP403 as the vector. By
searching
its synthetic pathway, we
found the key enzyme folKE and its coding genes folK and folE. Then, we inserted the gene
folKE into our designed
plasmids to complete the construction and synthesis of plasmids. In view of biosafety, we
designed a kill switch which
is activated under hypoxic conditions, include a Hypoxia-inducible promoter, a RBS, a coding
of SrpR repressor that
inhibits transcription of the SrpR-dependent promoter and a terminator.

1. Zhang, S.-M., et al. (2002). Folate,
vitamin B6, and vitamin B12 intakes in relation to
risk of stroke among men.
Stroke, 33(1), 196-202.

2. Reynolds, E. (2006). Vitamin B12, folic
acid, and the nervous system. The Lancet
Neurology, 5(11), 949-960.

**Build:**

![](https://static.igem.wiki/teams/5238/engine-success/3-3.png)

The entire plasmid include the coding of tetrahydrofolate-folKE as our target product to bind
to target Lilrb3 for the
treatment of Alzheimer's disease. And a suicide switch, include a Hypoxia-inducible
promoter, a RBS, a coding of SrpR
repressor that inhibits transcription of the SrpR-dependent promoter and a terminator. (See
more in design
safety-suicide switch)

Regrettably, we encountered difficulties when attempting to concatenate the suicide switch
with the target gene folKE
due to time constraints, which prevented us from successfully constructing the plasmid and
transforming it into
*Lactobacillus plantarum*. Consequently, in the subsequent experimental validation, we
utilized the plasmid
pSIP403-P9-folKE, which expresses only the target gene.

**TEST:**

According to our experimental procedures, we successfully transformed plasmids
into L168 , confirmed through PCR and
agarose gel electrophoresis. After culturing L168 , we extracted RNA and
measured metabolites from
the supernatant. We then conducted reverse transcription and quantitative PCR (qPCR) to
quantify gene expression levels
in L168 , both with and without the pSIP403-P9-folKE plasmid. The experiment assessed the
relative expression levels of
two genes using qPCR. The results, shown in the figure 1, indicate that the expression of
folK and folE was
significantly higher in L168 containing the pSIP403-P9-folKE plasmid compared to the control
group (Fig. 1).

![](https://static.igem.wiki/teams/5238/engine-success/figure1.png)

Figure 1. Relative Expression Analysis of genes folK and folE by qPCR.

Moreover, we employed Liquid Chromatography-Mass Spectrometry (LC-MS) to analyze THF
production in the supernatant of
L168 .The results clearly show that THF levels significantly increased in L168 with the
pSIP403-P9-folKE plasmid compared
to the control group, confirming that the folKE gene was effectively expressed in L168 and
produced THF as anticipated
(Fig. 2).

![](https://static.igem.wiki/teams/5238/f984d2d335ef440bed6eba2a2440c79.webp)

Figure 2. Relative levels of THF between two groups by LC-MS.

You can find more information on the **Results** page.

**Learn:**

**Application of Molecular Docking and Interaction Experiments:** The demonstration of
tetrahydrofolate's (THF)
effectiveness in targeting the LilrB3 receptor, associated with AD pathology, through
molecular docking simulations and
molecular interaction experiments, provides a scientific foundation for developing novel
therapeutic strategies. This
approach not only deepens our understanding of the mechanisms underlying AD but also paves
the way for innovative
treatment modalities.

**Utilization of Genetic Engineering:** The construction of a plasmid containing the THF
synthesis genes (folKE), coupled
with a suicide switch mechanism, allows for the design of an engineered bacterial strain
that produces THF under
specific conditions, such as intestinal hypoxia. This strategy is not only beneficial for AD
treatment but also offers
new perspectives for the treatment of other diseases.

**Experimental Validation:** The successful transformation of plasmids into L168 ,
confirmed
through PCR and agarose gel
electrophoresis, followed by RNA extraction, metabolite measurement, and quantitative PCR
(qPCR) to quantify gene
expression levels, validates the increase in gene expression and THF production. These
experimental results not only
confirm the effectiveness of the genetic engineering strategy but also lay the groundwork
for further clinical
applications.

**Safety and Specificity:** The inclusion of a suicide switch ensures the self-destruction
of the
engineered bacterial
strain in non-target environments (e.g., high-oxygen environments outside the intestine),
which helps to mitigate
potential risks associated with the release of genetically engineered organisms into the
environment.

**Future Research Directions:** This study offers a fresh perspective on AD treatment but
also
indicates areas that require
further investigation, such as the long-term effects of THF in vivo, safety assessments, and
potential synergistic
effects with other treatment methods.

In future experiments, we will strive to validate the functionality of the hypoxia-inducible
suicide switch and continue
to explore the protocols for constructing engineered *Lactobacillus plantarum* L168
strains,
endeavoring to successfully
integrate the target gene with the suicide switch.

# **SYSTEM 2:inosine--gsk**

**Design:**

![](https://static.igem.wiki/teams/5238/engine-success/3.png)

Inosine, a purine nucleoside, has shown potential therapeutic effects in the
treatment of depression. It acts as a
neuroprotective agent by promoting axonal regeneration and enhancing neuronal survival.
Studies have indicated that
inosine can 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. 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[2].

We identified the sequence of the key gene gsk in the synthesis of inosine, and designed both
upstream and downstream
primers for gsk. The selected vector for this purpose was pSIP403. Subsequently, we designed
and constructed the
pSIP403-P9-gsk plasmid in the DH5α prior to introducing it into the sensory Lactobacillus
plantarum L168 to produce
inosine. Moreover, we also add the kill switch mentioned above into the vector for
biosafety.

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. 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.

**Build:**

![](https://static.igem.wiki/teams/5238/engine-success/2-2.png)

We identified the sequence of the key gene gsk in the synthesis of inosine,
and designed both upstream and downstream
primers for gsk. The selected vector for this purpose was pSIP403. Subsequently, we designed
and constructed the
pSIP403-P9-gsk plasmid in the DH5α prior to introducing it into the sensory Lactobacillus
plantarum L168 to produce
inosine. Moreover, we also add the kill switch mentioned above into the vector for
biosafety. (See more in design
safety-suicide switch).

Regrettably, we encountered difficulties when attempting to concatenate the suicide switch
with the target gene gsk due
to time constraints, which prevented us from successfully constructing the plasmid and
transforming it into
*Lactobacillus plantarum*. Consequently, in the subsequent experimental validation, we
utilized the plasmid
pSIP403-P9-gsk, which expresses only the target gene.

**TEST:**

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, 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).

![](https://static.igem.wiki/teams/5238/engine-success/figure3.png)

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). You can find more
information on the Results page.

![](https://static.igem.wiki/teams/5238/engine-success/figure4.png)

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

**Learn:**

Neuroprotective Properties of Inosine: Inosine's function as a neuroprotective agent is
underscored by its capacity to
stimulate axonal regeneration and bolster neuronal survival. This highlights the
significance of neurotrophic factors in
the maintenance and recuperation of neural health, which is essential for emotional
well-being.

Modulation of Neurotransmitter Levels: The research indicates that inosine has the potential
to alleviate depressive
symptoms by modulating neurotransmitter levels. This suggests that the equilibrium of
neurotransmitters plays a pivotal
role in the pathophysiology of mood disorders, and interventions that target these levels
may offer therapeutic
advantages.

Molecular Docking and Interaction Experiments: The study reveals that inosine can bind to the
neuronal nitric oxide
synthase (nNOS) receptor, a novel discovery that could pave the way for more precise
treatments with a reduced incidence
of side effects. This approach exemplifies the utility of molecular biology in the
identification and validation of new
therapeutic targets.

Gut-Brain Axis and Depression: The focus on the gut-brain axis and the exploitation of small
molecules produced by the
gut microbiota to target the brain presents a promising strategy for developing treatments
that can overcome the
blood-brain barrier. This approach could lead to the development of more efficacious and
rapidly acting antidepressants.

Experimental Validation: The successful transformation of the plasmid into Lactobacillus
plantarum L168 and the subsequent validation through PCR and agarose gel electrophoresis,
along with the quantification of gene expression and inosine production, establish a robust
experimental framework. These methodologies confirm the operability of the engineered system
and the generation of the therapeutic molecule of interest.

Future Directions: The study's promising outcomes also underscore the necessity for further
research. This includes the
exploration of the long-term effects of inosine on neural health, the refinement of delivery
methods for molecules
produced by the gut microbiota, and the exploration of potential synergies with existing
treatment modalities.

In future experiments, we will strive to validate the functionality of the hypoxia-inducible
suicide switch and continue
to explore the protocols for constructing engineered *Lactobacillus plantarum* L168
strains,
endeavoring to successfully
integrate the target gene with the suicide switch.

# **SYSTEM 3:theanine-glnA**

**Design:**

L-theanine is a unique amino acid found in tea leaves, constituting 1-2% of their dry weight.
It is known for its
various health benefits, including the improvement of learning and memory, relaxation,
anti-cancer properties, and
neuroprotection. Chemically similar to glutamate, L-theanine acts as a competitive
antagonist at glutamate receptors
such as NMDAR and AMPAR, albeit weakly, thus inhibiting neuronal death. It also regulates
the glutamate-glutamine cycle
by inhibiting glutamine transport and promoting the expression of the glutamine transporter
protein slc38a1, providing
neuroprotection against neurodegenerative diseases[1].

L-theanine degrades into glutamate in normal cells, subsequently increasing intracellular
concentrations of glutamine
and glutathione. Glutathione is a crucial antioxidant, reducing oxidative damage,
maintaining striatal neurotransmitter
homeostasis, and inhibiting nitric oxide production. In the context of Alzheimer's disease
(AD), L-theanine has been
shown to alleviate cognitive dysfunction and neurotoxicity induced by beta-amyloid by
reducing oxidative damage and
inactivating the ERK/p38 kinase and NF-κB pathways[2].

Furthermore, L-theanine also demonstrates potential effects in the treatment of depression.
It may improve mood and
sleep by increasing serotonin, dopamine, and GABA levels in the brain, which are closely
related to mood regulation.
L-theanine may also reduce excitotoxicity caused by excess glutamate by its antagonistic
action on glutamate receptors,
which could be particularly beneficial for patients with depression.

In summary, L-theanine, as a small molecule, shows potential not only in neuroprotection and
the treatment of AD but
also as a promising candidate for the treatment of depression and other neurological
disorders.

We identified the sequences of the key genes glnA of L-theanine, and designed both upstream
and downstream primers for
them. The selected vector for this purpose was pSIP403. Subsequently, we designed and
constructed the plasmid in the
DH5α prior to introducing it into the sensory *Lactobacillus plantarum* L168 to produce
theanine.

1. Chen SQ, Wang ZS, Ma YX, et al.
Neuroprotective Effects and Mechanisms of Tea Bioactive
Components in
Neurodegenerative Diseases. Molecules. 2018;23(3):512. doi:10.3390/molecules23030512

2. Kim TI, Lee YK, Park SG, et al.
l-Theanine, an amino acid in green tea, attenuates
beta-amyloid-induced cognitive
dysfunction and neurotoxicity: reduction in oxidative damage and inactivation of ERK/p38
kinase
and NF-kappaB pathways.
Free Radic Biol Med. 2009;47(11):1601-1610. doi:10.1016/j.freeradbiomed.2009.09.008

**Build:**

![](https://static.igem.wiki/teams/5238/engine-success/55.png)

We identified the sequences of the key genes glnA of L-theanine, and designed both
upstream and
downstream primers for
them. The selected vector for this purpose was pSIP403. Subsequently, we designed and
constructed
the plasmid in the
DH5α prior to introducing it into the sensory *Lactobacillus plantarum* L168 to produce
theanine.

### TEST:

We successfully transformed the plasmid into *Lactobacillus plantarum* L168 , and
validation
through PCR
and agarose gel
electrophoresis confirmed this achievement. Following this, we cultured Lactobacillus
plantarum L168
and conducted RNA
extraction, while the supernatant was used for metabolite analysis.
We then
performed reverse
transcription and quantitative polymerase chain reaction (qPCR) to quantify the gene
expression
levels in Lactobacillus
plantarum L168 , both with and without the recombinant plasmid. The qPCR results imply
that our
designed plasmid
significantly upregulated the expression of key genes for theanine in Lactobacillus
plantarum L168
compared to the
control group (Fig. 5). You can find more information on the Results page.

![](https://static.igem.wiki/teams/5238/engine-success/figure5.png)

Figure 5. Relative Expression Analysis of genes glnA by qPCR.

**Learn:**

We successfully employed the glnA gene to produce theanine within *Lactobacillus
plantarum*
L168 .
Utilizing PCR and
agarose gel electrophoresis techniques, the team confirmed the successful transformation
of the
target gene within
*Lactobacillus plantarum* L168 . Furthermore, through the application of quantitative
polymerase chain
reaction (qPCR)
technology, we quantified the gene expression levels, demonstrating that the designed
plasmid
significantly enhanced the
expression of key genes associated with theanine. By producing theanine within
Lactobacillus
plantarum L168 , this paves
the way for potential clinical applications, particularly in the development of novel
therapeutics
for Alzheimer's
Disease (AD) and depression.

# **SYSTEM 4:Niacin--pncA**

**Design:**

Niacin, also known as vitamin B3, is an essential nutrient that offers various health
benefits,
including lowering
cholesterol, alleviating arthritis, and enhancing brain function. In the body, niacin is
converted into niacinamide,
which forms part of coenzyme I and coenzyme II. These coenzymes are involved in tissue
respiration, the oxidation
process, and the anaerobic breakdown of carbohydrates. Niacinamide inhibits the formation of
melanin, prevents rough
skin, and aids in the repair of damaged cells. It is also indispensable for the synthesis of
sex
hormones.

Recent studies have highlighted the potential of niacin, also known as vitamin B3, in
treating
Alzheimer's Disease (AD)
and depression. Researchers from the Indiana University School of Medicine explored how
niacin
modulates microglial
responses to amyloid plaques in animal models of AD. They discovered that niacin, when
administered in laboratory
models, restricted the progression of AD. This finding identifies a potential new
therapeutic
target for AD that can be
regulated through the FDA-approved drug Niaspan.

We identified the sequences of the key genes pncA of Niacin,, and designed both upstream and
downstream primers for
them. The selected vector for this purpose was pSIP403. Subsequently, we designed and
constructed the plasmid in the
DH5α prior to introducing it into the sensory *Lactobacillus plantarum* L168 to produce
niacin.

**Build:**

![](https://static.igem.wiki/teams/5238/engine-success/66.png)

We identified the sequences of the key genes pncA of Niacin,, and designed both upstream and
downstream primers for
them. The selected vector for this purpose was pSIP403. Subsequently, we designed and
constructed the plasmid in the
DH5α prior to introducing it into the sensory *Lactobacillus plantarum* L168 to produce
niacin.

**TEST:**

We successfully transformed the plasmid into *Lactobacillus plantarum* L168 , and
validation
through PCR and agarose gel
electrophoresis confirmed this achievement. Following this, we cultured Lactobacillus
plantarum L168 and conducted RNA
extraction, while the supernatant was used for metabolite analysis. We
then performed reverse
transcription and quantitative polymerase chain reaction (qPCR) to quantify the gene
expression levels in Lactobacillus
plantarum L168 , both with and without the recombinant plasmid. The qPCR results imply that
our designed plasmid
significantly upregulated the expression of key genes for niacin in *Lactobacillus
plantarum*
L168 compared to the control
group (Fig. 6). You can find more information on the **Results** page.

![](https://static.igem.wiki/teams/5238/engine-success/figure6.png)

Figure 6. Relative Expression Analysis of genes pncA by qPCR.

**Learn:**

We successfully employed the pncA gene to produce niacin within *Lactobacillus
plantarum* L168 .
Utilizing PCR and agarose
gel electrophoresis techniques, the team confirmed the successful transformation of the
target gene within Lactobacillus
plantarum L168 . Furthermore, through the application of quantitative polymerase chain
reaction (qPCR) technology, the
team quantified the gene expression levels, demonstrating that the designed plasmid
significantly enhanced the
expression of key genes associated with niacin. By producing niacin within Lactobacillus
plantarum L168 , this study
paves the way for potential clinical applications, particularly in the development of novel
therapeutics for Alzheimer's
Disease (AD) and depression.

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RESULTS

![](images/back.png)

* [system1: AD tetrahydrofolate--folKE](#section1)
* [system2: MDD inosine--gsk](#section2)
* [system3&4: theanine--glnA,niacin--pncA](#section3)

# Result of system1: AD tetrahydrofolate--folKE

**SPR
Biacore
molecular interaction**

In System 1, we initially conducted Surface Plasmon Resonance (SPR) Biacore molecular
interaction assays to screen which
small molecule could better bind to the LilrB3 receptor. Based on the simulated molecular
docking results provided by
the modeling group (for details, see [model](https://2024.igem.wiki/njmu-china/model#section4)), we
selected candidate substances
that are more suitable for
human use and have fewer side effects: tetrahydrofolate and beta-carotene.

We procured LilrB3 protein, tetrahydrofolate, and beta-carotene small molecules, as well as
GM-5 chips, and performed
the experiments on the SPR Biacore molecular interaction instrument. Initially, we
immobilized the LilrB3 protein onto
the GM-5 chips using the instrument. Subsequently, we prepared solutions of tetrahydrofolate
and beta-carotene at
corresponding concentration gradients for experimental analysis.

In the experiment with tetrahydrofolate and LilrB3 protein, the results indicated that the
interaction between the
LilrB3 protein and tetrahydrofolate is consistent with the modeling predictions, with a
dissociation constant (KD) value
of 3.183 × 10^-8 M. (Figure 1-2)

![](https://static.igem.wiki/teams/5238/result/1.png)

**Figure 1.** Result of interaction between lilrb3 protein and
Tetrahydrofolate-1

![](https://static.igem.wiki/teams/5238/result/2-1.png)

**Figure 2.** Result of interaction between lilrb3 protein and
Tetrahydrofolate-2

This indicates that tetrahydrofolate can effectively bind to the LilrB3 protein.

However, the results for beta-carotene were not as expected. As the concentration increases,
the binding rate decreases,
indicating nonspecific binding. This suggests that in this system, there may be no
significant interaction between
beta-carotene and the LilrB3 protein. This could be due to the lipophilic nature of
beta-carotene, which may not be
conducive to its binding with the LilrB3 protein under the conditions used in this system.
(Figure 3)

![](https://static.igem.wiki/teams/5238/result/3.png)

**Figure 3.** Result of interaction between lilrb3 protein and
beta-carotene

Based on the aforementioned results, we have decided to select tetrahydrofolate as the
binding molecule for the LilrB3
protein.

Our objective is to enhance the expression of tetrahydrofolate (THF) to target the LilrB3
receptor. As a critical target
in Alzheimer's disease (AD), the receptor LilrB3 strongly binds to APOE4. By disrupting the
APOE4-LilrB3 binding, this
system may relieve the formation and deposit of amyloid protein to ameliorate AD pathology.

![](https://static.igem.wiki/teams/5238/result/4.png)

Figure 4. Tetrahydrofolate Module Mechanism Diagram [1]

We choose *Lactobacillus plantarum* L168 as the chassis and pSIP403 as the vector. By searching
its synthetic pathway, we
found the key enzyme folKE and its coding genes folK and folE. Then, we inserted the gene
folKE into our designed
plasmids to complete the construction and synthesis of plasmids. In view of biosafety, we
designed a kill switch which
is activated under hypoxic conditions, include a Hypoxia-inducible promoter, a RBS, a coding
of SrpR repressor that
inhibits transcription of the SrpR-dependent promoter and a terminator.

**DNA level**

*Lactobacillus plantarum* L168, a strain we obtained from our PI, Xingyin Liu's lab, has been
proven to be able to
alleviate social behavior deficits and reduce gut inflammation in animal models.[2,3]

We constructed the pSIP403-P9-folKE in DH5α, transformed it into *Lactobacillus plantarum*
L168, and extracted the plasmid
to prove the success of our transformation.

Initially, we designed primers to linearize the plasmid and amplify the target gene from the
genome of L168
via PCR. The vector utilized in this process was pSIP403. For homologous recombination, we
strategically designed
primers and conducted PCR using the homology arm located on the plasmid backbone.

| Primer | Sequence |
| --- | --- |
| HWY86F | tctagactcgaggaattcggtacc |
| HWY86R | atctaaaatctccttgtaatagtattttatagaatacatatatgctggc |
| HWY69F | atactattacaaggagattttagatATGGCAAGTAGGGAAGAACGG |
| HWY97R | cgaattcctcgagtctagaCTACTTTGCGATTCGCTGTAAGAATTCT |

Then we transformed it into E.coli DH5α. To confirm the efficacy of our transformation, we
devised primers, conducted
plasmid PCR and colony PCR, and subsequently analyzed the results via agarose gel
electrophoresis.

**Function
Verification**

We achieved successful plasmid transformation into L168, and the validation through PCR and
agarose gel electrophoresis
confirmed the success of this process. Subsequently, we cultured L168 and performed RNA
extraction. At the same time, the culture supernatant was employed for metabolite measurement.

Subsequently, reverse transcription and quantitative polymerase chain reaction (qPCR)
experiments were conducted to
quantify the gene expression levels of L168 that contained the pSIP403-P9-folKE plasmid or
not.

As shown in the figure 5, these are our qPCR results.

![第一张图片](https://static.igem.wiki/teams/5238/result/5.png)
![第二张图片](https://static.igem.wiki/teams/5238/result/6.png)

Figure 5. Relative Expression Analysis of genes folK and folE by qPCR.

The left group represents two different transcription levels of the gene folK. In this group,
the right column shows the
baseline L168 in its normal state, serving as the control group, which harbors the empty
vector pSIP403. We can see the
successful expression of the gene folK in L168 from the left column with the significantly
increased level of the gene
folK. The right group represents two different transcription levels of the gene folE,
indicating the preliminary effect
of the gene folE in L168.

The above experiment analyzed the relative expression levels of two genes using qPCR. The
figure illustrates that the
relative expression of two genes (folK and folE) was significantly upregulated in
pSIP403-P9-folKE plasmid in L168
compared to the control group (p ＜ 0.0001).

We also used Liquid Chromatograph Mass Spectrometer (LC-MS) to examine the production of THF
in the supernatant of L168.

As shown in the figure 6, these are our LC-MS results.

![](https://static.igem.wiki/teams/5238/f984d2d335ef440bed6eba2a2440c79.webp)

Figure 6. Relative levels of THF between two groups by LC-MS.

It's easy to see the level of THF increased dramatically in L168 containing the
pSIP403-P9-folKE plasmid compared to the
control group, demonstrating that as expected, the folKE gene was successfully
expressed in
L168 and produced THF.

[1] By Figdraw.

[2] Chen K, Luan X, Liu Q, et al.
Drosophila
Histone Demethylase KDM5 Regulates Social
Behavior through Immune Control
and Gut Microbiota Maintenance. Cell Host Microbe. 2019;25(4):537-552.e8.
doi:10.1016/j.chom.2019.02.003.

[3] Zhang Q, Zhao Q, Li T, et al.
*Lactobacillus plantarum*-derived indole-3-lactic acid
ameliorates colorectal
tumorigenesis via epigenetic regulation of CD8+ T cell immunity. Cell Metab.
2023;35(6):943-960.e9.
doi:10.1016/j.cmet.2023.04.015.

# Result of system2: MDD inosine--gsk

**SPR Biacore
molecular interaction**

In System 2, we initially conducted Surface Plasmon Resonance (SPR) Biacore molecular
interaction
assays to screen which
small molecule could better bind to the LilrB3 receptor. Based on the simulated molecular
docking results provided by
the modeling group (for details, see model 【放model链接】), we selected candidate substances
that
are more suitable for
human use and have fewer side effects: inosine, uridine 5'-monophosphate and flavin adenine
dinucleotide.

We procured nNOS protein, inosine, uridine 5'-monophosphate and flavin adenine dinucleotide
small
molecules, as well as
GM-5 chips, and performed the experiments on the SPR Biacore molecular interaction
instrument.
Initially, we immobilized
the nNOS protein onto the GM-5 chips using the instrument. Subsequently, we prepared
solutions
of inosine, uridine
5'-monophosphate and flavin adenine dinucleotide at corresponding concentration gradients
for
experimental analysis.

In the experiment with inosine and nNOS protein, the results indicated that the interaction
between the nNOS protein and
inosine is consistent with the modeling predictions, with a dissociation constant (KD) value
of
2.524×10-6, with chi2
within a credible range.(Figure 1-2)

![](https://static.igem.wiki/teams/5238/result/8.png)

Figure 1. Result of interaction between nNOS protein and inosine-1

![](https://static.igem.wiki/teams/5238/result/9.png)

Figure 2. Result of interaction between nNOS protein and inosine-2

This indicates that inosine can effectively bind to the nNOS protein.

However, the results for uridine 5'-monophosphate were not as expected. As the concentration
increases, the binding rate
decreases, indicating nonspecific binding. This suggests that in this system, there may be
no
significant interaction
between uridine 5'-monophosphate and the nNOS protein.

![](https://static.igem.wiki/teams/5238/result/10.png)

Figure 3. Result of interaction between nNOS protein and uridine
5'-monophosphate-1

Repeating the experiment with increased uridine concentration, the results are still
unsatisfactory.

![](https://static.igem.wiki/teams/5238/result/11.png)

Figure 4. Result of interaction between nNOS protein and uridine
5'-monophosphate-2

![](https://static.igem.wiki/teams/5238/result/12.png)

Figure 5. Result of interaction between nNOS protein and uridine
5'-monophosphate-3

After our discussion with our teacher, we thought that the high refractive index of uridine
itself might be the reason
it's not suitable as a component of the mobile phase. Consequently, the SPR molecular
interaction assay may not
effectively capture the binding affinity between uridine and the protein.

Ultimately, we proceeded to screen the last candidate small molecule, flavin adenine
dinucleotide
(FAD), for its
molecular interaction with neuronal nitric oxide synthase (nNOS) protein. However, we
encountered some challenges:
flavin adenine dinucleotide is lipid-soluble and was dissolved in DMSO. Consequently, we
utilized a PBS buffer system
containing 0.02% DMSO for our experiments.

![](https://static.igem.wiki/teams/5238/result/13.png)

Figure 6. Result of interaction between nNOS protein and flavin adenine
dinucleotide-1

![](https://static.igem.wiki/teams/5238/result/14.png)

Figure 7. Result of interaction between nNOS protein and flavin adenine
dinucleotide-2

The results indicated that the outcomes were not satisfactory. The lack of significant
interaction observed in this
buffer suggests that the experimental results might have been affected by DMSO buffer.

Based on the aforementioned results, we have decided to select inosine as the binding
molecule
for the nNOS protein.

Our objective is to enhance the expression of inosine to target the neuronal nitric oxide
synthase (nNOS) receptor.
Increased SERT-nNOS coupling in the dorsal raphe nucleus is a significant trigger for
depression, so we overexpress gsk
to produce inosine, which will bind to the nNOS receptor to uncouple SERT from nNOS.

![](https://static.igem.wiki/teams/5238/result/15.png)

We identified the sequence of the key gene gsk in the synthesis of inosine, and designed both
upstream and downstream
primers for gsk. The selected vector for this purpose was pSIP403. Subsequently, we designed
and
constructed the
pSIP403-P9-gsk plasmid in the DH5α prior to introducing it into the sensory Lactobacillus
plantarum L168 to produce
inosine. Moreover, we also add the kill switch mentioned above into the vector for
biosafety.

**DNA level**

*Lactobacillus plantarum* L168, a strain we obtained from our PI, Xingyin Liu's lab, has been
proven to be able to
alleviate social behavior deficits and reduce gut inflammation in animal models.[1,2]

We generated the pSIP403-P9-gsk plasmid in DH5α, subsequently performing its transformation
into *Lactobacillus plantarum*
L168 and conducting plasmid extraction to confirm the successful transformation.

Initially, we designed primers to linearize the plasmid and amplify the target gene from the
genome of L168
via PCR. The vector utilized in this process was pSIP403. For homologous recombination, we
strategically designed
primers and conducted PCR using the homology arm located on the plasmid backbone.

| Primer | Sequence |
| --- | --- |
| HWY86F | tctagactcgaggaattcggtacc |
| HWY86R | atctaaaatctccttgtaatagtattttatagaatacatatatgctggc |
| HYY96F | cgaattcctcgagtctagaTTAACGATCCCAATAACTTTCTTCCAAACTATCT |
| HWY87R | atactattacaaggagattttagatATGAAGTTTCCAGGTAAACGGAAGAGT |

Then we transformed it into E.coli DH5α. To confirm the efficacy of our transformation, we
devised primers, conducted
plasmid PCR and colony PCR, and subsequently analyzed the results via agarose gel
electrophoresis.

**Function
Verification**

We achieved successful plasmid transformation into L168, and the validation through PCR and
agarose gel electrophoresis
confirmed the success of this process. Subsequently, we cultured L168 and performed RNA
extraction. At the same time, the culture supernatant was employed for metabolite measurement.

Subsequently, reverse transcription and quantitative polymerase chain reaction (qPCR)
experiments were conducted to
quantify the gene expression levels of L168 that contained the pSIP403-P9-gsk plasmid or
not.

As shown in the figure 8, these are our qPCR results.

The right column (L168) represents the baseline L168 in its normal state, serving as the
control group. The qPCR results
elucidate that our designed plasmid effectively and significantly upregulated the gene
expression of inosine in L168.

![](https://static.igem.wiki/teams/5238/result/2-8.png)

Figure 8. Relative Expression Analysis of gene gsk by qPCR.

We also used Liquid Chromatograph Mass Spectrometer (LC-MS) to examine the production of
inosine in the supernatant of
L168.

As shown in the figure 9, these are our LC-MS results.

![](https://static.igem.wiki/teams/5238/result/2-9.png)

Figure 9. Relative levels of inosine between two groups by LC-MS.

It’s easy to see the level of inosine increased dramatically in L168 containing the
pSIP403-P9-gsk plasmid compared to
the control group, demonstrating that as expected, the gsk gene was successfully expressed
in L168 and produced inosine.

[1] Chen K, Luan X, Liu Q, et al. Drosophila
Histone Demethylase KDM5 Regulates Social
Behavior through Immune Control
and Gut Microbiota Maintenance. Cell Host Microbe. 2019;25(4):537-552.e8.
doi:10.1016/j.chom.2019.02.003.

[2] Zhang Q, Zhao Q, Li T, et al.
*Lactobacillus plantarum*-derived indole-3-lactic acid
ameliorates colorectal
tumorigenesis via epigenetic regulation of CD8+ T cell immunity. Cell Metab.
2023;35(6):943-960.e9.
doi:10.1016/j.cmet.2023.04.015.

# Result of system3&4: theanine--glnA,niacin--pncA

There is sufficient research evidence indicating that L-theanine and niacin have alleviating
effects on depression and
Alzheimer's disease, both of which also exist in the supernatant of L168 according to our
previous experimental data.
The key gene of theanine synthesis glnA and the key gene of niacin pncA were independently
integrated into two different
plasmids, namely pSIP403-P9-glnA and pSIP403-P9-pncA.
We identified the sequences of the key genes glnA and pncA, and designed both upstream and
downstream primers for them.
The selected vector for this purpose was pSIP403. Subsequently, we designed and constructed
the two plasmids in the DH5α
prior to introducing it into the sensory *Lactobacillus plantarum* L168 to produce theanine
and niacin.

**DNA level**

*Lactobacillus plantarum* L168, a strain we obtained from our PI, Xingyin Liu's lab, has been
proven to be able to
alleviate social behavior deficits and reduce gut inflammation in animal models.[1,2]

We generated and verified the pSIP403-P9-glnA plasmid and pSIP403-P9-pncA plasmid in DH5α,
subsequently performing their
transformation into two groups of *Lactobacillus plantarum* L168 and conducting plasmid
extraction to confirm the
successful transformation.

Initially, we designed primers to linearize the plasmid and amplify the target genes from the
genome of L.plantarum L168
via PCR. The vector utilized in this process was pSIP403. For homologous recombination, we
strategically designed
primers and conducted PCR using the homology arm located on the plasmid backbone.

Then we transformed it into E.coli DH5α. To confirm the efficacy of our transformation, we
devised primers, conducted
plasmid PCR and colony PCR, and subsequently analyzed the results via agarose gel
electrophoresis.

**Function
Verification**

We achieved successful plasmid transformation into L168, and the validation through PCR and
agarose gel electrophoresis
confirmed the success of this process. Subsequently, we cultured L168 and performed RNA
extraction. At the same time, the culture supernatant was employed for metabolite measurement.

Subsequently, reverse transcription and quantitative polymerase chain reaction (qPCR)
experiments were conducted to
quantify the gene expression levels of L168 that contained the recombinant plasmid or not.

As shown in the figure 1, these are our qPCR results. The left group represents the
transcription level of glnA, and the
right one represents pncA. The qPCR results indicate that our designed plasmid effectively
and significantly upregulated
the key gene expression of both theanine and niacin in L168 compared to the control group
respectively.

![第一张图片](https://static.igem.wiki/teams/5238/result/3-1.png)
![第二张图片](https://static.igem.wiki/teams/5238/result/3-11.png)

Figure 1. Relative Expression Analysis of genes glnA and pncA by
qPCR.

[1] Chen K, Luan X, Liu Q, et al.
Drosophila Histone Demethylase KDM5 Regulates Social Behavior through Immune Control
and Gut Microbiota Maintenance. Cell Host Microbe. 2019;25(4):537-552.e8.
doi:10.1016/j.chom.2019.02.003.

[2] Zhang Q, Zhao Q, Li T, et al.
*Lactobacillus plantarum*-derived indole-3-lactic acid ameliorates colorectal
tumorigenesis via epigenetic regulation of CD8+ T cell immunity. Cell Metab.
2023;35(6):943-960.e9.
doi:10.1016/j.cmet.2023.04.015.

CONTACT US

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