We built our constructs using a hybrid cloning approach that combines the strengths of Golden Gate ( BsaI , Reclone syntax) for Level 0 and 1 assemblies, and RFC10 BioBrick assembly for Level 2 assembly .
In the Level 0 and 1 assemblies, we employed the L1 TU1 lacZ vector for mammalian cells and the pJUMP modular cloning framework for bacterial cloning, which follows the Reclone standard syntax. This system provides a standardized set of backbones and domestication rules, ensuring iGEM compatibility while enabling efficient multi-part Golden Gate assembly with BsaI .
All genetic parts were ordered as synthetic DNA fragments from Twist Bioscience and IDT. Instead of pre-synthesizing fragments with Type IIS restriction sites and overhangs, we ordered them in their basic form without overhangs. The assembly overhangs were designed directly into the cloning primers. This strategy provides greater flexibility: if an overhang requires redesign or correction, only the primer needs to be replaced, avoiding the need to reorder entire gene fragments.
(A) Type IIS sites for Golden Gate assembly ( BsaI for Level-0→Level-1),
(B) The Reclone overhangs based on Reclone syntax, which are appropriate for each part category (promoter, RBS, CDS, terminator),
In addition, for our Level 0, we ordered integrated fragments of promoters, RBSs, and terminators, separated by documented BioBrick restriction sites. Because such short regulatory elements cannot always be synthesized individually by IDT or Twist, this design ensures accurate ordering, simplifies downstream cloning, and maintains modularity for future part extraction.
1- Level 0 parts: All basic components (promoters, ribosome binding sites, coding sequences, linkers, and terminators) were overhanged following Reclone syntax.
2- Level 1 transcriptional units (TUs): Using BsaI Golden Gate, individual Level 0 parts were combined to generate functional TUs.
To construct a transcriptional unit for conditional expression of CO-BERA under the control of the oxidative stress–inducible promoter KatA, but with an integrated rep-operator site. This ensures tighter regulation by allowing repression from the Rep repressor circuit until specific lung conditions (H₂O₂ + acidic pH) relieve inhibition.
Forward primer: (CO-BERA + rep-operator) Forward
Reverse primer: (CO-BERA + rep-operator) Reverse
Primers were designed with BsaI recognition sites and compatible overhangs for Golden Gate assembly into a Level-1 backbone.
CO-BERA + rep-operator fragment: was amplified using (F / R).
Digested with BsaI and assembled into a Level-1 backbone following Reclone syntax.
This construct contains a Rep-operator–tuned CO-BERA expression unit, which combines:
Oxidative stress induction (KatA promoter)
Together, this enables AND logic control, so that CO-BERA is expressed only in inflamed lungs where H₂O₂ is high and pH is reduced.
To construct a transcriptional unit expressing the Rep repressor, which provides continuous repression of the KatA promoter (via rep-operator binding). ensuring that CO-BERA is only expressed when both oxidative stress (H₂O₂) and acidic pH relieve repression.
Forward primer (F3-F)
Reverse primer (F3-R)
Forward primer (RepR-F)
Reverse primer (RepR-R)
Forward primer (F1-F)
Reverse primer (F1-R)
Each primer was designed with BsaI recognition sites and compatible overhangs for Golden Gate assembly.
Amplified from Fragment 3 using (F3-F / F3-R).
Extracted from Fragment 3 via SacI / KpnI digestion, domesticated with BsaI for Level-1 assembly.
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted via XbaI / SalI digestion, domesticated with BsaI .
Amplified with (RepR-F / RepR-R) and domesticated with BsaI .
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted via BamHI / XbaI digestion, domesticated with BsaI .
Regulatory elements (P32 / RBS / TrrnB) were combined with Rep repressor CDS in a Golden Gate ( BsaI ) reaction. The resulting construct generates a Rep repressor transcriptional unit, which maintains repression of the KatA promoter until acidic pH in inflamed airways inhibits LacR and relieves this control.
To construct a transcriptional unit expressing the Lac repressor (LacR), which forms the pH-responsive repression. Under the control of the P170-CP25 promoter, LacR ensures repression of the rep-operator in acidic pH (Less than 7) which inhibit the repression of expression of CO-BERA (Activation)
Forward primer (F3-F)
Reverse primer (F3-R)
Forward primer (LacR-F)
Reverse primer (LacR-R)
Forward primer (F1-F)
Reverse primer (F1-R)
Each primer was designed with BsaI recognition sites and compatible overhangs for Golden Gate assembly following the Reclone syntax.
Amplified from Fragment 3 using (F3-F / F3-R).
Extracted from Fragment 3 via EcoRI / SacI digestion, domesticated with BsaI for Level-1 assembly.
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted from Fragment 1 via XbaI / SalI digestion, domesticated with BsaI .
Amplified with (LacR-F / LacR-R) and domesticated for BsaI Golden Gate assembly.
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted via BamHI / XbaI digestion, domesticated with BsaI .
Regulatory elements (P170-CP25 / RBS / TrrnB) were combined with LacR CDS in a single Golden Gate ( BsaI ) reaction. The resulting construct generates a LacR transcriptional unit, ensuring tight repression of the Rep operator until pH-triggered inhibition of LacR expression occurs.
To engineer a modular system for localizing and loading CO-BERA RNA into bacterial membrane vesicles (MVs) using transmembrane protein (TMP)–linked RNA-binding proteins (RBPs). (DUF4811–L7Ae) This complex was expressed under the constitutive promoter to ensure an effective loading system.
Forward primer (F1-F)
Reverse primer (F1-R)
DUF4811–L7Ae (DUF–F / DUF–R)
Each primer was designed with BsaI and compatible overhangs for Golden Gate assembly.
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted from Fragment 1 via EcoRI / SacI digestion, domesticated with BsaI for Level 1 assembly.
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted from Fragment 1 via XbaI / SalI digestion, domesticated with BsaI for Level 1 assembly.
DUF4811–L7Ae: amplified and digested with BsaI .
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted from Fragment 1 via BamHI / XbaI digestion, domesticated with BsaI .
Regulatory elements (P11 / RBS / TrrnB) combined with DUF4811–L7Ae in a Golden Gate ( BsaI ) reaction
Generated RNA loading constructs, which express a TMP–L7Ae fusion under P11 control.
We assemble two Level-1 transcriptional units (TUs)
(A) CO-BERA expression
(B) Loading circuit into the pJUMP43-2A Level-2 backbone using a classical RFC10-style restriction/ligation system
Digest CO-BERA TU with EcoRI / SpeI.
Digest Loading TU with XbaI / PstI.
Digest pJUMP43-2A backbone with EcoRI / PstI. Because SpeI and XbaI produce compatible 4-base cohesive ends, inserts will ligate in the chosen order and form a mixed scar at the Spe/Xba junction that will not be re-cut by either enzyme.
To enhance therapeutic RNA delivery efficiency, we designed an endosomal escape module using a mutated form of Listeriolysin O (LLO), expressed under the oxidative stress–responsive KatA promoter, and compatible with the pJUMP modular system.
Fragment 1 Forward/Reverse
LLO Forward / Reverse
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted from Fragment 1 via BstI / HindIII digestion, domesticated with BsaI for Level 1 assembly.
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted from Fragment 1 via XbaI / SalI digestion, domesticated with BsaI .
LLO coding sequence amplified using (LLO-F / LLO-R).
Directly digested with BsaI for Level 1 assembly.
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted from Fragment 1 via BamHI / XbaI digestion, domesticated with BsaI .
All regulatory elements (P-KatA / RBS / TrrnB) combined with LLO CDS in a Golden Gate ( BsaI ) reaction.
Generated the endosomal escape circuit, driving expression of LLO under the oxidative stress–responsive KatA promoter
To enhance the biosafety of our engineered Lactobacillus plantarum system, we designed a toxin–antitoxin (TA) System. This genetic circuit ensures that engineered bacteria survive only under controlled conditions. The antitoxin promoter drives expression of the protective antitoxin, while in the absence of the required regulatory signal, the toxin dominates, leading to self-elimination of escaped bacteria.
Forward primer (F1-F)
Reverse primer (F1-R)
Forward primer (TA-F)
Reverse primer (TA-R)
All primers were designed with BsaI recognition sites and standardized overhangs for Golden Gate assembly.
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted from Fragment 1 via SacI / KpnI digestion.
Domesticated with BsaI for Level 1 assembly.
Amplified with TA-F / TA-R primers.
Digested with BsaI and prepared for Level 1 assembly.
Amplified from Fragment 1 using (F1-F / F1-R).
Extracted from Fragment 1 via BamHI / XbaI digestion.
Domesticated with BsaI for Level 1 assembly.
Combined Antitoxin promoter / TA operon / TrrnB terminator in a Golden Gate ( BsaI ) reaction.
Generated a safety circuit where L. plantarum viability is conditional and controlled, preventing survival outside of the intended lung environment.
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Create a mammalian expression cassette that reports TSLP mRNA knockdown by CO-BERA. The construct expresses a single open reading frame GFP-T2A-TSLP under a CMV Promoter and terminates with a poly(A) signal. If the CO-BERA siRNA correctly targets the GFP-TSLP transcript, GFP fluorescence will be lost; if not, GFP will be expressed (T2A separates GFP from TSLP at translation).
Fragment 3 (CMV Promoter) Forward/Reverse
Fragment 1 (PolyA) Forward/Reverse
GFP Forward/Reverse
T2A + His + TSLP Forward/Reverse
All primers include BsaI sites and Reclone-compatible overhangs for downstream Level-1 Golden Gate
CMV Promoter (Fragment 3):amplify with F3-F/F3-R → Extracted from the fragment using XbaI / BstI. Domestication for Level-1 via BsaI .
Poly(A) terminator (Fragment 1): amplified with F1-F/F1-R → Extracted from the fragment using KpnI / BamHI. Domestication with BsaI for Level-1.
GFP CDS: amplify with GFP-F/GFP-R: amplify with GFP-F/GFP-R → domesticate with BsaI (prepare as Level-0 CDS part).
T2A + His-TSLP CDS:amplify with T2A-TSLP-F/T2A-TSLP-R → domesticate with BsaI .
Assemble TU: Combine CMV Promoter, GFP CDS, T2A+His+TSLP CDS, and PolyA terminator in a BsaI Golden Gate reaction following Reclone overhang ordering to produce a single Level-1 plasmid containing the transcriptional unit.
Deliver CO-BERA (via purified bacterial MVs or positive control siRNA) to cells expressing GFP-T2A-TSLP .
Correct targeting: decreased GFP fluorescence + reduced TSLP protein signal.
Incorrect/no targeting: GFP fluorescence persists, and TSLP remains detectable.
In addition to our main therapeutic constructs, we designed a set of further circuits to validate individual modules of our project. These circuits allowed us to troubleshoot failures in conditioning, RNA loading, or CO-BERA expression. By modularizing each step, we ensured that technical issues could be rapidly diagnosed and alternative strategies tested.
If the dual-gate system (pH + H₂O₂) fails to behave as intended or proves overly leaky/complex in early validation, this backup removes the pH gate and relies solely on the oxidative-stress (H₂O₂) sensor (pKatA) to control CO-BERA expression. This reduces circuit complexity while still providing disease-responsive control (activated in inflamed airways where H₂O₂ is elevated). The backup is intended for short-term functional validation of CO-BERA activity and delivery before reintroducing pH gating for stricter safety control.
Forward primer: pKatA_coBERA_F
Reverse primer: pKatA_coBERA_R
Amplify the fragment using pKatA_coBERA_F / pKatA_coBERA_R.
Digest the PCR product (or Level-0 clone) with BsaI to produce Reclone-compatible overhangs.
Perform a Golden Gate ( BsaI ) reaction combining the domesticated fragment with the Level-1 acceptor pJUMP23-1A backbone to generate the Level-1 transcriptional unit.
Acts as a contingency if the dual-gate system (pH + H₂O₂) behaves unpredictably during validation.
Maintains conditional expression: CO-BERA is produced in oxidative (inflamed) environments where H₂O₂ is elevated, while reducing circuit complexity for early functional tests.
Useful to establish baseline: RNA production, loading into BMVs, and downstream silencing in mammalian reporter assays.
To provide a backup strategy in case the conditional promoter (KatA, H₂O₂-inducible) fails to drive reliable expression of CO-BERA. This circuit ensures constant expression of CO-BERA under a constitutive promoter (J23119).
Non-conditioned CO-BERA expression fragment (J23119 promoter – CO-BERA – C/D Box – TrrnB terminator).
Forward primer:CO-BERA expression (no condition) Forward
Reverse primer:CO-BERA expression (no condition) Reverse.
Amplify the non-conditioned CO-BERA expression fragment using the designed primers.
Digest the amplified fragment with BsaI for Level 1 Golden Gate assembly.
Clone into the Level 1 backbone to generate a complete transcriptional unit.
If KatA fails to respond properly to oxidative stress, CO-BERA will still be expressed.
Provides a baseline for testing RNA stability, export into vesicles, and downstream silencing efficiency.
To provide a backup strategy in case the loading system (TMP–L7Ae mediated export into vesicles) fails. Instead of relying on bacterial vesicle delivery, this circuit enables direct expression of CO-BERA inside human cells.
fragment (promoter – CO-BERA – terminator, optimized for mammalian expression).
Forward primer: CO-BERA expression in HEK Forward
Reverse primer: CO-BERA expression in HEK reverse
Amplify the CO-BERA expression fragment using the designed primers.
Digest with BsaI for the Level 1 Golden Gate assembly.
Clone into a Level 1 backbone to produce a mammalian expression unit.
This circuit acts as a failure safeguard for loading
If DUF4811–L7Ae-mediated MV loading is inefficient, CO-BERA can still be tested directly in mammalian cells.
Provides a benchmark for verifying CO-BERA stability and function in eukaryotic cytoplasm without bacterial delivery
Useful for validating siRNA function against the target (TSLP) independently of the bacterial chassis.
