◈Principle◈

It has been reported that lot of children with ASD have elevated levels of heavy metal ions in their plasma, further lead to dysfunction of mitochondrial. Hence, we proposed an engineered bacteria reducing metal ions level of plasma might be improve function of mitochondrial. We found metallothionein (MT), a common protein utilized to remove metal ions, by searching the literature$^6$$^,$$^7$. MT is a family of low molecular weight proteins that have a single peptide chain containing 61-68 amino acids, of which 20 are cysteines, distributed in two domains α and β clusters, which bind to a total of 7 divalent metal ions$^8$. The fact that metallothionein has a considerable affinity for divalent metal ions provides viable strategies for preventing heavy metal poisoning$^9$. We used the human metallothionein MT1A gene to express metallothionein$^9$. After careful consideration, we decided to link MT to the Lpp-OmpA hybrid display system to create a membrane-anchored recombinant protein for high efficiency$^6$$^,$$^1$$^0$.

We use The MerR family TFs as the sensor. They are a large class of bacterial proteins with unique physiological functions and molecular actions: under normal conditions, they function as transcription repressors, but rapidly transform into transcription activators in response to an imbalance of cellular metal ions$^1$$^1$$^,$$^1$$^2$. Specifically, RNA polymerase preferentially transcribes from the MerR promoter in the absence of heavy metal ions and MerR, hence increasing the quantity of MerR present in the cell. In the absence of heavy metal ions, the MerR protein is linked to DNA in the repressor conformation, sustaining repression of the promoter. The binding of heavy metal ions to one of the two binding sites on MerR induces a conformational change that puts MerR into the activating conformation, thereby allowing these sequences to interact productively with the RNA polymerase σ70 subunit to form an open transcriptional complex, thereby initiating transcription$^1$$^1$.

In addition, for biosafety, we designed a killing switch which is inducted by an inexpensive and non-toxic monosaccharide L-arabinose. Promoter PBAD is frequently used for killing switch due to s moderately high expression levels and tight regulation of transcription$^1$$^3$. MazF is an endoribonuclease that cleaves RNAs at ACA sites and causes the death of microbe, mediates killing of cells without causing lysis of bacterial cells. We inserted the toxic protein MazF in downstream of the PBAD promoter$^1$$^4$$^,$$^1$$^5$. Therefore, upon achieving the expected effect, the expression of MazF will lead to the host bacteria lysis.

◈Gene circuits◈

For the chassis, we choose Escherichia coli Nissle 1917, one of the most investigated probiotic bacteria that are non-pathogenic to the human body$^1$$^6$. For plasmids, we use pET-28a(+) vector. The pET System is the most powerful system yet developed for the cloning and expressing of recombinant proteins in E. coli$^1$$^7$.

Studies indicated that exposure to lead and mercury is associated with the development of autism$^1$$^8$. To sense mercury or lead ions, we chose the MerR and PbrR promoters and connected them with a "OR" gate. When the host ingests mercury or lead ions, the recombinant metallothionein will be expressed and attached to the bacterial membrane, adsorbing heavy metals and preventing heavy metal poisoning.

Regarding the killing switch, in the absence of arabinose, the AraC binds to certain sites of the DNA, preventing RNA polymerase from binding to pBAD and transcribing. With arabinose, the binding sites of AraC and DNA change, and RNA polymerase can bind to pBAD, initiating transcription, expressing MazF$^1$$^3$$^-$$^1$$^5$$^,$$^1$$^9$.

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$^2$$^0$. When sodium alginate encounters divalent cations (such as Ca 2+), its phase changes from liquid to cross-linked gel particles. However, alginate beads are not highly resistant to acid, and although they can complete the release of probiotics in the small intestine, they sacrifice a portion of the probiotics' protective ability in gastric acid.

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$^2$$^1$. Therefore, in order to further improve the survival rate of bacteria in gastric juice, pancreatic juice, and other digestive fluids, we decided, based on the alginate encapsulation of probiotics, to use freeze-drying technology to coat probiotics with whey protein. When the cells are encapsulated, yogurt can be a good carrier of probiotics. Protected bacteria that are alive at the time of consumption will survive through the gastrointestinal tract and reach the intestine in a viable state.

◈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$^2$$^2$. Significant elevations in lactate have also been documented in the urine of autistic children$^2$$^3$.

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$^2$$^4$.

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$^2$$^5$.

◈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.