Our approach focuses on developing a novel therapeutic strategy for asthma by engineering Lactobacillus plantarum to express siRNA under specific conditions, particularly during asthma flare-ups. This approach aims to enhance the bacterial endosomal escape and improve the overall safety of the therapeutic system (more details are mentioned in our safety page).

To achieve our objectives, we used two plasmids. The first plasmid was the pMG36E plasmid that was engineered for CO-BERA expression and the loading system within Lactobacillus plantarum. The construction of pMG36E was accomplished through a hierarchical assembly strategy, initially involving the integration of Level 1 parts into the pJUMP23-1A vector. These Level 1 components will be subsequently assembled to form Level 2 parts, which will be then ligated into the pJUMP46-2A vector. These parts will be assembled forming level 2 parts, which will be ligated into pJUMP46-2A. The second plasmid was the L1 TU1 lacZ vector, which was tailored for GFP-TSLP expression in human cell lines. This will allow for the assessment of therapeutic efficacy of our approach on TSLP mRNA in the human cells.

Each critical step in the development process has undergone rigorous laboratory validation to confirm the functionality and efficiency of our therapeutic system, thereby providing robust proof of concept (For more details Proof of concept page).

The genetic fragments which are essential for our circuit were procured from Integrated DNA Technologies (IDT) and Twist, while some foundational basic parts were sourced from standard distribution kits. Fragment orders were placed in the second half of August. Notably, the basic parts of our circuit were ordered as three fragments.

Dry lab

Simulation of DUF4811-Linker-L7Ae complex

We made a molecular dynamics simulation to validate the best transmembrane protein in the binding stability with the RNA binding protein (L7Ae). So we made the simulation for two transmembrane proteins ,DUF4811 and FOLDASE PrsA, within the Lactobacillus Plantarum membrane. We extracted the Root Mean Square Deviation (RMSD) and the Root Mean Square Fluctuation (RMSF) graph for both. DUF4811 complex RMSD ranged within 1.5 A and RMSF ranged within 0.1A which ensures our complex stability compared to the other Transmembrane protein.

The RMSD graph shows that L7Ae protein is highly stable with RMSD < 2 Å which is suitable for binding with a C/D box. While the C/D box RNA RMSD graph shows that C/D box move relative to the L7Ae protein, sharp rise at the beginning to ~2.5 Å as the RNA is settling into the K-turn recognition site, while the average RMSD 3-6 Å suggests the RNA kink angle may be fluctuating while the overall K-turn architecture remains bound to L7Ae (for more details refer to modelling page software).After 4 ns, the graph shows a decrease and stabilisation in RMSD ~3.5 Å, suggesting that the complex is settling into a more stable configuration.

Simulation of different C/D box copies with L7Ae

To connect the CO-BERA scaffold with L7Ae protein (RNA-binding protein) it needs a C/D box. So, to determine how many C/D box copies we did Rna analysis, Molecular dockings ,and molecular dynamic simulations which showed that one C/D box has the best scores in RNA ensemble diversity, docking score=-218.63, RMSD (root mean square deviation), RMSF( root mean square fluctuation) graphs as indication for stability.To connect the CO-BERA scaffold with L7Ae protein (RNA-binding protein) it needs a C/D box. So, to determine how many C/D box copies we did Rna analysis, Molecular dockings ,and molecular dynamic simulations which showed that one C/D box has the best scores in RNA ensemble diversity, docking score=-218.63, RMSD (root mean square deviation), RMSF( root mean square fluctuation) graphs as indication for stability.

Simulation of CO-BERA C/D box with the protein complex (l7Ae, linker protein and DUF4811)

To ensure the delivery of the CO-BERA scaffold to the bacterial membrane forming membrane vesicle to reach the lung cell, we need to do molecular docking and molecular dynamic simulation to the CO-BERA scaffold and the complex of proteins it will bind to (L7Ae, Linker and DUF4811).

The result graphs show Root Mean Square Deviation (RMSD) between 3.0-5.0 Å (0.30-0.50 nm), which is within the acceptable range for RNA-protein complex, and it falls in the optimal range for the functionality of our complex as the duplex siRNA will unwind forming 2 siRNA strand, also is essential for RISC loading and Dicer processing.

Since the graph shows a progressive increase in RMSD we need to ensure that the CO-BERA scaffold will remain attached to our protein complex (L7Ae - linker - DUF4811) and ensure accurate loading into the membrane vesicle. Therefore, we made a simulation at the bond area between C/D box L7Ae (as mentioned in model two). That shows a stabilised graph with RMSD ~3.5 Å after 4 ns, suggesting that the complex is settling into a more stable configuration

The RMSF graph show values variation from 0.8-5.5 Å, the RMSF values of 0.8-2.0 Å representing stable duplex core segments that maintain overall structural integrity during processing, RMSF values of 2.0-3.0 Å indicating controlled duplex breathing necessary for thermodynamic asymmetry assessment the extremely flexibility peak at residue 50 (RMSF=5.5 Å) which corresponds to the duplex cleavage site by DICER, the higher flexibility the higher enzyme accessibility and strand displacement. So, the RMSF balanced flexibility shows effective RNA interference pathway activation

Simulatiion Argonaute enzyme with Guide siRNA

Argonaute enzyme is a core protein in the RNA interfering pathway, which guides the RISC complex to the TSLP (Thymic Stromal Lymphopoietin) mRNA to cleave the mRNA and silence the gene expression. So, we did molecular dynamics simulation to detect the interaction of Argonaute enzyme with our Guide siRNA.

Results

Analysed the with MDTRAJ which gave us this RMSD and RMSF graph which show the stability of the argonaute protein with RMSD range around 2-2.7 Å after an initial equilibration phase indicating the stability of the protein , while the guide siRNA shows higher RMSD values (4-5.5 Å) which is expected, as RNA is generally more flexible than protein and as referred to (Department of Biotechnology, School of Biosciences and Technology, Vellore Institute of Technology, Vellore, 632014, Tamil Nadu, India), which have done simulation similar to ours of siRNA with argonaute, indicates that the average RMSD of the simulation was 0.568 nm (5.68 Å), indicating a quasi-stable state with moderate conformational rearrangements. And that the more dynamic nature of the AGO-siRNA complex may be biologically relevant, as previous studies have shown that structural flexibility and RNA strain contribute to efficient guide strand binding and RISC loading.

The RMSF of the Argonaute protein most resides show stable RMSF with the range 0.5-2.0 Å.The peak around (CYS272) may be related to catalytic function of the protein or a surface loop or an active site loop that maintain conformational flexibility.

The RMSF graph of the siRNA shows relatively uniform stability across most nucleotides with RMSF values around 2.0-2.8 Å. A21 shows the highest flexibility (~4.3 Å) as its the 3' end of the guide strand and A1 also shows elevated flexibility (~3.0 Å) as its the 5' end.

Wet lab

Fragments preparation

The experimental workflow commenced with the resuspension of all lyophilized DNA fragments. This included fragments 1, 2, and 3, which were procured from Integrated DNA Technologies (IDT) and Twist Bioscience, as well as lyophilized fragments sourced from distribution kits, specifically the CMV promoter, RBS, Terminator, and the mammalian vector.

The parts isolated from the distribution kits:

Each fragment was reconstituted by combining 100 µL of distilled water with the lyophilized material in its respective tube. Then, the tube was shaken until the pellet was completely dissolved. Subsequently, the tube was thoroughly shaken until the entire pellet was completely dissolved. Following reconstitution, 100 µL of each fragment solution was transferred to a distinct, labeled 1.5 mL Eppendorf sterile tube and stored at -20°C for short-term preservation.

PCR amplification

PCR amplification of our fragments necessitated the preparation of lyophilized primers. This involved reconstituting the primers by mixing thoroughly 10 µL of their stock solution to 90 µL of distilled water. Following primer preparation, 25 µL of a 2x concentrated Master Mix was dispensed into three separate PCR tubes. Subsequently, 2 µL of the working solution of both forward and reverse primers for each respective fragment were added and thoroughly mixed. Finally, 0.5 µL of the template fragment was introduced into each tube, and the tubes were transferred to the PCR thermal cycler. The PCR program was set to an annealing temperature of 55 °C for 1 minute. Subsequently, sequence verification was required to validate the size of the amplified fragments.

Gel electrophoresis

Verification of the amplified fragments and pMG36E plasmid’s size was performed using gel electrophoresis, a process executed in three distinct stages: gel preparation, sample loading and electrophoresis, followed by visualization.

Gel preparation began by dissolving 0.75 g of agarose powder in 50 mL of TAE buffer inside a microwavable flask, followed by thorough mixing. The mixture was then heated in a microwave for 1 minute until the agarose was completely dissolved. To this molten agarose, 1.5 µL of Ethidium bromide was added.

The gel solution was then poured into a casting tray equipped with a well comb to create sample loading wells. The gel was allowed to solidify completely at room temperature for 20-30 minutes prior to use.

Once solidified, the agarose gel was carefully placed into an electrophoresis box, which was subsequently filled with TAE buffer and a sufficient amount of ethidium bromide to completely submerge the gel. A molecular weight ladder, containing DNA fragments of known sizes, was loaded into the first lane of the gel. Following this, 2 µL of loading dye was mixed with 5 µL of each PCR product, and these prepared samples were then loaded into the remaining wells. An electric current was subsequently applied to the gel, and the power source was disconnected once the loading dye had migrated approximately 75-80% of the way down the gel. Finally, the gel was carefully transferred to a UV transilluminator to visualize the separated DNA fragments and analyze the results.

Following gel electrophoresis, it was observed that the amplification of fragment 3 was unsuccessful. Additionally, re-amplification of fragments 1 and 2 was necessary to yield sufficient copy numbers for subsequent downstream molecular procedures.

Purification of PCR products

Following PCR amplification, DNA fragments were purified using a spin column protocol. This involved mixing 200 µL of RNA lysis buffer to the PCR product by pipetting. Subsequently, 40 µL of isopropanol was added to the mixture by inversion. This mixture was then transferred to a spin column and allowed to incubate for 5 minutes, facilitating the binding of DNA to the column's membrane. Unbound material was removed by centrifugation for 30 seconds.

The bound DNA was then washed with 500 µL of wash buffer AW2, which was followed by another centrifugation step to ensure complete removal of the buffer. The column was then air-dried for 2 minutes to evaporate residual ethanol. Finally, the purified DNA fragments were eluted with 50 µL of nuclease-free water into a new Eppendorf tube, rendering them suitable for subsequent transformation procedures.

Transformation

To assess the functionality of various M13 primer variants for subsequent experimental procedures, a parallel bacterial transformation protocol was performed. In this procedure, 5 µL of purified DNA fragments were introduced into 20-50 µL of Jm109 competent cells within a Falcon tube. The mixture was gently agitated by flicking the tube's bottom and subsequently incubated on ice for 10 minutes. A heat shock was then applied by transferring the transformation tubes to a 42°C water bath for 45 seconds, immediately followed by a 2-minute incubation on ice. Subsequently, 250-1,000 µL of pre-warmed LB medium was added to the transformed bacteria, which was then incubated with shaking at 37°C for 30 minutes to facilitate recovery and expression of antibiotic resistance genes. Furthermore, 5 µL of Ampicillin was added to the liquid medium. Finally, the cultures were incubated overnight at 37°C with shaking to select transformed colonies. Initially, the transformation process encountered a failure, necessitating a repeat. The transformation was successfully repeated using the identical protocol, thereby confirming the functionality of the M13 primers.

Repeated PCR amplification

To ensure sufficient yield for downstream applications, PCR amplification of fragments 1 and 2 were repeated using the previously described protocol. In contrast, fragment 3 had failed to amplify initially and required optimization through gradient PCR. This involved testing six different annealing temperatures to identify optimal amplification conditions. Furthermore, the functionality of M13 primer variants was confirmed by their successful use in the PCR amplification of the 1-D5 plasmid, demonstrating their suitability for sequencing and other applications.

Plasmid and DNA Isolation and concentration measurement

Plasmid and DNA isolation from the transformed competent cells was performed in three distinct steps: cell lysis, DNA binding and washing, and elution.

The isolation process commenced with the lysis of bacterial cells. Initially, 200 µL of resuspension buffer was added to rehydrate the bacterial pellet, followed by thorough mixing via pipetting. Subsequently, 50 µL of alkaline lysis buffer was added to the mixture and incubated for 5 minutes to ensure complete lysis of the cell membranes. To neutralize this lysis reaction, 350 µL of N3 buffer was added and mixed by inversion.

The resulting mixture was then centrifuged for 4 minutes, separating the plasmid DNA-containing supernatant from cellular debris. This supernatant was then carefully transferred to a spin column, allowing the plasmid DNA to bind to the silica membrane.

Subsequently, the spin column underwent centrifugation to ensure the robust binding of plasmid DNA while concurrently washing through impurities. For purification, 500 µL of wash buffer AW1 was sequentially added and centrifuged through the column, followed by an additional 500 µL of wash buffer AW2 and subsequent centrifugation.The column was then subjected to air-drying for 2 minutes to eliminate any residual ethanol. Finally, the purified plasmid DNA was eluted with 50 µL of nuclease-free water into a new Eppendorf tube, followed by a 3-minute incubation period to fully rehydrate the DNA and facilitate its dissociation from the membrane, thereby maximizing the final yield. The eluted DNA was then centrifuged for 3 minutes to pellet any remaining impurities. Ultimately, the concentration of both the DNA fragments and the plasmid was quantified using a Nanodrop spectrophotometer. Following this, both DNA fragments and the plasmid were isolated in preparation for subsequent digestion by restriction enzymes.

Digestion

Following the isolation of our genetic fragments, specific restriction enzymes were employed to generate compatible "sticky ends" for directional cloning according to Moclo assembly. Each genetic part was treated with a distinct pair of restriction enzymes. Concurrently, the vector was digested with the identical pair of restriction enzymes to ensure compatibility for subsequent ligation. The digested products then underwent a two-stage PCR process: an initial heat activation step at 37 °C for 1 hour, followed by a heat-inactivation step. Significantly, this step involved the digestion and subsequent isolation of each basic part, ensuring they were ready for the next stages of assembly.

Gel electrophoresis

To confirm the successful digestion and appropriate sizing of our genetic components, the digested parts were rigorously verified using gel electrophoresis. The protocol followed for this verification was identical to that previously established for fragment analysis. This step was critically important for the subsequent construction of the backbone vector. At this stage, each individual part intended for use in our experiment, encompassing those derived from the three initial DNA fragments as well as other fragment components, underwent meticulous size verification. These verified parts included the following:

Basic parts of the Lactobacillus plantrum circuits:

Twist parts

All genetic parts enumerated below were procured from Twist Biosciences, with orders placed during the latter half of August. These parts are responsible for conditioned expression of CO-BERA, loading system, endosomal escape and toxin-anti-toxin system. Each part's successful acquisition was confirmed by comparing the observed band sizes from PCR amplification with their theoretically predicted lengths, as determined by their respective designed primers.

P11 Promoter

The P11 Promoter exhibited PCR bands exceeding 50 base pairs (bp), precisely matching the theoretically estimated length of 60 bp derived from its designed primers. This congruence confirms the successful procurement and amplification of this promoter.

Anti-toxin Promoter

PCR amplification of the Anti-toxin promoter yielded bands greater than 75 bp, which aligns perfectly with the theoretically calculated length of 92 bp based on its designed primers. This result unequivocally demonstrates the successful acquisition of this promoter.

SV40 Poly A Signal

The SV40 Poly A signal, upon PCR amplification, produced bands exceeding 100 bp. This observed length is consistent with the theoretical estimation of 122 bp derived from its designed primers, thereby confirming its successful acquisition.

TrrnB Terminator

PCR amplification of the TrrnB Terminator resulted in bands larger than 50 bp, which is in agreement with the theoretically predicted length of 72 bp based on its designed primers. This validates the successful procurement of this terminator

RBS (Ribosomal Binding Site)

The RBS part, following PCR amplification, showed bands exceeding 10 bp, precisely matching its theoretically estimated length of 11 bp as determined by its designed primers. This confirms the successful acquisition of the RBS.

T7 Promoter

PCR amplification of the T7 promoter yielded bands greater than 10 bp, which is consistent with the theoretically calculated length of 18 bp based on its designed primers. This result confirms the successful procurement of this promoter.

P-kat A Promoter

The P-kat A promoter, upon PCR amplification, produced bands exceeding 50 bp. This observed length aligns with the theoretically estimated length of 66 bp derived from its designed primers, thereby confirming its successful acquisition.

P32 Promoter + lac R operon

PCR amplification of the P32 promoter + lacR operon resulted in bands larger than 150 bp, which is in excellent agreement with the theoretically predicted length of 188 bp based on its designed primers. This validates the successful procurement of this combined genetic element.

Pkat A + Rep Repressor

The Pkat A + Rep repressor part, following PCR amplification, showed bands exceeding 75 bp, precisely matching its theoretically estimated length of 83 bp as determined by its designed primers. This confirms the successful acquisition of this regulatory element.

Pkat A + Operator

PCR amplification of the Pkat A + operator yielded bands greater than 75 bp, which is consistent with the theoretically calculated length of 86 bp based on its designed primers. This result confirms the successful procurement of this regulatory element.

T7 + SVTR Kozak

The T7 + SVTR Kozak part, upon PCR amplification, produced bands exceeding 25 bp. This observed length aligns with the theoretically estimated length of 38 bp derived from its designed primers, thereby confirming its successful acquisition.

P170-Cp Promoter

PCR amplification of the P170-Cp promoter resulted in bands larger than 250 bp, which is in excellent agreement with the theoretically predicted length of 326 bp based on its designed primers. This validates the successful procurement of this promoter.

P32 Promoter with lac Operon

The P32 promoter with lac operon, following PCR amplification, showed bands exceeding 150 bp, precisely matching its theoretically estimated length of 188 bp as determined by its designed primers. This confirms the successful acquisition of this regulatory element.

IDT Parts

The following genetic fragments were ordered from Twist Biosciences during the latter half of August. Successful acquisition and amplification of each fragment were confirmed by comparing the observed band sizes from PCR with their theoretically predicted lengths, as determined by their respective designed primers.

T2A + His tag + TSLP

PCR amplification of the T2A + His tag + TSLP fragment yielded bands exceeding 500 bp, which is in good agreement with the theoretically estimated length of 566 bp derived from its designed primers. This confirms the successful procurement and amplification of this fusion construct.

secE + L7Ae

The secE + L7Ae fragment, upon PCR amplification, produced bands larger than 500 bp, aligning with the theoretically calculated length of 552 bp based on its designed primers. This result demonstrates the successful acquisition of this fragment.

GFP (Green Fluorescent Protein)

PCR amplification of the GFP gene resulted in bands exceeding 500 bp, consistent with the theoretically predicted length of 717 bp based on its designed primers. This validates the successful procurement of this fluorescent protein gene.

CO-BERA Expression Non-Conditioned Circuit

The CO-BERA expression non-conditioned circuit, following PCR amplification, showed bands greater than 750 bp, which is in agreement with the theoretically estimated length of 908 bp derived from its designed primers. This confirms the successful acquisition of this genetic circuit.

Conditioned CO-BERA + CL-operator Circuit

PCR amplification of the Conditioned CO-BERA + CL-operator circuit yielded bands larger than 250 bp, aligning with the theoretically calculated length of 450 bp based on its designed primers. This result demonstrates the successful acquisition of this regulatory circuit.

Conditioned CO-BERA No Operator Circuit

The Conditioned CO-BERA no operator circuit, upon PCR amplification, produced bands exceeding 250 bp, consistent with the theoretically predicted length of 430 bp based on its designed primers. This validates the successful procurement of this genetic circuit.

Duf4811-L7Ae

PCR amplification of the Duf4811-L7Ae fragment resulted in bands greater than 750 bp, which is in good agreement with the theoretically estimated length of 867 bp derived from its designed primers. This confirms the successful acquisition of this protein-encoding fragment.

CO-BERA Circuit + Rep Operator

The CO-BERA circuit + Rep operator, following PCR amplification, showed bands greater than 500 bp, which is in agreement with the theoretically estimated length of 508 bp derived from its designed primers. This confirms the successful acquisition of this genetic circuit.

CO-BERA + Terminator

PCR amplification of the CO-BERA + Terminator fragment yielded bands larger than 250 bp, aligning with the theoretically calculated length of 422 bp based on its designed primers. This result demonstrates the successful acquisition of this combined genetic element.

Basic parts of the human cell circuits

The following genetic fragments were ordered from Twist Biosciences at the beginning of September. Successful acquisition and amplification of each fragment were confirmed by comparing the observed band sizes from PCR with their theoretically predicted lengths, as determined by their respective designed primers.

TSLP (Thymic Stromal Lymphopoietin)

The TSLP gene, upon PCR amplification, produced bands exceeding 250 bp, consistent with the theoretically predicted length of 494 bp based on its designed primers. This validates the successful procurement of the TSLP gene.

Toxin Anti-toxin

PCR amplification of the Toxin anti-toxin fragment resulted in bands greater than 500 bp, which is in good agreement with the theoretically estimated length of 615 bp derived from its designed primers. This confirms the successful acquisition of this protein-encoding fragment.

Lac Repressor

The Lac repressor gene, following PCR amplification, showed bands greater than 1000 bp, which is in agreement with the theoretically estimated length of 1083 bp derived from its designed primers. This confirms the successful acquisition of this regulatory protein gene.

Rep Repressor

PCR amplification of the Rep repressor gene yielded bands larger than 500 bp, aligning with the theoretically calculated length of 660 bp based on its designed primers. This result demonstrates the successful acquisition of this regulatory protein gene.

LLO (Listeriolysin O)

The LLO gene, upon PCR amplification, produced bands exceeding 1500 bp, consistent with the theoretically predicted length of 1587 bp based on its designed primers. This validates the successful procurement of this protein-encoding gene.

Foldase and L7Ae

PCR amplification of the Foldase and L7Ae fragments resulted in bands greater than 1250 bp, which is in good agreement with the theoretically estimated length of 1293 bp derived from its designed primers. This confirms the successful acquisition of this protein-encoding fragment.

Ligation (level 0 assembly)

Following the size verification of each basic genetic part, a ligation reaction was subsequently performed to assemble Level 0 constructs. Specifically, the basic parts designated for Lactobacillus plantarum circuits were ligated into the pUC19 destination vector, whereas the basic parts for human cell circuits were ligated into the pT1-01 destination vector.

For each reaction, a precisely measured 5 µL of the digested PCR product (insert) was combined with 10 µL of the corresponding digested destination vector. Moreover, the reaction mixture was supplemented with 2 µL of ligase buffer, 1 µL of T4 DNA ligase enzyme, and 1 µL of ATP, thereby establishing a final insert-to-vector volume ratio of approximately 1:2. The ensuing mixture was then incubated at room temperature for 1 hour to facilitate the enzymatic joining of the DNA fragments. This crucial step represented the Level 0 assembly, during which individual target fragments, obtained via PCR and subsequently digested, were successfully incorporated into their respective destination vectors.

Purification of ligated parts

Following the ligation reactions, the newly assembled DNA fragments were purified using a spin column protocol, which is identical to that employed for earlier fragment purification. Specifically, 200 µL of RNA lysis buffer was added to the ligated product and thoroughly mixed by pipetting. Subsequently, 40 µL of isopropanol was introduced and mixed by inversion. This entire mixture was then transferred to a spin column and allowed to incubate for 5 minutes, ensuring efficient binding of the DNA to the column's silica membrane. Unbound contaminants were removed by centrifugation for 30 seconds. The bound DNA was then subjected to a wash step with 500 µL of wash buffer AW2, followed by another centrifugation to spin the buffer through the column. The column was then air-dried for 2 minutes to eliminate any residual ethanol. Finally, the purified ligated DNA fragments were eluted with 50 µL of nuclease-free water into a fresh Eppendorf tube, preparing them for subsequent transformation procedures.

Transformation

The purified ligated parts were then introduced into Jm109 competent cells via bacterial transformation, following the previously established protocol, with the aim of amplifying these newly constructed plasmids. The initial transformation attempt unfortunately failed for reasons that were not immediately apparent. Consequently, the transformation into Jm109 competent cells was repeated under the same protocol. This repeated transformation proved successful, providing sufficient quantities of the desired plasmids for subsequent experimental procedures.

Isolation and Colony PCR

To confirm successful transformation and the presence of the desired plasmids, colony PCR was performed. The results from colony PCR confirmed positive transformation events, indicating that bacterial colonies harbored the target plasmids. Subsequently, the confirmed plasmids were extracted from these colonies using the previously established plasmid isolation protocol, ensuring success of level 0 assembly and their availability for the next experimental stages.

Digestion and Fragment Isolation for Level 1 Assembly

Following plasmid isolation, each individual genetic part, now harbored within its respective plasmid, was subjected to restriction enzyme digestion. Each part was carefully digested with its compatible restriction enzyme pair to generate specific "sticky ends," which are essential for directional ligation in the subsequent assembly steps. These precisely digested parts were then prepared for the Level 1 assembly.

Ligation (Level 1 Assembly)

The Level 1 assembly involved the ligation of Lactobacillus plantarum Level 0 genetic parts into five distinct destination vectors: pJUMP23-1A, pJUMP29-1B, pJUMP29-1C, pJUMP29-1D, and pJUMP29-1E. Concurrently, human cell basic parts were assembled into the L1 TU1 lacZ destination vector.

For each construct, the ligation reaction was prepared. Specifically, 5 µL of the digested insert was combined with 10 µL of its corresponding digested destination vectors. Furthermore, the reaction mixture was supplemented with 2 µL of ligase buffer, 1 µL of T4 DNA ligase enzyme, and 1 µL of ATP, maintaining a final component ratio of 10 µL vector to 5 µL insert volume. Subsequently, this ligation mixture was incubated at room temperature for 1 hour to facilitate the enzymatic joining of the genetic components.

It is noteworthy that the ligation of Lactobacillus plantarum basic parts into four of the five destination vectors failed, with successful ligation observed exclusively with pJUMP23-1A. Consequently, this step represents the critical Level 1 assembly stage, characterized by the directed incorporation of individual, pre-assembled (Level 0) genetic parts into pJUMP23-1A to generate a more complex genetic circuit.

Purification of ligated parts

Following the Level 1 ligation reactions, the newly assembled DNA fragments were purified using a spin column protocol identical to the method previously established for earlier fragment purification. This rigorous purification step ensured the removal of unwanted enzymes, unincorporated nucleotides, and other reaction byproducts, thereby preparing the DNA for optimal performance in subsequent transformation procedures.

Transformation

The purified Level 1 ligated parts were subsequently introduced into Jm109 competent cells through bacterial transformation. This process rigorously followed the previously established protocol, aiming to amplify the newly constructed plasmids. This transformation successfully led to the isolation of transformed colonies, thereby providing a sufficient quantity of the desired plasmids for all downstream experimental procedures.

Isolation and Colony PCR

To confirm successful transformation and the presence of the desired plasmids, colony PCR was performed. The results from colony PCR confirmed positive transformation events, indicating that bacterial colonies harbored the target plasmids. Subsequently, the confirmed plasmids were extracted from these colonies using the previously established plasmid isolation protocol, ensuring success of level 1 assembly and their availability for the next experimental stages.