In System One, we engineered plasmid pSIP403-N-acyltransferase-PnisA-nisRK-erY, into which the gene encoding the pivotal enzyme N-acyltransferase was incorporated. This modification allowed the plasmid to facilitate the expression of ferrichrome, which subsequently binds to anti-GAD within the human body, thereby reducing the concentration of anti-GAD. This reduction in anti-GAD levels is aimed at achieving therapeutic effects for autism.
In System Two, we designed plasmid pSIP403-hemA-PnisA-nisRK-erY, wherein the hemA gene was inserted. This alteration enabled the plasmid to express protoporphyrin, which binds to anti-MBP within the human body, consequently leading to a decrease in anti-MBP concentration. The intended outcome of this reduction is the treatment of autism.
- System 1
- System 2
◈ferrichrome◈
In System One, our objective is to enhance the expression of ferrichrome. This enhancement facilitates the binding of anti-GAD within the brain to ferrichrome, consequently reducing the levels of anti-GAD expression in the patient's cerebral system.
We choose Lactobacillus Plantarum L168 as the chassis and pSIP403 as the vector, and search its synthetic pathway and find the key enzyme N-acyltransferase. Then, we inserted the genes of N-acyltransferase into our designed plasmids to complete the construction and synthesis of plasmids.

◈protoporphyrin◈
For anti-MBP, we selected protoporphyrin as the small molecule metabolite for antibody degradation. During our investigation, we identified the rate-limiting enzyme, glutamyl-tRNA reductase, within the synthetic pathway.
We identified the gene sequence hemA, encoding the key enzyme glutamyl-tRNA reductase, and designed both upstream and downstream primers for hemA. The selected vector for this purpose was pSIP403. Subsequently, we designed the pSIP403-hemA-PnisA-nisRK-erY plasmid and introduced it into the sensory Lactobacillus plantarum L168. As an additional safety measure, we incorporated a suicide switch that could be activated by a transferable nisin-controlled expression (NICE) system, utilizing the combination of the nisA promoter and nisRK regulatory genes.

In general, if probiotics want to colonize the intestine, they must first pass through
harsh conditions such as gastric and pancreatic juices to increase the number of viable
bacteria that reach the small intestine. Therefore, the bioavailability of probiotics
can be significantly increased through the use of suitable delivery techniques, such as
microencapsulation of probiotics with suitable materials. The so-called
microencapsulation is that animal and plant cells are wrapped in beaded microcapsules,
preventing enzymes and other biological macromolecules and cells from escaping but
allowing tiny molecules and medium-sized nutrients to freely enter the microcapsules, so
as to achieve the aim of culture and protection. According to studies, calcium
alginate has been widely utilized for the microencapsulation of live bacteria
since it is inexpensive, non-toxic, and simple to apply
To increase alginate's resilience to acidic environments, we decided to add whey
protein, a combination of globular proteins extracted from whey. These proteins
are partially resistant to pepsin digestion and shield probiotics prior to their
delivery to the target site. Several research have demonstrated that whey protein
can be used as an encapsulating material to improve the physical and chemical
stability of alginate particles.The pellet proved capable of preserving probiotic
activity after a three-hour in vitro incubation in simulated gastric juice

◈Principle◈
To detect the mitochondrial function of autistic children, we select lactate as
the biosensor. When mitochondrial dysfunction occurs, the activity of pyruvate
dehydrogenase is inhibited, and pyruvate cannot enter the tricarboxylic acid cycle.
Insufficient ATP production inhibits the activity of pyruvate carboxylase, hence
inhibiting gluconeogenesis. Consequently, huge quantities of pyruvate are converted to
lactate, resulting in a large increase in the concentration of lactate in the body,
which leads to a significant increase in lactate in the urine. A study indicated that
lactate is a good biomarker in clinical biochemical metabolites in children with ASD
Currently, numerous lactate detection devices have been created. The majority of them
include enzymatic reactions involving lactate oxidase and lactate dehydrogenase connected
to amperometric detection or electrochemical biohybrid oxygen sensing based on natural
bacteria metabolism. However, many biosensing technologies are either insensitive or
expensive, limiting their application and adoption
To detect lactate concentrations, we constructed a whole-cell biosensor primarily mainly
include the lldPRD operon. Studies have shown that the lldPRD operon of Escherichia
coli is involved in L-lactate metabolism, which is induced by the growth of this compound.
The lldPRD operon (formerly named lct) of Escherichia coli is responsible for aerobic
l-lactate metabolism. It consists of three genes that form a single transcription unit that
can be induced to initiate in L-lactic acid, the lldR gene of which encodes the regulatory
protein lldR. Regulatory protein LldR regulates lactate metabolism mainly through its dual
effects, that is, lldPRD is an inhibitor or activator. Studies have reported that LldR
binds O1 and O2 in the absence of L-lactate, which may lead to DNA loops and transcriptional
repression. Binding of L-lactate to LldR promotes conformational changes that may disrupt DNA
loops, thereby forming transcriptional open complexes

◈Gene circuits◈
We chose Escherichia coli DH5α as the chassis and pSB4K5 as the vector. Constitutively, P9 promoter initiated the expression of lldR. When lactate is introduced, it can bind to the lldR and activate the P11 promoter24, allowing the transcription of the lacZ reporter gene, whose transcript product is β-Galactosidases. On the test strip, X-gal reacted with β-Galactosidases to create an insoluble blue product, causing a color shift.
On the test paper, the immobilized galactoside reacted with -Galactosidases to create an insoluble blue product, causing a color shift.
