iGEM 2025 — Team afcm-egypt

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Notebook Page{% endblock %}
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![Hero Section Image](https://static.igem.wiki/teams/5545/hero/section-hero-recovered-06.webp)

![](https://static.igem.wiki/teams/5545/img/right-side-lung.svg)

# Notebook 2025

## Overview

This year, our journey was meticulously documented, capturing every triumph, every setback, and every pivotal moment across our wet lab experiments, computational modeling, software development, and crucial stakeholder engagements, including our extensive troubleshooting efforts

January

Week 1 (18th of January to 24th of January)
Dry lab

Our efforts focused on a comprehensive analysis of previous iGEM projects and extensive reading of synthetic biology literature.

Week 2 (25th of January to 31st of January)
Dry lab

We identified asthma as a significant global health issue, noting the pressing unmet needs in treatment adherence and the management of severe type 2 asthma.

Human Practice

We began designing application forms for the recruitment of new team members.

February

Week 3 (1st of February to 7th of February)
Dry lab

We continued to review previous iGEM projects, shifting our focus towards primary synthetic biology literature to develop a therapeutic approach for asthma.

Human Practice

We gathered opinions from over 15 asthmatic patients, helping us understand the challenges they face in daily life and with medication adherence, which was instrumental in shaping our solution to patient needs.

Integrated Human Practice

Our team visited Al-Abassya Chest Hospital, where we held a productive meeting with Dr. Sameh Ahmed. This discussion provided valuable insights into the pathogenesis, symptoms, triggers, and current treatment modalities of asthma.

Week 4 (8th of February to 14th of February)
Dry lab

The entire team engaged in extensive discussions, brainstorming numerous ideas for the development of a potential approach to asthma treatment.

Human Practice

The recruitment of new team members was successfully completed.

Week 5 (15th of February to 21st of February)
Human Practice

An urgent team meeting was convened to make a final decision on the core technology to be used in our approach.

Dry lab

We settled on developing a therapeutic approach specifically targeting IL-13.The recruitment of new team members was successfully completed.

Week 6 (22nd of February to 28th of February)
Dry lab

We deepened our literature analysis of IL-13, and meticulously reviewed previous iGEM projects related to this cytokine.

After understanding asthma pathogenesis, we shifted our focus to TSLP. Notably, TSLP is an upstream cytokine that drives both type 2 and non-type 2 inflammatory pathways, thus offering a broader therapeutic target.

March

Week 7 (1st of March to 7th of March)
Human Practice

Our team attended the Pulmo-Port Said Conference, where we actively explored the competitor landscape and identified potential partnership opportunities.

Integrated Human Practice

We organized a meeting with Dr. Ahmed Beshir at the conference to delve into patient adherence problems, side effects of current treatment, and emerging therapeutic approaches such as TSLP inhibitors.

Week 8 (8th of March to 14th of March)
Dry lab

Our team intensified its literature analysis concerning TSLP and began brainstorming various technologies that could be incorporated into our approach.

Integrated Human Practice

We arranged another meeting with Dr. Ahmed Bashir to discuss the latest advancement in asthma treatment, particularly anti-TSLP therapy. Our goal was to explore solutions that could reduce medication frequency, minimize side effects, and improve the quality of life for patients.

Week 9 (15th of March to 21st of March)
Dry lab

After careful consideration, we decided to utilize Antisense Oligonucleotides (ASOs) to knock down the TSLP activity at the transcriptional level.

We designed specific ASO sequences engineered to target and bind to particular mRNA sequences (TSLP mRNA), thereby inhibiting its expression.

Week 10 (22nd of March to 28th of March)
Modelling

A comprehensive ASO model was developed and performed to validate its ability to effectively degrade TSLP mRNA, primarily through RNase H-dependent mechanisms.

Integrated Human Practice

Our team attended a meeting with Dr. Mohamed Hussein, who clarified the drawbacks of ASO, emphasizing the need for chemical modification and potential conflict with philosophical principles. These insights prompted us to explore alternative targeting of TSLP.

Week 11 (29th of March to 4th of April)
Dry lab

We embarked on a renewed review of synthetic biology literature and sought expert opinions. We reaffirmed our commitment to developing a therapeutic approach targeting TSLP.

We shifted our focus toward CO-BERA, recognizing its advantage in not requiring chemical modification to achieve its function and its ability to be expressed endogenously from bacteria.

Human Practice

Our innovative approach was named “PRESS”.

April

Week 12 (5th of April to 11th of April)
Modelling

The CO-BERA model was completed, which validated its ability to degrade TSLP mRNA effectively with the aid of essential enzymes that exist in human cells. The result of this model significantly outperformed those of the ASO model.

Software

Our team initiated a molecular dynamics simulation by understanding our project parameters and identifying the most suitable tool for simulating our complexes. This involved preparing DNA sequences for each protein within the complex and determining additional parameters required for accurate simulation.

Integrated Human Practice

We joined a meeting with DR. Atef, who validated siRNA as an effective silencing mechanism and supported using the Golden Gate method at Assembly.

Week 13 (12th of April to 18th of April)
Dry lab

We investigated compatible technologies for CO-BERA delivery to the distal airway.

We decided to utilize liposomal vesicles for direct CO-BERA delivery into the distal airway tract.

Integrated Human Practice

We joined a meeting with Dr. Ragda to validate the process of CO-BERA-siRNA interactions, aiming to reduce off-targeting probabilities.

Week 14 (19th of April to 25th of April)
Dry lab

We conducted a detailed literature analysis of liposomal vesicles, which revealed their short half-life in vivo and the necessity for repeated administration.

Integrated Human Practice

We organized a meeting with Dr.Hazman, who expressed reservations about using liposomes. He reassured us about the potential of bacterial vehicles and clarified potential side effects and sources for purchasing suitable bacterial strains.

Week 15 (25th of April to 2nd of May)
Dry lab

Our delivery system was revised to explore the use of Corynebacterium species.

Human Practice

We attended World Asthma Day, gathering crucial feedback on asthma prevalence, patient perspectives, and suitable inhaler designs to further refine our “PRESS” approach.

May

Week 16 (3rd of May to 9th of May)
Dry lab

We transitioned from Corynebacterium abundance to a respiratory microbiome called Lactobacillus plantarum.

We thought about the ideal device for engineered Lactobacillus plantarum delivery.

Human Practice

We attended the 5th Arab Sustainable Development Week, which encouraged us to align PRESS to serve the SDGs goals.

Software

We prepared our parameters and topology files using the CHARMM-GUI Package, and proceeded to simulate these files using Gromacs software.

Week 17 (10th of May to 16th of May)
Dry lab

Our team settled on an aerosol device for the delivery of our engineered bacteria.

Software

Gromacs simulation progressed through eight distinct steps for each complex, comprising one minimization, six equilibration phases, and one production step.

Integrated Human Practice

Dr. Souzan Fouad joined us in a meeting to clarify the drawbacks of nebulizers and recommended using Dry Powder Inhalers (DPIs) as a superior delivery method.

We had a valuable meeting with Dr. Tarek Salim, who outlined the differences between inhaling devices, confirming DPIs as more patient-friendly and safer. He also recommended advanced technology, specifically an artificial lung, for preclinical testing.

SDGs

We focused on SDG 3 – Good Health and Well-being to reduce the worldwide burden of asthma, offering a treatment like PRESS.

Week 18 (17th of May to 23rd of May)
Dry lab

Based on extensive literature review, experimental data, and expert opinions, our dry lab definitively settled on the usage of Dry Powder Inhalers (DPIs).

We began researching a spray drying method for bacterial drying.

Modelling

We developed a spray drying model using MATLAB to predict bacterial viability, which unfortunately indicated low bacterial viability and poor storage features.

Week 19 (24th of May to 30th of May)
Human Practice

To address the drying challenge, our team members visited EIPICO Pharmaceutical Factory, where discussions led to the selection of freeze-drying as the ideal drying method for preserving bacterial viability.

Integrated Human Practice

We attended a meeting with Dr. Wagida Anwar (IRB), who emphasized the critical importance of patient safety, biosecurity, and ethical standard considerations for PRESS research.

Week 20 (31st of May to 6th of June)
Dry lab

Following a comprehensive review of expert opinions and experimental data, our dry lab confirmed the selection of the freeze-drying process.

Modelling

We developed a freeze-drying model using MATLAB to predict the powder potency by calculating the number of viable bacteria per milligram of powder, which was successfully completed.

Integrated Human Practice

We discussed with Mr. Yahia Badawy, who clarified Egyptian regulatory steps for releasing a new drug to market, starting from preclinical studies.

June

Week 21 (7th of June to 13th of June)
Dry lab

We recognized that a small amount of CO-BERA could be efficiently loaded into membrane vesicles, so we began searching for suitable RNA-binding proteins (RBPs) for CO-BERA loading.

Integrated Human Practice

We joined a meeting with Mr. Rejeesh (FDA Consultant), who highlighted the importance of preclinical studies, cytotoxicity testing, and sustainable planning for FDA approval.

Education

We planned through our educational program to manage visits to all age groups and different occupations. Aiming to raise asthma awareness and introduce synthetic biology aligned with life skills.

Week 22 (14th of June to 20th of June)
Dry lab

We initially settled on Cold Shock Proteins (CSPs) for RNA loading into the membrane vesicles.

After further literature analysis and review, we shifted our focus from CSPs toward synthetic TMP–linker–RBP.

Software

Molecular dynamics simulation for CO-BERA-CD box complex with a protein consisting of RNA binding protein and linker (L7Ae) and transmembrane binding protein (Dough).

We tested the membrane orientation and stability of synthetic TMP–linker–RBP fusions through in-silico studies with SWISS-Model, CHARMM-GUI, and GROMACS simulations.

Week 23 (21st of June to 27th of June)
Dry lab

Our team aimed to target NO for the conditioned expression of CO-BERA, ensuring its release only in the context of asthma.

Modelling

NO conditioning model developed using CO-LAB, demonstrated the possibility of CO-BERA release in the presence of NO, a known biomarker of asthma. However, it also revealed the impossibility of using this switch in Lactobacillus plantarum (LB) due to the absence of necessary activator factors.

Integrated Human Practice

We met with Dr. Mohamed Anany to identify the strength of PRESS in solving adherence problems; he also highlighted its potentially high cost and safety concerns.

Education

We conveyed to New Academy kindergarten to teach kids there the proper inhaler technique through songs and educate them about asthma triggers using activities and our children's book. Additionally, we emphasized the emergency steps for asthma attacks.

Week 24 (28th June to 4th July)
Dry lab

We shifted our focus toward utilizing H₂O₂ to stimulate CO-BERA expression, as H₂O₂ levels are known to increase in the inflammatory response of asthma.

Modelling

H2O2 conditioning model developed using CO-LAB, proved the high efficiency of CO-BERA under increased H2O2 conditions, which is specific to airway inflammation.

Software

We further tested H₂O₂ conditioning through in-silico studies, confirming that H₂O₂ successfully achieved spatial and temporal control of gene expression in stressed bacterial environments.

Integrated Human Practice

We held a meeting with Mr. Mohamed Mahrous (AstraZeneca) to demonstrate the viability of PRESS’s and explore a possible future collaboration for global market entry.

Education

We organized a visit to Al-Salam preparatory school, where we detailed the improper inhaler technique side effects, and then we clarified the proper inhaler technique.

July

Week 25 (5th of July to 11th of July)
Dry lab

We discovered that CO-BERA experienced significant degradation before achieving its intended function, prompting us to research methods for enhancing endosomal escape.

Modelling

We developed an H2O2 DIFFUSION model in the bacteria using CO-LAB that confirmed an increase in the diffusion rate of H₂O₂ into the bacteria, reaching above the threshold of pKat promoter.

SDGs

We committed to SDG 13 – Climate Action to reduce emissions harming our planet’s climate from normal inhalers.

Week 26 (12th of July to 18th of July)
Dry lab

Following a thorough literature review and analysis, we settled on Listerolysin O (LLO), recognizing its ability to disrupt endosomal membranes and enhance endosomal escape.

Wet lab

Concurrently, the wet lab team initiated comprehensive training sessions to enhance technical proficiency, thereby preparing actively for subsequent scientific discussions and strategic planning meetings.

Integrated Human Practice

We organized a meeting with Dr. Asmaa Mohamed, who expressed reassurance about PRESS’s potential to reduce psychological burdens and enhance patients’ well-being.

We had a meeting with Engineers El Sebay & Mohammed, who provided valuable tips regarding our asthma-friendly city design, including air filter placement and renewable energy usage.

Human Practice

Our team visited El-Zagazig University Hospital to collect feedback from doctors and patients, which identified concerns about fear of bacterial use, emphasizing the need for robust safety measures and effective educational awareness campaigns.

Education

We made specific materials for family members and used them through our visits, such as a child's book, a parent's side book, and a song and game about asthma management.

Week 27 (19th of July to 25th of July)
Dry lab

We decided to utilize a mutated form of LLO, as this variant is considered safer and prevents the pathogenicity of normal LLO.

Our overarching goal remains to develop an approach with the highest safety level, leading us to investigate technologies that could further enhance the safety of our project.

Software

We conducted simulations for Listeriolysin O under two different pH levels within the lipid membrane of phagosomes in the lungs of human cells.

An in-silico protein model was also employed to validate the lower toxicity and sustained structural stability of both LLO and its mutated form.

Education

In the New Vision School secondary school, we explained asthma pathophysiology.

SDGs

We supported SDG 11 – Sustainable Cities and Communities to design a more convenient and healthier environment for asthmatic patients.

Week 28 (26th of July to 1st of August)
Dry lab

Our team finalized the decision to incorporate a toxin-antitoxin system to enhance the biosafety and controllability of our probiotic platform.

Software

We awaited the LLO simulation results to confirm its stability at various PH levels.

Wet lab

Our wet lab team thoroughly discussed the proposed methodologies with experts, leading to the finalization of a comprehensive laboratory plan.

Education

Through our visit to STEM Gharbiya, we discussed the differences between current asthma medications and the pros and cons of various inhaler types.

SDGs

We pursued SDG 7 – Affordable and Clean Energy to lower energy pollution related to fossil fuel consumption, which is a main trigger for asthma.

August

Week 29 (2nd of August to 8th of August)
Modelling

The double conditioning model for CO-BERA expression confirmed that the CO-BERA concentration exceeded 8 Micromole and PH below 7. This highly specific activation profile is ideal for our target population, suffering from severe uncontrolled asthma

SDGs

We contributed to SDG 15 – Life on Land to protect green landscapes, which provide clean air that reduces asthma prevalence worldwide.

Education

During our meeting with Public & Geriatrics, we learned more about the struggles that asthma patients face in their lives and learned the current treatments' limitations and side effects. Additionally, we introduced asthma to the public and heard their thoughts about it.

Week 30 (9th of August to 15th of August)
Wet lab

Detailed lab protocols were drafted in collaboration with Dr. Atef and Dr. Abdullah, and team members received ongoing training in fundamental lab techniques.

Education

We visited many asthma patients across different high-risk asthma occupations such as farming, bakeries, brick factories, spinning and weaving factories, painters, cleaners, wood factories, pet shops, and sand quarries. Through these visits, we stressed wearing PPE and educated them about their occupations’ triggers.

Week 31 (16th of August to 22nd of August)
Wet lab

All necessary fragments, materials, and primers required for the experiments were ordered from IDT and Benchling.

Education

We visited pharmacies, the main suppliers, to present our therapeutic approach & receive feedback to further tweek our project.The purpose was also to incrase public engagement further.

SDGs

We embraced SDG 4 – Quality Education to help children with asthma have a normal education in life with PRESS.

Week 32 (23rd of August to 29th of August)
Education

We visited Future Blossoms Kindergarten, where we educated the children on human body structures and parts, and some life skills, such as teamwork and respect for nature.

SDGs

We aligned with SDG 5 – Gender Equality to relieve the burden on the shoulders of women, who are primary caregivers, allowing them to concentrate on their career growth.

Week 33 (30th of August to 5th of September)
Wet lab

Initial lab procedures commenced with the resuspension of DNA fragments 1, 2, and 3, followed by their amplification using Polymerase Chain Reaction (PCR).

The size verification of all amplified fragments was performed via gel electrophoresis, after which these fragments were transformed into E. coli JM109 competent cells.

Education

At Future Leaders Primary School, we explained asthma, its symptoms, and lungs using models and gave an intro about DNA and microbes. Additionally, they developed critical thinking skills and learned how to solve problems.

Human Practice

Pharmaconex Event: selecting HPMC as the ideal DPI capsule, compared to alternatives.

September

Week 34 (6th of September to 12th of September)
Wet lab

PCR amplification of Fragments 1 and 2 was repeated to achieve optimal yields. Concurrently, Fragment 3's PCR amplification was optimized to determine the ideal annealing temperature. Additionally, M13 primer variants were tested for functionality through PCR amplification of the 1-D5 plasmid.

Transformed bacteria were cryopreserved by creating glycerol stocks for long-term storage.

Education

In our visit to a local educational center, we briefly explained DNA, the central dogma, and genes. Not to forget, we detailed for them the scientific research method and cycle & the difference between observation and inference.

Week 35 (13th of September to 19th of September)
Wet lab

Stored bacterial stocks were subsequently revived and cultured to expand pure populations containing the desired recombinant plasmid.

Both the target DNA fragments and the destination vector were isolated and then digested with compatible restriction enzymes. The digested fragments and vectors were subsequently ligated to form a Level 0 assembly, which was then transformed into E. coli JM109 competent cells.

Education

During our visit to Green Heights School, we discussed with students synthetic biology, gene expression, and siRNA. In addition, we introduced them to safety and research ethics.

Week 36 (20th of September to 26th of September)
Wet lab

The transformation of the Level 0 ligated parts into E. coli JM109 competent cells was repeated due to initial unreliable results.

The assembled fragments were isolated via PCR amplification, and their correct size was verified by gel electrophoresis in preparation for the next assembly level.

Education

Through our Zoom meeting with ASU students, we talked about synthetic biology applications, plasmid design, and genetic circuits. Also, we explained to them the importance of dry lab precautions and ethical validation.

Week 37 (27th of September to 3rd of October)
Wet lab

Following size verification, these ligated parts and the pJUMP23-1A plasmid were digested with the same restriction enzymes. A subsequent ligation reaction was performed, combining the digested components with ligase buffer, T4 DNA ligase, and ATP to create the Level 1 assembly. These ligated constructs were then purified and transformed into E. coli JM109 competent cells.

Education

By gathering with the public at a local society club, we were able to introduce the concept of using synthetic biology in therapeutics. Through our discussion session, we identified their doubts and corrected their misconceptions.

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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![Hero Section Image](https://static.igem.wiki/teams/5545/hero/section-hero-recovered-04.webp)

![](https://static.igem.wiki/teams/5545/img/right-side-lung.svg)

# Introduction

**Asthma** is a chronic inflammatory disease that affects over 300 million individuals in the world and is a significant leading cause of morbidity and a reduction in quality of life. Although there exist available options of inhaled corticosteroids, β2-agonists and biologics like anti-IL-5 or anti-IgE antibodies, there remain numerous patients who are left with refractory symptoms and airway remodeling. Difficult to manage especially are those cases of **severe asthma** and often with steroid-resistant disease.

## From Ideas to Design

In the early stages of our project, each member of the team was involved in the exploration of different technological platforms available for the therapy of **Asthma**. The suggested concepts represented a wide range of thought such as viral vectors and lipid nanoparticles for siRNA delivery, to antibody-related biologics and small molecules. After several discussions, the team compared these options in terms of safety, specificity, ease of engineering, and long-term applicability.

 Throughout the prolonged journey, we experienced numerous challenges that continually shaped our platform. We based upon extensive literature research, the integration of numerous experts, in silico software validation, and ultimately, experimental validation in the laboratory. Each of these hurdles is addressed throughout our DBTL cycles, which resulted in critical modification of our PRESS

![Figure: progress of our project](https://static.igem.wiki/teams/5545/soft/timeline-1-2.webp)

References

* Rabe APJ, Loke WJ, Gurjar K, Brackley A, Lucero-Prisno III D, et al. Global Burden of Asthma, and Its Impact on Specific Subgroups: Nasal Polyps, Allergic Rhinitis, Severe Asthma, Eosinophilic Asthma. J Asthma Allergy. 2023;16:1097-1113. doi:10.2147/JAA.S41814519.
* Mahesh PA, et al. Molecular mechanisms of steroid-resistant asthma. Explor Asthma Allergy. 2023;1:174-185. doi:10.37349/eaa.2023.00018.
* Global, regional, and national prevalence of asthma in 2019: a systematic analysis and modelling study. J Glob Health. 2022;12:04009. DOI 10.7189/jogh.12.04052.

# Iteration 1

Choosing the Therapeutic Target

Press the button on the right side

**Design**
**At the beginning of our project**, we first aimed to silence **Interleukin-13 (IL-13)** because it is one of the key **cytokines** and has an important role in the **Asthma** cascade. Blocking **IL-13** was expected to reduce **airway hyperresponsiveness**, **mucus production**, and **remodeling**, making it a rational **therapeutic target** (1,2).

**Build**
**Our first design** strategy therefore, focused on engineering an **RNA therapeutic** against **IL-13**, with the goal of preventing its downstream **inflammatory effects** in severe **Asthma**.

**Test**
Through an extended **literature review**, we discovered important **limitations:**

* **IL-13** primarily regulates **type-2 eosinophilic Asthma**, but it does not adequately cover other clinically important phenotypes, such as **non-type 2 (neutrophilic) Asthma** (1).
* **IL-13** silencing alone would not effectively tackle **corticosteroid-resistant Asthma** (2).

**Learn**
Upon further analysis and after meeting with **Dr/ Ahmed Beshir**, we realized that targeting **IL-13** offered limited **clinical benefit** and might exclude patients with **non-type-2 asthma**. As a result, we redirected our attention to **Thymic Stromal Lymphopoietin (TSLP)**, an upstream **cytokine** that drives both **type-2** and **non-type-2 inflammatory pathways** and is strongly implicated in **corticosteroid resistance** (3,4). By shifting our **therapeutic target** from **IL-13** to **TSLP**, we enhanced both the **clinical relevance** and the potential impact of our project.

![Figure: TSLP is wider in effect than IL-13](https://static.igem.wiki/teams/5545/parts/eng11.webp)

Design

Build

Test

Learn

References

* Dowdy SF. Endosomal escape of RNA therapeutics: How do we solve this rate-limiting problem? RNA. 2023 Apr;29(4):396-401. doi: 10.1261/rna.079507.122. Epub 2023 Jan 20. PMID: 36669888; PMCID: PMC10019367
* Corren J, Lemanske RF Jr, Hanania NA, Korenblat PE, Parsey MV, Arron JR, et al. Lebrikizumab treatment in adults with asthma. N Engl J Med. 2011;365(12):1088–98. doi:10.1056/NEJMoa1106469
* Gauvreau GM, O'Byrne PM, Boulet LP, Wang Y, Cockcroft D, Bigler J, et al. Effects of an anti-TSLP antibody on allergen-induced asthmatic responses. N Engl J Med. 2014;370(22):2102–10. doi:10.1056/NEJMoa1402895
* Menzies-Gow A, Corren J, Bourdin A, Chupp G, Israel E, Wechsler ME, et al. Tezepelumab in Adults and Adolescents with Severe, Uncontrolled Asthma. N Engl J Med. 2021;384(19):1800–9. doi:10.1056/NEJMoa2034975

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# Iteration 2

Selecting the Silencing Technology

Press the button on the right side

**Design**
After choosing **TSLP** as our **molecular target**, we then started to think about how to silence it and which level of the **central dogma** we should target. Our first thought was to use **Antisense Oligonucleotides (ASOs)** to turn down its activity at the **post transcriptional level**. We designed **ASOs** to target and bind specific **mRNA sequences (TSLP mRNA)** and inhibit its expression (1).

**Build**
**Our strategy** was based on utilizing **ASOs** as the primary **therapeutic element** within **engineered vectors**. The rationale was that **ASOs**, delivered via **synthetic liposomal vesicles**, would hybridize with **TSLP mRNA** in **host cells** and suppress its expression (2).

**Test**
After Finishing our model and upon feedback from **Dr/ Mohammed Hussein** that highlighted several substantial limitations:

* **ASOs** require significant **chemical modifications**, such as alterations to their backbone or sugar moieties, to remain stable and effective **in vivo** (3,4).
* These necessary modifications are not naturally **biosynthesized**, making them difficult to produce at scale (5).

In addition to the result of our **modeling** (As shown in this fig. ), the **Bar graph** below compares **TSLP knockdown efficiency** across four conditions: **untreated (Natural), Antisense Oligonucleotides (ASO), BERA, and CO-BERA.**

![Figure: Bar compares TSLP knockdown efficiency](https://static.igem.wiki/teams/5545/parts/eng-2.webp)

**ASO treatment achieved 70.0% knockdown**, outperforming the Natural baseline (15%) but falling short of **BERA (82.5%)** and **CO-BERA (99.7%)**. These results, combined with **scalability** and **stability concerns**, led to the strategic pivot away from **ASO-based silencing** toward **biologically encoded delivery platforms**.
Given these challenges, this strategy proved impractical and necessitated reevaluation.

**Learn**
From these findings, we concluded that **ASOs** were not feasible for our project. So we decided to search for another **silencing system**. As a result of discussions with **Dr/ Raghda**, we shifted toward using **CO-BERA** which has a **dual-function siRNA system**.
Unlike **ASOs**, **siRNAs** can exploit the natural **RNAi pathway** without requiring **chemical modifications** for activity, making them more suitable for **biological production** and more reliable as a **therapeutic component** (6–8). Advances in **RNA bioengineering** (6,7) support **CO-BERA** as a scalable and effective solution.

![Figure: CO-BERA difference from ASO ](https://static.igem.wiki/teams/5545/parts/eng-22.webp)

Design

Build

Test

Learn

References

* Holgate ST. Pathogenesis of asthma. Clin Exp Allergy. 2008;38(6):872–97.
* Barnes PJ. Targeting cytokines to treat asthma and chronic obstructive pulmonary disease. Nat Rev Immunol. 2018;18(7):454–66.
* Crooke ST, Witztum JL, Bennett CF, Baker BF. RNA-targeted therapeutics. Cell Metab. 2018;27(4):714–39.
* Østergaard ME, Southwell AL, Kordasiewicz HB, Watt AT, Skotte NH, Doty CN, et al. Chemical modification of PS-ASO therapeutics reduces cellular protein binding and improves the therapeutic index. Nat Biotechnol. 2020;38(12):1431–9.
* Wang F, Zuroske T, Watts JK. RNA therapeutics on the rise. Nat Rev Drug Discov. 2020;19(7):441–2.
* Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, et al. Molecular engineering of functional siRNA agents. ACS Synth Biol. 2012;1(10):576–84.
* Chen W, Zhou Y, Shi J, Xu S, Chen B, Sun H, et al. Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs. Appl Microbiol Biotechnol. 2019;103(5):2059–69.

◆ ◆ ◆

# Iteration 3

Choosing the Delivery Platform

Press the button on the right side

**Design**
Following the selection of **CO-BERA** as our **therapeutic agent**, we turned our attention to the **delivery system**. Our initial plan was to use **liposomes** as carriers. **Liposomes** are widely used for **drug delivery** because they can encapsulate **nucleic acids** and fuse with **host cell membranes**, enabling **intracellular delivery** (1).

**Build**
We created a **liposome** to be our **delivery vehicle**, where **CO-BERA** molecules would be packaged and administered directly to the lungs. The rationale was that **liposomes** might offer a **controlled release system**, enabling local delivery to **airway epithelial cells** (2).

**Test**
However, a deeper evaluation and more **literature review** showed significant Limitations:

* Conventional **liposomal formulations** tend to be **cleared rapidly** or degraded at the site of administration; specialized approaches (e.g., **PEGylation** or **LNP formulations**) can be used to improve stability but add to formulation complexity and cost (3).
* They needed **repeated administration**, particularly for **chronic respiratory diseases** where **mucociliary clearance** and local degradation reduce **residence time** (4).
* Large-scale, stable encapsulation of **RNA molecules** into **liposomes** remains technically a problem and costly (5).

**Learn**
These limitations made **liposomes** unsuitable as our **delivery platform**. We therefore shifted toward using **Corynebacterium** as a **living chassis**. Unlike **liposomes**, bacteria can act as continuous "factories," capable of producing **therapeutic agents**. This approach promised longer persistence, reduced dosing frequency, and adaptability compared to **non-living delivery systems** (6).

![Figure: Liposomes and Bacterial vector](https://static.igem.wiki/teams/5545/soft/factory-hq.webp)

Design

Build

Test

Learn

References

* Sercombe L, Veerati T, Moheimani F, Wu SY, Sood AK, Hua S. Advances and Challenges of Liposome Assisted Drug Delivery. Front Pharmacol. 2015;6:286.
* Allen TM, Cullis PR. Liposomal drug delivery systems: From concept to clinical applications. Adv Drug Deliv Rev. 2013;65(1):36–48.
* Pattni BS, Chupin VV, Torchilin VP. New Developments in Liposomal Drug Delivery. Chem Rev. 2015;115(19):10938–66.
* Hua S. Advances in nanoparticulate drug delivery approaches for respiratory disorders. Expert Opin Drug Deliv. 2014;11(6):845–56.
* Kulkarni JA, Witzigmann D, Chen S, Cullis PR, van der Meel R. Lipid Nanoparticle Technology for Clinical Translation of siRNA Therapeutics. Acc Chem Res. 2019;52(9):2435–44.
* Bergenfelz C, Do P, Larsson L, Ivarsson H, Malmborn K, Håkansson AP. Corynebacteria from the respiratory microbiota modulate inflammatory responses and are associated with a reduced pneumococcal burden in the lungs. Front Cell Infect Microbiol. 2025;14:1530178. doi:10.3389/fcimb.2024.1530178.

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# Iteration 4

Selecting the Final Chassis

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**Design**
After narrowing down our **delivery system**, we considered **Corynebacterium** as the chassis for **therapeutic production**. It is a **Gram-positive bacterium** capable of secreting **vesicles** and can be engineered for **nucleic acid expression** (1).

**Build**
We explored **Corynebacterium** as the microbial "factory" that would continuously release our therapeutic **CO-BERA** into the **lung environment**. Its **non-pathogenic strains** have been used in **biotechnology**, making it a candidate worth evaluating (1).

**Test**
Further research, however, highlighted limitations:

* **Corynebacterium** lacks strong evidence of direct **clinical benefit** in the **respiratory tract**.
* It has limited **immunomodulatory activity**, providing no additional **therapeutic effect** beyond acting as a carrier.
* Its **colonization dynamics** in the lung are less characterized compared to **probiotic organisms** (2).

**Learn**
After more research about **probiotic organisms** in the **respiratory tract**, we decided to use **Lactobacillus plantarum**, a **probiotic species** naturally associated with the **gut–lung axis**. Unlike **Corynebacterium**, **L. plantarum** not only used as a **delivery chassis** but also provides intrinsic **therapeutic benefits**, including:

* **Anti-inflammatory effects** through modulation of **NF-κB** and **MAPK pathways** (3).
* Reduction of **IgE** and **pro-inflammatory cytokines**, key markers of **Asthma** (4).
* Enhancement of host **antioxidant defense mechanisms**, improving **respiratory function** (5).

Thus, **Lactobacillus plantarum** provides a **dual-action advantage**: **firstly**, it acts as a chassis for **CO-BERA delivery**. **Secondly**, the **L.plantarum** can be a **therapeutic agent** due to its **anti-inflammatory function**. This made it the ideal choice for our final system.

![Figure: Lactobacillus over Corynebacterium](https://static.igem.wiki/teams/5545/parts/eng-4.webp)

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References

* Becker J, Wittmann C. Systems and synthetic metabolic engineering for amino acid production – the heartbeat of industrial strain development. Curr Opin Biotechnol. 2012;23(5):718-26.
* Bergenfelz C, Do P, Larsson L, Ivarsson H, Malmborn K, Håkansson AP. Corynebacteria from the respiratory microbiota modulate inflammatory responses and associate with a reduced pneumococcal burden in the lungs. Front Cell Infect Microbiol. 2025;14:1530178. doi:10.3389/fcimb.2024.1530178.
* Lee J, Kim Y, Yun HS, Kim JG, Oh S, Kim SH. Genetic and proteomic analysis of anti-inflammatory functions of **Lactobacillus plantarum** 88 in IL-10-deficient mice. J Appl Microbiol. 2016;120(6):1700-12.
* Zhou Y, Cui Y, Qu X. **Lactobacillus plantarum** inhibits intestinal inflammation and regulates gut microbiota in DSS-induced colitis mice. Exp Ther Med. 2019;18(3):2243-51.
* Plaza-Díaz J, Ruiz-Ojeda FJ, Gil-Campos M, Gil A. Mechanisms of action of probiotics. Adv Nutr. 2019;10(suppl\_1):S49–S66.

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## Optimizing Reliability

As the project progressed, we found that **therapeutic RNA expression** was insufficient to ensure **reliable performance**. Further optimization of **accuracy**, **efficiency**, and **safety** was required for our system. The **gene expression** should become more specific, the **RNA cargo** delivered efficiently inside **host cells**, and a **safety system** incorporated to prevent possible unintended risks.

Through design iterations, we added further layers of regulation for **conditional expression**, investigated means for enhancing **intracellular delivery** and functionality, and incorporated **biocontainment** through engineering to enhance safety. These improvements transformed the original idea into a much more robust, targeted, and responsible **therapeutic platform**.

# Iteration 5

Precision Expression through Oxidative & pH Control

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**Design**
From initial design of expression of our approach we decided to placed **CO-BERA** under the control of the **inducible promoter**, which is activated by **inflamed lung**. We expected this design to provide **disease-specific activation** in **asthma**.

**Build**
In our system, we implemented an **inflammation-dependent promoter system** for **CO-BERA expression**. Specifically, we chose **H₂O₂-sensitive promoters (KatA promoter)** that respond to **disease hallmarks**, since elevated **oxidative stress (H₂O₂)** changes in **inflamed airways**. The **KatA promoter**, which is repressed under normal conditions by **PerR**. However, it is induced upon the presence of **reactive oxygen species** which was chosen as one of the key switches.

![Figure: H2O2 response system](https://static.igem.wiki/teams/5545/soft/h2o2.webp)

**Test**
However, both our modeling and **literature reviews** revealed that **pKatA** shows **basal leakage** under **non-stress conditions**. Specifically, **PerR repression** was not absolute, meaning **residual transcription** could occur even in **healthy environments**. This leakage risked unintended **CO-BERA** expression, raising concerns for **off-target activity**.
shown Conditioning model

![Figure: conditioned Daula expression](https://static.igem.wiki/teams/5545/parts/eng-5.webp)
Time-course analysis of **pKAT mRNA expression (mM)** under varying concentrations of **hydrogen peroxide (H₂O₂)**, ranging from basal (no H₂O₂) to **0.15 mM**. Expression levels increased proportionally with **H₂O₂ concentration**, plateauing after ~5 hours. The **basal expression level (13.36 mM)** accounted for **40.08%** of total activation, indicating **basal leakiness**. These data validate **pKAT** as a tunable **oxidative stress–responsive promoter** suitable but show issue for **conditional gene expression systems**.

**Learn**
From this, we concluded that **H₂O₂** alone was not sufficient for **precise therapeutic control**. A more stringent **dual-conditioning system** was needed to minimize **off-target effects**.

**Rebuild**
We engineered an additional **pH-sensitive layer** of repression:

* Inserted a **Rep operator** downstream of **pKatA**.
* The **Rep repressor** was placed under the constitutive **p32 promoter**.
* To introduce **pH sensitivity**, we integrated a **Lac operator** within **p32**.
![Mechanism of TSLP Silencing via CO-BERA-Mediated RNA Interference img](https://static.igem.wiki/teams/5545/design/ph7-7.webp)
* In **inflamed lungs (pH < 6.9)**, **LacR** is activated through the **PH sensitive promoter (P170-CP25)**, and then binds the **Lac operator**, and inhibits **p32**. This reduces **Rep levels**, relieving suppression of **KatA**.

Thus, **CO-BERA** expression became dependent on two concurrent **inflammatory signals**: elevated **H₂O₂** and **acidic** as mentioned in Approach design page.
![operator–repressor system](https://static.igem.wiki/teams/5545/img/design/expression.webp)

**Re-Test**
Based on our **logic- AND Gate model** and supportive literature reports on **dual-input promoters**, the reconstructed circuit showed:

* Markedly reduced **basal leakage** under **neutral pH / low oxidative stress**.
* Robust expression under combined **acidic pH** and **oxidative stress**, mimicking **asthmatic lung conditions**.

![Figure: H2O2 conditioned expression](https://static.igem.wiki/teams/5545/parts/eng-55.webp)

Description of **Dual-Input AND Gate Circuit Performance**

The **dual-input AND-gated genetic circuit** was reconstituted for **CO-BERA** expression under the combined stimuli of **pH** and **oxidative stress**. The **neutral pH** and **low oxidative stress** conditions representative of a **healthy lung** showed a markedly low **basal leakage** of the genetic circuit, thus effectively repressing the expression and thereby preventing any unwanted activation. Contrarily, elevated **H₂O₂** and **acidic pH** are the simultaneous conditions set up by **inflamed asthmatic airways**, and hence the circuit was fully turned on in transcription, with **CO-BERA mRNA level** spikes and settling to around **20 mM**. Such a **conditional response** assures the greatest fidelity of the **AND gate logic** to further limit **therapeutic gene expression** to a **disease-specific microenvironment**. These results are in agreement with **dual-input promoter** literature and thus establish the concept of the circuit for **pulmonary inflammation-targeted intervention**.

**Learn**
This iteration established a **dual-gated**, highly specific **expression system**. By integrating insights from both modeling and literature, we eliminated the **leakage problem** while maintaining strong **therapeutic activation** under **disease-relevant conditions**.

![Figure:Dual conditioning](https://static.igem.wiki/teams/5545/design/ph7-9.webp)

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References

* Ceroni F, Algar R, Stan GB, Ellis T. Quantifying cellular capacity identifies gene expression designs with reduced burden. Nat Methods. 2015;12(5):415-8. doi:10.1038/nmeth.3339. PMID: 25849635; PMCID: PMC4207942.
* Lee JW, Helmann JD. Functional specialization within the Fur family of metalloregulators. Biometals. 2007;20(3-4):485-99.
* Mongkolsuk S, Helmann JD. Regulation of inducible peroxide stress responses. Mol Microbiol. 2002;45(1):9-15.
* Faulkner MJ, Helmann JD. Peroxide stress elicits adaptive changes in bacterial metal ion homeostasis. Antioxid Redox Signal. 2011;15(1):175-89.

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## About our loading

While developing our **RNA therapeutic system**, we recognized that **loading efficiency** into **bacterial membrane vesicles (MVs)** is a major challenge. Although **Lactobacillus plantarum** can naturally release vesicles that carry **biomolecules**, only a small fraction of the engineered RNA (**CO-BERA**) was successfully packaged. This limitation reduces the **therapeutic payload** and weakens **clinical applicability**. Therefore enhancing the proportion of **RNA molecules** loaded into vesicles became a key design goal. To address this, we explored **biological strategies** that could actively guide RNA toward **vesicle formation sites**, rather than relying on passive or random encapsulation.

# Iteration 6

Increasing RNA Loading Efficiency

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**Design**
After successfully designing our **therapeutic RNA scaffold (CO-BERA)**, with further search, we noticed that only a small fraction of the RNA could be efficiently loaded into **bacterial membrane vesicles**. To overcome this limitation, we looked for strategies that would enhance **RNA localization** to vesicles.

**Build**
We proposed to use **RNA-binding proteins (RBPs)** to guide **CO-BERA** closer to the **vesicle formation sites**. Literature pointed to the **Cold Shock Protein (CSP) family** in **Lactobacillus**, which are naturally present and capable of **binding RNA**.

**Test**
Initial analysis suggested and after deep search we found that however, **CSP** could indeed bind RNA, but its role in **vesicle loading** was uncertain. As it is **cytoblasmic protein** which is not ensure the loading efficacy.

**Learn**
We decided to search more about another RNA binding protein and integrated to membrane could serve as a **localization factor** and ensure packaging of RNA into vesicles.

![Figure: RNA loading process](https://static.igem.wiki/teams/5545/parts/eng8-ezgif-com-gif-to-webp-converter.webp)

![Figure: Enhanced loading efficiency](https://static.igem.wiki/teams/5545/parts/eng88-ezgif-com-gif-to-webp-converter.webp)

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# Iteration 7

Searching for a Membrane-Bound RNA Binding System

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**Design**
We first considered **Cold Shock Proteins (CSPs)** as **RNA-binding proteins (RBPs)** for loading **CO-BERA** into **membrane vesicles**. However, literature revealed that **CSPs** are **cytoplasmic** rather than **membrane-bound**, meaning they would not ensure accurate localization of RNA into vesicles. We then searched for **RBPs** naturally linked to **transmembrane proteins (TMPs)** in **Lactobacillus plantarum**, but found no published evidence of such systems in **Gram-positive bacteria**. This pushed us to pursue an **engineered solution**.

**Build**
We decided to design **protein complex** consisting of the **membrane-anchored foldase PrsA** linked to **L7Ae** (a well-characterized **RBP**) through a **flexible linker**. The idea was that **PrsA** would anchor the construct to the membrane while **L7Ae** bound the **RNA cargo**.

**Test**
The **PrsA–linker–L7Ae construct** was modeled using **SWISS-Model**, **CHARMM-GUI**, and tested through **molecular dynamics simulations** in **GROMACS**. These simulations demonstrated **unstable orientation** and **poor anchoring** in the membrane, suggesting that **PrsA** was not a suitable choice for this application.

![Figure: Loading using L7Ae](https://static.igem.wiki/teams/5545/parts/foldd.webp)

 The **RMSD plot** of the **Foldase PrsA complex** shows a steady increase over the **5 ns simulation**, indicating progressive **structural deviation** from the initial conformation. This reflect **dynamic flexibility** or **instability** under simulated conditions.

**Learn**
From this outcome, we learned that the choice of **TMP** is crucial, as not all anchors provide **structural stability** or proper **membrane integration** when fused to **RBPs**.

**Rebuild**
We selected **DUF4811**, a **transmembrane protein** found in **MV** of **Lactobacillus** and designed **DUF4811–linker–L7Ae** fusion constructs for testing.

![Figure: Loading using L7Ae](https://static.igem.wiki/teams/5545/model2025/duffif.webp)

this is 3D structure prediction of DUF4811-L7Ae

**Re-Test**
Computational modeling with **SWISS-Model and CHARMM-GUI**, followed by **GROMACS** molecular dynamics simulations, confirmed **stable membrane orientation**.

![Figure: Loading using L7Ae](https://static.igem.wiki/teams/5545/soft/comp.webp)

Figure: This density to RMSD graph shows the huge difference between the stability of two TMP linked to L7Ae (RBP)

**Learn**
This established **DUF4811–L7Ae** as a stable and reliable **RNA loading system**, supporting efficient localization of RNA to vesicles.

![Figure: On-demand release system](https://static.igem.wiki/teams/5545/designn1/on-demandrelease3-ezgif-com-gif-to-webp-converter.webp)

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# Iteration 8

Optimizing C/D Box Motif for CO-BERA Loading Efficiency

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**Design**
To enhance the **loading efficiency** of **CO-BERA RNA** into **bacterial membrane vesicles (BMVs)**, we aimed to **optimize** the **C/D Box motif** configuration, which serves as the **recognition site** for the **L7Ae RNA-binding protein** [1]. Initially, we designed **CO-BERA** with **two C/D Box motifs** at the **3′ end**, inspired by the **iGEM 2024 HBMU-Taihe team**, which reported that **diagnostic RNAs** with **two C/D Boxes** achieved **superior vesicle loading** [8].

**Build**
We designed our **genetic circuit** based on using **two C/D Box motifs** at the **3′ end** of **CO-BERA** to facilitate **L7Ae binding** and **BMV packaging** [2]. The **CO-BERA construct** was incorporated into an **L. plantarum expression vector**, compatible with our **DUF4811-L7Ae fusion protein** for **RNA localization** to **BMVs** [3].

**Test**
After conducting **molecular dynamics simulations** using **CHARMM-GUI** and **OpenMM**, we found that a **single C/D Box motif** exhibited a **higher docking score** and **lower ensemble diversity** compared to the **two C/D Box** configurations [4, 5]. The **simulations** suggested that **two motifs** may introduce **RNA folding constraints**, reducing **loading efficiency** into **BMVs**.

![Figure: simulation of 2 C/D Box copies with L7Ae using OpenM molecular dynamics simulation](https://static.igem.wiki/teams/5545/soft/rna-relative-to-protein-cd2la7e.webp)

Figure: C/D Box motif molecular dynamics simulation results

**Learn**
The **simulation results** indicated that a **single C/D Box motif** may outperform the **two C/D Box** design for **CO-BERA loading** into **BMVs** due to **enhanced L7Ae accessibility**. To **validate** this finding empirically, we designed and ordered **DNA fragments** encoding **CO-BERA constructs** with **varying numbers** of **C/D Box motifs** (**one**, **two**, and **three**) from **Twist Bioscience**. These **fragments** include **C/D Box sequences** at the **3′ end** of **CO-BERA** and are designed for **Golden Gate cloning** into our **L. plantarum expression vector** [6]. Our **lab experiments** will involve **transforming** these **constructs** into **Lactobacillus plantarum**, **inducing BMV production**, and **quantifying RNA loading efficiency** via **qRT-PCR** to confirm the **optimal C/D Box configuration** [7].

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References

* Fabbiano F, Notarangelo M, Corsi J, et al. **RNA packaging** into **extracellular vesicles**: An orchestra of **RNA-binding proteins**? J Extracell Vesicles. 2020;9(1):12043. doi:10.1002/jev2.12043.
* Rozhdestvensky TS, Tang TH, Tchirkova IV, Brosius J, Bachellerie JP, Hüttenhofer A. **Binding** of **L7Ae protein** to the **K-turn** of **archaeal snoRNAs**: a shared **RNA binding motif** for C/D and H/ACA box snoRNAs in Archaea. Nucleic Acids Res. 2003;31(3):869–877. doi:10.1093/nar/gkg175.
* Velez M, Arluison V. Does the **Hfq Protein** Contribute to **RNA Cargo Translocation** into **Bacterial Outer Membrane Vesicles**? Pathogens. 2025;14(4):399. doi:10.3390/pathogens14040399.
* Jo S, Kim T, Iyer VG, Im W. **CHARMM-GUI**: A web-based graphical user interface for **CHARMM**. J Comput Chem. 2008;29(11):1859–1865. doi:10.1002/jcc.20945.
* Eastman P, Swails J, Chodera JD, et al. OpenMM 7: Rapid development of high performance algorithms for molecular dynamics. PLoS Comput Biol. 2017;13(7):e1005659. doi:10.1371/journal.pcbi.1005659.
* Engler C, Kandzia R, Marillonnet S. A one pot, one step, **precision cloning method** with high throughput capability. PLoS One. 2008;3(11):e3647. doi:10.1371/journal.pone.0003647.
* Bustin SA, Benes V, Garson JA, et al. The **MIQE guidelines**: Minimum information for publication of **quantitative real-time PCR** experiments. Clin Chem. 2009;55(4):611–622. doi:10.1373/clinchem.2008.112797.
* iGEM HBMU-Taihe 2024. **GPC3 CDS–2×C/D Box RNA packaging construct**. iGEM Registry Part:BBa\_K5073025. Available from: https://parts.igem.org/Part:BBa\_K5073025.

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## About our endosomal escape

After establishing the optimized **conditional expression** and **packaging** of our **CO-BERA therapeutic system** within engineered **Lactobacillus plantarum**, we aimed to ensure that the **therapeutic RNA** could reach its **functional site** inside **host cells**. While previous iterations optimized **sensing**, **regulation**, and **delivery** to **airway epithelial cells**, **intracellular release** remained a challenge. Once **bacterial membrane vesicles** deliver **CO-BERA** into the **host cell**, most **RNA cargos** become trapped within **endosomes**, which reduces their **silencing efficiency**. Therefore, the next cycle was to enhance **endosomal escape** to maximize **cytoplasmic delivery** of **CO-BERA** and ensure its full **therapeutic potential**.

# Iteration 9

Enhancing **endosomal escape** with Mutated LLO

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**Design**
Although our **CO-BERA system** was successfully loaded and conditionally expressed, there was one significant limitation; **therapeutic RNAs** delivered through **bacterial membrane vesicles** face **endosomal trapping** once inside **host cells**. Literature shows that only **< 1% of RNA cargos** naturally escape endosomes which severely limits **therapeutic efficacy** (1). To overcome this barrier, we sought a strategy for enhancing **endosomal escape**.

**Build**
After searching, we found **Listeriolysin O (LLO)**, the **pore-forming protein** from **Listeria monocytogenes** that has the ability to disrupt **endosomal membranes** and help in **endosomal escape** (2).

**Test**
Several studies mentioned the risk of using **wild LLO** due to its **pathogenicity**, as **LLO** is one of the main factors enabling **Listeria monocytogenes** to escape the **phagosome** and establish infection inside **host cells**(3,4).

So using the wild protein directly could mimic **pathogen-like activity**, raising **biosafety concerns**.

**Learn**
So we decided to use a **less virulent version** of this protein that is less harmful and useful in therapy.

**Rebuild**
To ensure **safety** and avoid **pathogenic effects**, we designed and incorporated a **mutated LLO**. To be safe and prevent pathogenicity, We used an engineered **LLO variant** shown in the literature to have strongly reduced **pathogenic activity** while retaining **pH-dependent pore formation** compared to **wild-type LLO** (2,3). This allowed us to preserve its **endosome-disrupting function** while eliminating potential **pathogenesis**.

**Re-Test**
Our analysis of existing literature confirmed that engineered **LLO mutants** retain the ability to mediate **pH-dependent pore formation** in endosomes, without the risks associated with the **wild-type protein** (3)..

**Learn**
With the integration of **mutated LLO**, our system gained a powerful mechanism for **endosomal escape**, ensuring that a much higher fraction of **CO-BERA** reaches the cytoplasm, where it can effectively silence **target genes** (3,4). This step is important for enhancing the **efficiency** and **therapeutic potential** of our **probiotic-based delivery platform**.

![Wild LLo](https://static.igem.wiki/teams/5545/designn1/21-ezgif-com-gif-to-webp-converter.webp)

Wild LLO : Degradation of CO-BERA at lysosomal or lat endosomal pH as wild LLO become active at low pH.

![L461T LLO](https://static.igem.wiki/teams/5545/designn1/11-ezgif-com-gif-to-webp-converter.webp)

L461T LLO : early activation of Mutated LLO so preserve CO-BERA from degradation.

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References

* Roth AJ, Zoller N, Bakowska-Polak A, Behrens HM, Klussmann S. Delivery of RNA therapeutics: the great endosomal escape! RNA Biol. 2023;20(1):214–227. PMID: 36372998; PMCID: PMC9595607.
* Resnik N, Tratnjek L, Erdani-Kreft M, Kisovec M, et al. Engineering a pH-responsive pore-forming protein. Sci Rep. 2017;7:42231. doi:10.1038/srep42231.
* Carriero M, Lanni F, De Simone M, Falcigno L, Matarazzo MR, Pane F, et al. Carboxyl-terminal residues N478 and V479 required for hemolytic activity of listeriolysin O. Front Immunol. 2017;8:1439. doi:10.3389/fimmu.2017.01439.
* Geoffroy MC, Gaillard JL, Alouf JE, Berche P. Purification, characterization, and toxicity of the sulfhydryl-activated hemolysin listeriolysin O from Listeria monocytogenes. Infect Immun. 1987;55(7):1641-6. PMID: 3110068; PMCID: PMC260565.

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# Iteration 10

Advancing Biosafety with a Toxin–Antitoxin (TA) System

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**Design**
Although **Lactobacillus plantarum** is considered safe as a **probiotic** and naturally functions along the **lung–GIT axis** (1), our team identified a potential **biosafety concern**: the possibility of **bacterial escape** beyond the intended environment. Uncontrolled persistence of **engineered strains** outside the lung could pose risks to patients and the ecosystem. To mitigate this, we sought a **genetic containment mechanism**.

**Build**
We added a **Toxin–Antitoxin (TA) system** into our design. In this system, the **toxin and antitoxin** is continuously expressed within the **lung environment**, which neutralises each other, ensuring **bacterial survival** and **therapeutic function**. However, if bacteria escape outside the lung and exposed to (phosphate as in blood stream or Low temperature as in external environment), the **antitoxin (PemI)** which is less stable will be degraded, while the stable **toxin (PemK)** remains active—leading to **self-elimination** of the escaped bacteria (2,3).

**Test**
After our **model validation** and reviewed successful applications of **TA systems** in **synthetic biology**, where they act as **biological kill-switches** (3,4). Modeling confirmed that such a system could maintain **bacterial survival** in the lung while providing **kill-switches** against uncontrolled dissemination.

![](https://static.igem.wiki/teams/5545/model2025/modeltoxinandtoxin/f79d8790-baea-43fa-a3dc-78c9e7894c86.webp)

**Learn**
By integrating the **TA system**, we significantly advanced the **biosafety** and **controllability** of our **probiotic platform**. This ensured not only **therapeutic efficacy** but also compliance with **biocontainment standards**(5).

![Figure: Toxin antitoxin in blood](https://static.igem.wiki/teams/5545/designn1/toxinantitoxininblood3-ezgif-com-gif-to-webp-converter.webp)

![Figure: Toxin antitoxin in environment](https://static.igem.wiki/teams/5545/proof/toxinantitoxininenvironment11-ezgif-com-gif-to-webp-converter.webp)

The illustrations show the killing of our bacteria in unintended environment

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References

* Plaza-Díaz J, Ruiz-Ojeda FJ, Gil-Campos M, Gil A. Mechanisms of action of probiotics. Adv Nutr. 2019;10(suppl\_1):S49–66. doi:10.1093/advances/nmy063.
* Harms A, Brodersen DE, Mitarai N, Gerdes K. Toxins, targets, and triggers: an overview of toxin–antitoxin biology. Mol Cell. 2018;70(5):768–784. doi:10.1016/j.molcel.2018.01.003.
* Kato F, Yoshizawa S, Yamaguchi Y. New insights into toxin–antitoxin systems in bacteria: biochemistry and physiology. Front Microbiol. 2023;14:1178321. doi:10.3389/fmicb.2023.1178321
* Chan CTY, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol. 2016;12(2):82–86. doi:10.1038/nchembio.1979.
* Stirling F, Bitzan L, O'Keefe S, Redfield E, Oliver JW, Way J, Silver PA. Rational design of evolutionarily stable microbial kill switches. Mol Cell. 2017;68(4):686–697.e3. doi:10.1016/j.molcel.2017.10.033.

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## Optimizing Bacterial Delivery

Our goal is to deliver our engineered **Lactobacillus plantarum** effectively into the lungs, and this forms the basis of therapeutic treatment. The method of delivery should preserve bacterial viability and stability, provide efficient deposition in the respiratory tract, and be a viable option when considered from a practical point of view for patients. To address these requirements, we searched about different pulmonary delivery strategies and refined our approach through iterative design and evaluation. This process leads us toward a delivery system that balances effectiveness, safety, and scalability for real clinical application.

# Iteration 11

Delivery Method Selection

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**Design**
Initially, we aimed to deliver our **engineered Lactobacillus plantarum** using **aerosolization**. This approach is widely used for **liquid-based drugs** and was expected to ensure **rapid** and **broad dispersal** of our **bacteria** into the **lungs** (1,2).

**Build**
We considered **aerosol devices** that generate **fine droplets** capable of reaching the **lower respiratory tract**, making them a plausible option for **live biotherapeutic delivery** (2).

**Test**
Upon reviewing the literature and experimental data on **bacterial delivery methods**, several challenges with **aerosolization** emerged (3,4):

* **Shear stress** during nebulization reduces **bacterial viability**.
* **Short residence time** in the lung due to **mucociliary clearance**.
* Higher risk of **contamination** and reduced **stability** during storage.

![Figure: Particle size and GSD effect on lung deposition efficiency](https://static.igem.wiki/teams/5545/model12/17.webp)

Graph showing how particle size and geometric standard deviation (GSD) affect lung deposition efficiency. The current design (~3 μm MMAD, GSD = 2.0) achieves ~25% deposition, supporting its suitability for alveolar targeting in dry powder inhaler formulations.

**Learn**
Following discussions with **Dr/ Suzan**, we transitioned to **Dry Powder Inhalers (DPI)** as the **delivery platform**. DPIs provided several advantages (5):

* Better preservation of **bacterial viability** during **formulation** and **storage**.
* Longer **half-life** and **stability** of the **probiotic formulation**.
* Greater **patient compliance** and **applicability**, as **DPIs** are widely used in **Asthma management**.

This switch allowed us to design a **delivery system** that is not only **effective** but also **practical** and **scalable** for patients with **Asthma**.

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References

* Laube BL. The expanding role of aerosols in systemic drug delivery, gene therapy, and vaccination. *Respir Care*. 2005 Sep;50(9):1161-76. PMID: 16122400.
* Darquenne C. Deposition mechanisms. *J Aerosol Med Pulm Drug Deliv*. 2020 Apr;33(2):61-5. doi:10.1089/jamp.2019.1558.
* Carvalho TC, Peters JI, Williams RO 3rd. Influence of particle size on regional lung deposition – what evidence is there? *Int J Pharm*. 2011 Sep 30;406(1-2):1-10. doi:10.1016/j.ijpharm.2010.12.040.
* Loretz B, Schäfer J, Vogt L, Winter G, Lehr CM. Pulmonary delivery of therapeutic proteins. *J Aerosol Med Pulm Drug Deliv*. 2011 Jun;24(3):127-35. doi:10.1089/jamp.2010.0859.
* Vandenheuvel D, Meeus J, Lavigne R, Van den Mooter G. Instability of bacteriophages in spray-dried trehalose powders is influenced by the stress imposed by spray-drying. *Int J Pharm*. 2013 Jun 15;450(1-2):128-36. doi:10.1016/j.ijpharm.2013.04.020.

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# Iteration 12

preparation of the powder

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**Design**
To ensure effective delivery of our **engineered Lactobacillus plantarum** via **Dry Powder Inhalers (DPIs)**, we aimed to develop a **scalable** and **stable formulation** that preserves **bacterial viability** and ensures **efficient lung deposition**. Initially, we selected **spray-drying** as the **formulation method** due to its **fast**, **continuous**, and widely used **industrial process** for producing **dry formulations**, making it attractive for **scalable therapeutic production** [1]. We designed the process to generate **fine dry powder** suitable for **inhalation**, targeting a **mass median aerodynamic diameter (MMAD)** of **1–5 μm** for optimal **alveolar deposition** [2].

**Build**
We explored **spray-drying conditions** commonly used for **probiotics**, where **heated air** rapidly evaporates the solvent to produce a **fine dry powder** compatible with **DPI devices** [3]. We incorporated **protective excipients** like **trehalose** to stabilize **L. plantarum** during drying, aiming to minimize **thermal stress** and maintain **cell integrity** [4]. The process was designed to optimize parameters such as **inlet temperature (120–150°C)** and **feed rate** based on literature recommendations for **probiotic formulations** [5].

**Test**
Further research and literature-based modeling revealed significant drawbacks of **spray-drying** for our application:

* **High temperature exposure (120–150°C)** reduced **probiotic viability**, with **survival rates** dropping to **59.5%** due to **thermal stress** [6].
* **Rapid dehydration** damaged **bacterial membranes**, leading to compromised **cell integrity** [7].
* **Poor long-term survival rates** limited **clinical feasibility**, as powders showed reduced **stability** during storage [8].

As our model validated these findings, where **spray-dried L. plantarum** exhibited a **viability loss of ~40%** (measured via **colony-forming unit (CFU) counts**) compared to controls. In contrast, **freeze-drying** achieved a **survival rate of 72.7%**, a **13.2% improvement** over spray-drying, as confirmed by **in vitro assays**. These results aligned with literature highlighting **freeze-drying's superiority** for **probiotic preservation** [9].

![Figure: Comparison of probiotic survival rates between freeze-drying and spray-drying](https://static.igem.wiki/teams/5545/parts/eng-11.webp)

**Bar chart comparing probiotic survival rates following two drying methods: Freeze-Drying and Spray-Drying. Freeze-Drying achieved a higher survival rate (72.7%) compared to Spray-Drying (59.5%), with a noted advantage of +13.2%. These results support the selection of Freeze-Drying for DPI formulation, due to its superior preservation of bacterial viability and reduced process-related risks.**

**Learn**
Based on our **modeling results**, **preliminary experimental data**, and guidance from **Dr. Ahmed Abdelsabour**, we transitioned to **freeze-drying (lyophilization)** as the preferred method for **DPI formulation**. Freeze-drying offers:

* **Superior preservation** of **bacterial viability** compared to spray-drying, minimizing **thermal** and **osmotic stress** [6].
* **Lower stress** on **bacterial membranes** during processing, maintaining **cell integrity** [9].
* **Compatibility** with **protective excipients** (e.g., **trehalose**, **mannitol**) that enhance **powder stability** and **aerosol dispersion** [10].

This choice ensures that **Lactobacillus plantarum** remains **viable** throughout **production**, **storage**, and **delivery**, aligning with **patient-friendly DPI systems** used in **asthma management**.

Design

Build

Test

Learn

References

* Sosnik A, Seremeta KP. Advantages and challenges of the spray-drying technology for the production of pure drug particles and drug-loaded polymeric carriers. *Adv Colloid Interface Sci*. 2015;223:40–54. doi:10.1016/j.cis.2015.05.003.
* Labiris NR, Dolovich MB. Pulmonary drug delivery. Part I: Physiological factors affecting therapeutic effectiveness of aerosolized medications. *Br J Clin Pharmacol*. 2003;56(6):588–599. doi:10.1046/j.1365-2125.2003.01892.x.
* Broeckx G, Vandenheuvel D, Claes IJ, Lebeer S, Kiekens F. Drying techniques of probiotic bacteria as an important step towards the development of novel pharmabiotics. *Int J Pharm*. 2016;505(1-2):303–318. doi:10.1016/j.ijpharm.2016.04.002.
* Morgan CA, Herman N, White PA, Vesey G. Preservation of micro-organisms by drying; a review. *J Microbiol Methods*. 2006;66(2):183–193. doi:10.1016/j.mimet.2006.02.017.
* Huang S, Vigneswari S, Ng IS, Chai C, Chen PT. Spray-drying of probiotics and its application in food and healthcare industries. *Crit Rev Food Sci Nutr*. 2022;62(23):6325–6342. doi:10.1080/10408398.2021.1906624.
* Santivarangkna C, Kulozik U, Foerst P. Inactivation mechanisms of lactic acid starter cultures preserved by drying processes. *J Appl Microbiol*. 2008;105(1):1–13. doi:10.1111/j.1365-2672.2007.03670.x.
* Peighambardoust SH, Golshan Tafti A, Hesari J. Application of spray drying for preservation of lactic acid bacteria. *Food Rev Int*. 2011;27(1):80–102. doi:10.1080/87559129.2010.496197.
* Carvalho AS, Silva J, Ho P, Teixeira P, Malcata FX, Gibbs P. Effects of various sugars added to growth and drying media upon thermotolerance and survival throughout storage of freeze-dried *Lactobacillus delbrueckii* ssp. *bulgaricus*. *Biotechnol Prog*. 2004;20(1):248–254. doi:10.1021/bp034165n.
* Leslie SB, Israeli E, Lighthart B, Crowe JH, Crowe LM. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. *Appl Environ Microbiol*. 1995;61(10):3592–3597. doi:10.1128/aem.61.10.3592-3597.1995.
* Maa YF, Prestrelski SJ. Biopharmaceutical powders: Particle formation and formulation considerations for inhalation delivery. *Curr Pharm Biotechnol*. 2000;1(3):283–302. doi:10.2174/1389201003378897.

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# Iteration 13

Fragment Preparation & Validation

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**Design**

* Plan to amplify and domesticate **Fragments 1, 2 and 3** from **Twist DNA** for use in **CO-BERA** and **loading circuits**.
* Designed **primers** with overhangs for **restriction** and **Golden Gate cloning**.

**Build**

* Resuspended **fragments** and **primers**.
* **PCR amplification** of all fragments.
* Transformation of the **TagBFPV2 plasmid** into **E. coli Jm109** to validate the **M13 primer system**.

**Test**

* **Gel electrophoresis** confirmed the correct amplification of **Fragments 1 and 2**.
* **Fragment 3** gave inconsistent gradient bands.
* Transformation of **TagBFPV2** was successful, confirming that **M13 primers** work.

**Learn**

* **PCR of Fragment 3** required optimization → **annealing temperature** adjusted to **60 °C**.
* Validated workflow for **primer use** and **colony screening**.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/2.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/1.webp)

Figure: electrophoresis showing the size of fragments 1,2 and 3.

Design

Build

Test

Learn

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

![Hero Section Image](https://static.igem.wiki/teams/5545/hero/section-hero-recovered-08.webp)

![](https://static.igem.wiki/teams/5545/img/right-side-lung.svg)

# Resuspension

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**Purpose**
We started our wet lab steps by resuspending Fragment-1, Fragment-2, and Fragment-3 to amplify them using a PCR reaction. The Fragments carry different genetic parts that will be used in our project according to the following figures:

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/1.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/2.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/3.webp)

**Materials**

* DNA fragments.
* Distilled water.
* Pipette tips.
* PCR tubes.
* 96-well plate.
* Eppendorf tube.

**Methods**

* The DNA fragment locations were detected from the 96-well plate provided by Twist Bioscience.

+ Fragment-1 → A2
+ Fragment-2 → H1
+ Fragment-3 → D2

* The tip of the pipette penetrated the cover of the multiwell plate to avoid cross-contamination.
* The lyophilized fragments were diluted with 100 µL of distilled water.
* We ensured that each lyophilized tube was well sealed.
* Then, the lyophilized tube was mixed properly with the distilled water by shaking the tube forcefully until the pellet at the bottom was no longer visible.
* The tube was spun down to collect the residuals on the wall of the tube.
* 100 µL of the medium was transferred to a sterile 1.5 mL Eppendorf tube. Then, the original tube was stored at -20°C until use (short-term storage).
* Finally, each Eppendorf tube was labeled.
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/4.webp)

Purpose

Materials

Methods

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# PCR

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PCR amplification of the resuspended fragments was performed to obtain sufficient copies for subsequent laboratory procedures.

**Materials**

* DNA templates (Fragment-1, Fragment-2, Fragment-3).
* Primers compatible with each fragment.
* Mastermix.
* Distilled water.
* PCR tubes.

**Methods**

* The lyophilized primers were resuspended by adding the following amount of distilled water to the lyophilized primers:

| Primer | concentrations | Stock solution |
| --- | --- | --- |
| Fragment-1 Forward | 24 nmol | 240 µL |

| Fragment-1 Reverse | 24.9 nmol | 250 µL |

| Fragment-2 Forward | 27.1nmol | 270 µL |

| Fragment-2 Reverse | 23.1 nmol | 230 µL |

| Fragment-3 Forward | 22.4 nmol | 224 µL |

| Fragment-3 Reverse | 20.1 nmol | 200 µL |

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/5.webp)
- Then, we prepared the working solution of each fragment by adding 10 µL of its stock solution to 90 µL of distilled water for a total of 100 µL for each primer. Then we mixed it properly.
- Aiming for a final pure volume of 50 µL, we added 25 µL of Master mix (2x concentration) for 3 PCR tubes.
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/6.webp)
- Then, we added 2 µL of the working solution of the forward and reverse primers for each fragment to the master mix. Subsequently, we mixed them properly.
- Finally, we added 0.5 µL of the template fragment.

| PCR tube (50 µL) | Forward primer (working solution) | Reverse primer (working solution) | Master mix | DNA Template | Distilled water |
| --- | --- | --- | --- | --- | --- |
| Fragment-1 | 2 µL | 2 µL | 25 µL | 0.5 µL | 20.5 µL |
| Fragment-2 | 2 µL | 2 µL | 25 µL | 0.5 µL  20.5 µL | |
| Fragment-3 | 2 µL | 2 µL | 25 µL | 0.5 µL  20.5 µL | |

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/7.webp)
- We put the final tubes into the PCR, and we set our program as follows:

* Annealing temperature: 55 °C.
* Elongation time: 1 min.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/8.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/90.webp)

Purpose

Materials

Methods

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# Gel electrophoresis

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**Purpose**
Gel electrophoresis was performed to verify the size of the synthesized fragments, ensuring their suitability for downstream applications.

**Materials**

* Agarose powder.
* TAE buffer.
* Ethidium bromide.
* Loading dye.
* Microwavable flask.
* Electrophoresis box.
* UV light device.

**Methods**

### Gel preparation

**Purpose of gel preparation**: To create a solid, porous matrix through which DNA fragments can travel.

* Ideally, the gel concentration should be 1.5 g of agarose powder in 100 ml of TAE buffer in a microwavable flask. To prepare our 50 ml tray volume. We had added 0.75 agarose powder to the 50 ml TAE buffer.
* Subsequently, it was mixed properly and heated in the microwave for 1 min until the agarose was completely dissolved.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/9.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/0000.webp)

* 1.5 µL of Ethidium bromide was added to the agarose. Ethidium bromide allows us to visualize the DNA bands under ultraviolet (UV) light.
* The gel was poured into a tray with a well comb (agarose was poured slowly to avoid bubble formation), which created wells for loading the samples.
* The gel was allowed to solidify completely at room temperature for 20-30 minutes before use.

### Sample loading and electrophoresis

**Purpose of sample loading and electrophoresis**: To introduce the DNA samples into the gel and use an electric current to separate the fragments.

* Once solidified, the agarose gel was placed in an electrophoresis box filled with TAE buffer. This buffer conducts electricity and maintains a stable pH, allowing the negatively charged DNA to migrate through the gel.
* Then, the gel box was filled with ethidium bromide until the gel was covered.
* Then, a molecular weight ladder, containing DNA fragments of known sizes, was loaded into the first lane of the gel. It serves as a reference to estimate the sizes of the PCR products.
* 2 µL of the loading dye was mixed with 5 µL of each of the PCR products before loading.
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/tasksrashidy/img-10.webp)
* Then,5 µL of the samples were loaded carefully into the remaining wells.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/12.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/11-e.webp)

* The electric current at 80 -150 V was applied to the gel electrophoresis, with the negative electrode near the wells. This causes the negatively charged DNA fragments to move toward the positive electrode. Smaller fragments travel faster and farther than larger ones.
* The run was stopped when the loading dye had moved approximately 75-80% of the way down the gel, which indicates sufficient separation of the DNA fragment (the run time was about 1-1.5h).
* After the run time, the electrodes were disconnected from the power source, and then the gel was carefully removed from the gel box.

### Visualization and interpretation of results

**Purpose of visualization**: To observe the separated DNA fragments and analyze the results.

* Finally, we transferred the gel into a UV light device to visualize our fragments and interpret the results by comparing the position of the PCR product bands to the bands of the molecular weight ladder.
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/2.webp)

Figure: electrophoresis showing the size of fragments 1,2 and 3.

Purpose

Materials

Methods

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# Gel purification

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**Purpose**
Gel purification is a molecular biology technique employed for the isolation and purification of a specific DNA fragment after agarose gel electrophoresis. This protocol is critical for various downstream applications, including cloning, PCR, and sequencing, as it effectively removes confounding contaminants such as unwanted DNA fragments, salts, and residual enzymes that could impede the fidelity and efficiency of subsequent experimental procedures.

**Materials**

* Agarose gel.
* gel extraction kit.
* Microcentrifuge tubes.
* Clean razor blade.
* UV light source.
* Microcentrifuge.
* Heating block or water bath.

**Methods**

### Excise the DNA Band

* During the interpretation of gel electrophoresis results, a protective glass plate was utilized to shield the UV transilluminator, and exposure time to UV light was minimized to mitigate potential DNA damage.
* The desired DNA band was precisely excised from the gel using a sterile razor blade, ensuring cuts were made as close as possible to the band to minimize the volume of co-excised agarose.
* The excised gel slice was then transferred into a pre-weighed microcentrifuge tube. Subsequently, the tube containing the gel slice was weighed to determine the exact mass of the agarose, which is crucial for calculating the appropriate volume of solubilization buffer required in the following step.

### Dissolve the Gel Slice

* A 3:1 volume ratio of solubilization buffer to excised gel slice (e.g., 300 µL of buffer for a 100 mg gel slice) was added to the microcentrifuge tube.
* The mixture was then inverted for fragments smaller than 5 kb or gently vortexed for larger fragments, with periodic mixing every few minutes to facilitate complete dissolution of the agarose.
* The tube was subsequently incubated at 50-60°C for approximately 5 minutes, or until the gel slice was entirely dissolved.
* A pH indicator was employed to confirm that the solution maintained a yellow color, signifying the optimal pH for efficient DNA binding. If necessary, a pH-adjusting solution was added to achieve the desired pH.

### Bind the DNA to the Spin Column

* A spin column was inserted into a collection tube, and the entire dissolved gel mixture was carefully transferred to the spin column.
* The column was then centrifuged at 13,000-17,000 x g for 1 minute to facilitate DNA binding to the silica membrane.
* Following centrifugation, the flow-through was discarded, and the spin column was re-inserted into the collection tube.

### Wash the DNA:

* Approximately 500-750 µL of wash buffer was added to the spin column, and the column was centrifuged again at 13,000-17,000 x g for 1 minute.
* The flow-through was discarded. Depending on the sensitivity of subsequent applications to residual salts, an additional wash step with wash buffer was performed.
* A final centrifugation step was executed at maximum speed (≥17,000 x g) for 1 minute to ensure complete drying of the silica membrane and to remove any residual ethanol, which is known to inhibit downstream enzymatic reactions.
* The spin column was then carefully removed from the collection tube, which was subsequently discarded along with any residual flow-through.

### Elute the DNA

* The spin column was meticulously transferred to a new, sterile microcentrifuge tube.
* 30-50 µL of Elution Buffer (pre-warmed to 50°C for enhanced yield) was dispensed directly onto the center of the column membrane, taking care to avoid contact with the pipette tip.
* The column was then allowed to sit at room temperature for 1-2 minutes, providing sufficient time for the DNA to rehydrate and dissociate from the membrane.
* Finally, the column was centrifuged at 13,000-17,000 x g for 1 minute to collect the purified DNA into the clean microcentrifuge tube.

### Quantify and Store the Purified DNA

* The concentration and purity of the eluted DNA were assessed using a spectrophotometer.
* The purified DNA was stored at 4°C for short-term experimental use or at -20°C for long-term preservation.

Purpose

Materials

Methods

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# Transformation

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**Purpose**
Following PCR, transformation of the recombinant plasmids into JM109 competent cells was performed to assess M13 functionality and to amplify the fragments for subsequent experiments.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/1.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/2.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/3.webp)

**Materials**

* DNA vector.
* Competent cells.
* Culture media.
* Ampicillin.
* Ice.
* Distilled water.
* Falcon tube.
* Transformation tubes.
* LB media.
* Shaker.

**Methods**

* The JM109 competent cells were taken out of -80°C and thawed on ice (approximately 20-30 mins).
* The agar plates that contain the antibiotic were removed from storage at 4°C and allowed to warm up at room temperature.
* The lyophilized DNA had been resuspended by adding 20 µL of distilled water to the DNA. Then, we mixed them properly.
* 5 µL of the resuspended DNA was added to 20-50 μL Jm109 competent cells in a Falcon tube. Then, it was gently mixed by flicking the bottom of the tube with your finger a few times.
* The competent cells/DNA mixture was incubated on ice for 10 min.
* Each of the transformation tubes was transferred to a hot path for 45 seconds, as the bottom 1/2 to 2/3 of the tube was placed in the path.
* Then, the tubes were turned back on ice for 2 minutes.
* 250-1,000 μl LB media was added to the transformed bacteria, and it was left to grow on the shaker for 30 minutes.
* Then, all the transformed bacteria were plated onto a 10 cm LB agar plate containing 5 µL of Ampicillin and were added to a liquid medium.
* Finally, we put our mixture on the media, and it was left on the shaker at 37°C overnight.

Purpose

Materials

Methods

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# PCR

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**Purpose**
PCR amplification was repeated for fragments 1 and 2 to achieve sufficient yield. Fragment 3 required gradient PCR to optimize annealing temperature. Additionally, the functionality of M13 primer variants was confirmed by their use in PCR amplification of the 1-D5 plasmid.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/1.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/2.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/3.webp)

**Materials**

* DNA fragments.
* Distilled water.
* Pipette tips.
* PCR tubes.
* 96-well plate.
* Eppendorf tube.

**Methods**

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/14.webp)
* We made another PCR reaction for fragment 1, 2 with the same protocol.
* Moreover, we ran a gradual PCR reaction for fragment 3 on 6 different annealing temperatures due to gradient bands shown yesterday.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/15.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/16.webp)

* Also, we ran a PCR reaction for plasmid (1-5d) with an elongation time of 1 min and an annealing temperature of 48°C to test the functionality of M13 primer variants (3 forward and 2 reverse) with a total of 6 probabilities.

| Primer | Code |
| --- | --- |
| M-13 Forward-1 | 1 |

| M-13 Reverse-1 | 2 |

| M-13 Reverse-2 | 3 |

| M-13 Forward-2 | 4 |

| M-13 Forward-3 | 5 |

- Each of the forward primers we had was tested with the two reverse primers.

| Forward primer | Reverse primer |
| --- | --- |
| M-13 Forward-1 (1) | M-13 Reverse-1 (2) |

| M-13 Forward-1 (1) | M-13 Reverse-2 (3) |

| M-13 Forward-2 (4) | M-13 Reverse-1 (2) |

| M-13 Forward-2 (4) | M-13 Reverse-2 (3) |

| M-13 Forward-3 (5) | M-13 Reverse-1 (2) || M-13 Forward-3 (5) | M-13 Reverse-2 (3) |

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/17.webp)

Purpose

Materials

Methods

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# Glycerol Stock

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**Purpose**
Glycerol stock preparation was performed to ensure the short and long-term cryopreservation of the transformed bacterial cultures.

**Materials**

* Bacterial culture.
* LB media.
* Glycerol stocks.
* Sterile water.
* Ampicillin.
* Eppendorf tube.

**Methods**

* After the steps of Overnight Liquid Culture Inoculation
* We took two portions of the actively growing overnight culture. This ensured a backup in case one stock is contaminated or lost.
* We took 2 glycerol stocks for our 1-5d transformed bacterial growth, each stock contained 500 μL of each overnight culture mixed with 500 μL of 50% glycerol in a 2 mL screw top tube.
* The solution was mixed gently by inverting the tube several times, ensuring the glycerol was evenly distributed among the bacteria.
* TThe prepared glycerol stock was snap frozen by placing the tubes into a liquid nitrogen bath. This rapid freezing technique helps prevent the formation of large, destructive ice crystals.
* After snap-freezing, the stock was transferred to a -80°C freezer for long-term storage. Where it can remain viable for many years.
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/18.webp)

Purpose

Materials

Methods

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# Activation protocol

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**Purpose**
To cultivate a high-density, pure culture of bacteria harboring the recombinant plasmid.

**Materials**

* LB media.
* Ampicilin.
* Plates of the transformed bacteria
* Shaker.

**Methods**

* 5 μL of ampicillin was added to 5 mL of LB media, ensuring that only the bacteria that successfully took up the ampicillin-resistant plasmid would survive and grow, while any untransformed bacteria would be killed.
* A colony from the plate was taken and seeded in the LB media, which guarantees that the entire liquid culture will be genetically uniform and contain the correct plasmid.
* The colony was left in the LB media in the shaker overnight. The overnight provides sufficient time for the bacteria to grow and reach a high cell density, while the agitation of the shaker provides aeration by continuously mixing oxygen into the media.
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/123.webp)

Purpose

Materials

Methods

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# Plasmid Extraction

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**Purpose**
Plasmid extraction was performed from amplified transformed cells via a three-step process involving cell lysis, DNA binding, and elution. The extracted plasmid was subsequently used for cloning.

**Materials**

* Tris-HCl buffer.
* EDTA.
* RNase A.
* Glucose.
* Wash buffers.
* Isopropanol.
* Alkaline lysis buffer.
* Resuspension buffer.
* N3 buffer.
* Silica membrane.

**Methods**

### Step (1): Lysis

* We started by lysing the bacterial cells.
* 200 µL of P1 buffer (resuspension buffer) was added to rehydrate the bacterial pellet, which often contains RNase A to degrade cellular RNA. Then the P1 buffer was mixed by pipetting.
* 50 µL of P2 buffer (alkaline lysis buffer) was added to disrupt the cell membrane and denature proteins and DNA, including the bacterial chromosome and the plasmid. Then the P2 was mixed by inverting.
* The P2 buffer was left for 5 minutes to allow complete lysis of the cell membrane.
* 350 µL of N3 buffer was added to neutralize the pH, leading to precipitation of the large chromosomal DNA and denatured protein, while the small, supercoiled plasmid DNA remains in solution. Then the N3 buffer was mixed by inverting.

### Step (2): Binding and washing

* The culture medium was centrifuged for 4 minutes to pellet the precipitated chromosomal DNA and cellular debris, leaving the plasmid DNA in the supernatant.
* The upper layer was transferred to a spin column to allow the plasmid DNA to bind to the column's silica membrane in the presence of the high salt concentration from the N3 buffer.
* Subsequently, the spin column was centrifuged, which forced the liquid (flow-through) containing cellular waste and other impurities through the column, leaving the plasmid DNA bound to the membrane.
* 500 µL of AW1 and 500 µL of AW2 (wash buffers) were added to remove any residual salts from the first wash.
* We let the mixture dry for 2 minutes, which allows for the removal of all traces of the wash buffers, especially the alcohol, which can interfere with subsequent steps.

### Step (3): elution

* The mixture was eluted with 50 µL water and placed in a new Eppendorf, which facilitated the release of the DNA from the silica membrane
* Then, a new Eppendorf tube was used to collect the clean DNA.
* Then, we waited for 3 minutes, which allowed the water to fully rehydrate the DNA and dissociate it from the membrane, increasing the final yield.
* Finally, the eluted DNA was centrifuged for 3 minutes.
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/19.webp)

Purpose

Materials

Methods

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# DNA purification

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**Purpose**
Following the initial plasmid extraction, a dedicated purification step was deemed essential to enhance the efficiency of subsequent cloning and assembly processes.

**Materials**

* Tris-HCl buffer.
* EDTA.
* RNase A.
* Wash buffers.
* Isopropanol.
* RNA lysis buffer.
* Resuspension buffer.
* Eppendorf tubes.

**Methods**

### Step 1: Binding

* 200 µL of RLT (RNA lysis buffer) was added to allow binding of DNA to the silica membrane in the spin column. Then it was mixed by pipetting.
* 40 µL of isopropanol was added, which promotes the binding of nucleic acids to the silica membrane by helping to dehydrate the DNA. Then it was mixed by inverting.
* The mixture was poured into a spin column.
* Then, the mixture was left for 5 minutes to allow the binding of the DNA to the column's membrane.

### Step (2): washing

* The mixture was centrifuged for 30 seconds, which allowed the removal of the unbound primers, enzymes, and salts.
* Then, the mixture was washed with 500 µL of AW2 (wash buffer), which removed any remaining salts and impurities.
* Then, the mixture was centrifuged to spin the buffer through the column and discard the flow-through.p>
* Subsequently, we left it dry for 2 minutes. This ensured that all residual ethanol from the wash buffer was removed from the column

### Step (3): elution

* The mixture was eluted with 50 µL water and placed in a new Eppendorf, which facilitated the release of the DNA from the silica membrane.
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/123.webp)

Purpose

Materials

Methods

◆ ◆ ◆

# Digestion

Press the button on the right side

**Purpose**
To digest fragments 1, 2, and 3 by specific restriction enzymes, thereby isolating specific genetic parts for subsequent use in the project. These genetic parts are:

### Fragment 1

| Part name | Restriction 1 | Restriction 2 | Buffer |
| --- | --- | --- | --- |
| 1- P11 promotor | Ecor1 | Sac1 | R2-1 |
| 2- Antitoxin promotor | Sac1 | Kpn1 | R2-1 |
| 3- SV40 Poly A signal | Kpn1 | BamH | R1-1 |
| 4- Trrn B terminator | BamH | Xbal | R2-1 |
| 5- RBS | Xbal | Sel1 | R3-1/span> |
| 6- T7 promotor | Sel1 | Pst1 | R3-1 |
| 7- P-kat A promoter | Pst1 | Hind 3 | R2-1 |

### Fragment 2

| Part name | Restriction 1 | Restriction 2 | Buffer |
| --- | --- | --- | --- |
| 1- P32 promoter + lac R operon | Sac1 | Kpn1 | R2-1 |
| 2- Pkat A + Rep repressor | Kpn1 | Pst1 | R1-1 |

### Fragment 3

| Part name | Restriction 1 | Restriction 2 | Buffer |
| --- | --- | --- | --- |
| 1- Pkat + operator | Hind3 | Xbal | R2-1 |
| 2- T7 + SVTR kozakr | Xbal | Ecor1 | R2-1 |
| 3- P170-Cp promotor | Ecor1 | Sac1 | R2-1 |
| 4- P32 promoter with lac operon | Sac1 | Kpn1 | R2-1 |

**Materials**

* Restriction enzymes.
* DNA aliquot.
* Nuclease-free water.
* Digestion vessels.

**Methods**

* Our genetic parts were digested using different pairs of restriction enzymes. The restriction enzymes were used in specific amounts with various elements, ensuring that each part has different "sticky ends" on each side for directional cloning.
* The vector was cut with the same pair of restriction enzymes used for a specific genetic part to make it compatible with each Part.
* We ran the digested vectors and PCR products on PCR, specifically the digestion protocol, which consists of 2 stages

The vector was cut with the same pair of restriction enzymes used for a specific genetic part to make it compatible with each Part.

| Parts reaction | Volume | Vector reaction | Volume |
| --- | --- | --- | --- |
| 1- PCR product (part) | 5 μL | Vector | 2 μL |
| 2- Restriction 1 | 1 μL | Restriction 1 | 1 μL |
| 3- Restriction 2 | 1 μL | Restriction 2 | 1 μL |
| 4- Buffer | 2 μL | Buffer | 2 μL |
| 5- H2O | 11 μL | H2O | 11 μL |

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/22.webp)

#### Incubation and inactivation

* Stage (1) Heat activation step to provide the optimal temperature (37 °C for 1 hr) for most restriction enzymes to cleave DNA efficiently.
* Stage (2) Heat-inactivation step to denature and kill the restriction enzymes at 85 °C, preventing them from cutting the DNA during the next ligation step.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/23.webp)

Purpose

Materials

Methods

◆ ◆ ◆

# Gel Electrophoresis

Press the button on the right side

**Purpose**
Gel electrophoresis was performed to verify the size of the fragments amplified during the repeated PCR reaction.

**Materials**

* Agarose powder.
* TAE buffer.
* Ethidium bromide.
* Loading dye.
* microwavable flask.
* electrophoresis box.
* UV light device.

**Methods**

We performed gel electrophoresis for all today's PCR products, as follows:

* Gel electrophoresis for the gradual PCR amplification of Fragment-3, and the amplified Fragments-1,2:
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/4.webp)
* Gel electrophoresis for the 1-d5 plasmid PCR product, which was performed using different M-13 primer variants to find the viable pair, and for the purified Fragments 1, 2, and 3 from yesterday’s and today’s reactions:
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/1.webp)

Purpose

Materials

Methods

◆ ◆ ◆

# Ligation

Press the button on the right side

**Purpose**
To ligate the excised genetic insert into its corresponding vector, forming a novel recombinant plasmid in a final reaction volume of 20 μL

**Materials**

* T4 DNA Ligase.
* Ligation Buffer (10X).
* Nuclease-free water.
* Vector DNA.
* Insert DNA.

**Methods**

* We added 5 μL of the digested PCR product to 10 μL of the digested corresponding vector with 2 μL of the buffer ligase, 1 μL ligase enzyme, and 1 μL ATP.
* The final component ratio was 2:1 of vector to insert volume (10 µL vector, 5 µL insert).
* Finally, this mixture was incubated at room temperature for 1 hour, which is the optimal temperature to form phosphodiester bonds, sealing the nicks in the DNA backbone.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/tasksrashidy/img-24.webp)

Purpose

Materials

Methods

◆ ◆ ◆

# Transformation

Press the button on the right side

**Purpose**
To introduce the ligated plasmids into JM109 competent cells to assess the viability of the recombinant constructs, following the established protocol.

**Materials**

* DNA vector.
* Competent cells.
* Culture media.
* Ampicillin.
* Ice.
* Distilled water.
* Falcon tube.
* Transformation tubes.
* LB media.
* Shaker.

**Methods**

* 5 μL of the ligated plasmids was added to Jm109 competent cells.
* Then, the heat shock protocol was performed to increase the efficiency of DNA uptake by bacterial cells as follows:

+ The mixture was left on the ice for 10 min, as the cold temperature stabilizes the cell membranes, making them more receptive to the plasmid DNA.
+ Then, the mixture was exposed to heat shock by being transferred to a hot bath for 45 seconds. This creates a thermal imbalance across the cell membrane, allowing the DNA to enter.
+ Then, they were turned back to the ice for 2 minutes, allowing for the recovery and stabilization of the bacterial membranes.

* 250 μL of LB media was added to each of the 13 tubes, as it provides nutrients for the cells to recover and begin expressing the genes on the newly acquired plasmid.
* Then, they were left on the shaker for 1 hour, ensuring proper aeration for the cells as they recover and begin expressing the antibiotic resistance gene on the plasmid.
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/tasksrashidy/img-25.webp)
* We melted the solid media.
* Then, 100 μL of Ampicillin and 50 μLof X-gal were added to the media, as the Ampicillin selects the bacteria that successfully took the plasmid, while the X-gal is used to differentiate between recombinant and non-recombinant plasmids.
* The media was poured on the plates.
* Then, 100 μLof Jm109 competent cells were added to the plates containing the selection media.
* We spread the cells using glass beads.
* Finally, the plates were incubated at 37 °C overnight, allowing the transformed bacteria to grow into colonies.
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/26.webp)

Purpose

Materials

Methods

◆ ◆ ◆

# Plasmid Extraction

Press the button on the right side

**Purpose**
Plasmid extraction was performed from amplified transformed cells via a three-step process involving cell lysis, DNA binding, and elution. The extracted plasmid was subsequently used for cloning.

**Materials**

* Tris-HCl buffer.
* EDTA.
* RNase A.
* Proteinase K.
* Wash buffers.
* Isopropanol.
* RNA lysis buffer.
* Resuspension buffer.
* Eppendorf tubes.

**Methods**

### Step (1): Lysis

+ We started by lysing the bacterial cells.
+ 200 µL of P1 buffer (resuspension buffer) was added to rehydrate the bacterial pellet, which often contains RNase A to degrade cellular RNA. Then the P1 buffer was mixed by pipetting.
+ 50 µL of P2 buffer (alkaline lysis buffer) was added to disrupt the cell membrane and denature proteins and DNA, including the bacterial chromosome and the plasmid. Then the P2 was mixed by inverting.
+ The P2 buffer was left for 5 minutes to allow complete lysis of the cell membrane.
+ 350 µL of N3 buffer was added to neutralize the pH, leading to precipitation of the large chromosomal DNA and denatured protein, while the small, supercoiled plasmid DNA remains in solution. Then the N3 buffer was mixed by inverting.

### Step (2): Binding and washing

+ The culture medium was centrifuged for 4 minutes to pellet the precipitated chromosomal DNA and cellular debris, leaving the plasmid DNA in the supernatant.
+ The upper layer was transferred to a spin column to allow the plasmid DNA to bind to the column's silica membrane in the presence of the high salt concentration from the N3 buffer.
+ Subsequently, the spin column was centrifuged, which forced the liquid (flow-through) containing cellular waste and other impurities through the column, leaving the plasmid DNA bound to the membrane.
+ 500 µL of AW1 and 500 µL of AW2 (wash buffers) were added to remove any residual salts from the first wash.
+ We let the mixture dry for 2 minutes, which allows for the removal of all traces of the wash buffers, especially the alcohol, which can interfere with subsequent steps.

### Step (3): elution

+ The mixture was eluted with 50 µL water and placed in a new Eppendorf, which facilitated the release of the DNA from the silica membrane
+ Then, a new Eppendorf tube was used to collect the clean DNA.
+ Then, we waited for 3 minutes, which allowed the water to fully rehydrate the DNA and dissociate it from the membrane, increasing the final yield.
+ Finally, the eluted DNA was centrifuged for 3 minutes.

Purpose

Materials

Methods

◆ ◆ ◆

# Gel electrophoresis

Press the button on the right side

**Purpose**
Gel electrophoresis was performed to verify the size of the extracted fragments, ensuring their suitability for downstream applications.

**Materials**

* Agarose powder.
* TAE buffer.
* Ethidium bromide.
* Loading dye.
* microwavable flask.
* electrophoresis box.
* UV light device.

**Methods**

### Gel preparation

**Purpose of gel preparation**: To create a solid, porous matrix through which DNA fragments can travel.

* Ideally, the gel concentration should be 1.5 g of agarose powder in 100 ml of TAE buffer in a microwavable flask. To prepare our 50 ml tray volume. We had added 0.75 agarose powder to the 50 ml TAE buffer.
* Subsequently, it was mixed properly and heated in the microwave for 1 min until the agarose was completely dissolved.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/9.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/0000.webp)

* 1.5 µL of Ethidium bromide was added to the agarose. Ethidium bromide allows us to visualize the DNA bands under ultraviolet (UV) light.
* The gel was poured into a tray with a well comb (agarose was poured slowly to avoid bubble formation), which created wells for loading the samples.
* The gel was allowed to solidify completely at room temperature for 20-30 minutes before use.

### Sample loading and electrophoresis

**Purpose of sample loading and electrophoresis**: To introduce the DNA samples into the gel and use an electric current to separate the fragments.

* Once solidified, the agarose gel was placed in an electrophoresis box filled with TAE buffer. This buffer conducts electricity and maintains a stable pH, allowing the negatively charged DNA to migrate through the gel.
* Then, the gel box was filled with ethidium bromide until the gel was covered.
* Then, a molecular weight ladder, containing DNA fragments of known sizes, was loaded into the first lane of the gel. It serves as a reference to estimate the sizes of the PCR products.
* 2 µL of the loading dye was mixed with 5 µL of each of the PCR products before loading.
* Then,5 µL of the samples were loaded carefully into the remaining wells.
![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/12.webp)
* The electric current at 80 -150 V was applied to the gel electrophoresis, with the negative electrode near the wells. This causes the negatively charged DNA fragments to move toward the positive electrode. Smaller fragments travel faster and farther than larger ones.
* The run was stopped when the loading dye had moved approximately 75-80% of the way down the gel, which indicates sufficient separation of the DNA fragment (the run time was about 1-1.5h).
* After the run time, the electrodes were disconnected from the power source, and then the gel was carefully removed from the gel box.

### Visualization and interpretation of results

**Purpose of visualization**: To observe the separated DNA fragments and analyze the results.

* Finally, we transferred the gel into a UV light device to visualize our fragments and interpret the results by comparing the position of the PCR product bands to the bands of the molecular weight ladder.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/3.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/dna-ladder.webp)

Figure: electrophoresis showing the size of the isolated parts

Purpose

Materials

Methods

◆ ◆ ◆

# Gel purification

Press the button on the right side

**Purpose**
Gel purification is a molecular biology technique employed for the isolation and purification of a specific DNA fragment after agarose gel electrophoresis. This protocol is critical for various downstream applications, including cloning, PCR, and sequencing, as it effectively removes confounding contaminants such as unwanted DNA fragments, salts, and residual enzymes that could impede the fidelity and efficiency of subsequent experimental procedures.

**Materials**

* Agarose gel.
* gel extraction kit.
* Microcentrifuge tubes.
* Clean razor blade.
* UV light source.
* Microcentrifuge.
* Heating block or water bath.

**Methods**

### Excise the DNA Band

* During the interpretation of gel electrophoresis results, a protective glass plate was utilized to shield the UV transilluminator, and exposure time to UV light was minimized to mitigate potential DNA damage.
* The desired DNA band was precisely excised from the gel using a sterile razor blade, ensuring cuts were made as close as possible to the band to minimize the volume of co-excised agarose.
* The excised gel slice was then transferred into a pre-weighed microcentrifuge tube. Subsequently, the tube containing the gel slice was weighed to determine the exact mass of the agarose, which is crucial for calculating the appropriate volume of solubilization buffer required in the following step.

### Dissolve the Gel Slice

* A 3:1 volume ratio of solubilization buffer to excised gel slice (e.g., 300 µL of buffer for a 100 mg gel slice) was added to the microcentrifuge tube.
* The mixture was then inverted for fragments smaller than 5 kb or gently vortexed for larger fragments, with periodic mixing every few minutes to facilitate complete dissolution of the agarose.
* The tube was subsequently incubated at 50-60°C for approximately 5 minutes, or until the gel slice was entirely dissolved.
* A pH indicator was employed to confirm that the solution maintained a yellow color, signifying the optimal pH for efficient DNA binding. If necessary, a pH-adjusting solution was added to achieve the desired pH.

### Bind the DNA to the Spin Column

* A spin column was inserted into a collection tube, and the entire dissolved gel mixture was carefully transferred to the spin column.
* The column was then centrifuged at 13,000-17,000 x g for 1 minute to facilitate DNA binding to the silica membrane.
* Following centrifugation, the flow-through was discarded, and the spin column was re-inserted into the collection tube.

### Wash the DNA:

* Approximately 500-750 µL of wash buffer was added to the spin column, and the column was centrifuged again at 13,000-17,000 x g for 1 minute.
* The flow-through was discarded. Depending on the sensitivity of subsequent applications to residual salts, an additional wash step with wash buffer was performed.
* A final centrifugation step was executed at maximum speed (≥17,000 x g) for 1 minute to ensure complete drying of the silica membrane and to remove any residual ethanol, which is known to inhibit downstream enzymatic reactions.
* The spin column was then carefully removed from the collection tube, which was subsequently discarded along with any residual flow-through.

### Elute the DNA

* The spin column was meticulously transferred to a new, sterile microcentrifuge tube.
* 30-50 µL of Elution Buffer (pre-warmed to 50°C for enhanced yield) was dispensed directly onto the center of the column membrane, taking care to avoid contact with the pipette tip.
* The column was then allowed to sit at room temperature for 1-2 minutes, providing sufficient time for the DNA to rehydrate and dissociate from the membrane.
* Finally, the column was centrifuged at 13,000-17,000 x g for 1 minute to collect the purified DNA into the clean microcentrifuge tube.

### Quantify and Store the Purified DNA

* The concentration and purity of the eluted DNA were assessed using a spectrophotometer.
* The purified DNA was stored at 4°C for short-term experimental use or at -20°C for long-term preservation.

Purpose

Materials

Methods

◆ ◆ ◆

# Digestion

Press the button on the right side

**Purpose**
To digest the transformed parts by specific restriction enzymes, thereby isolating specific genetic parts for the level one assembly.

**Materials**

* Restriction enzymes.
* DNA aliquot.
* Nuclease-free water.
* Digestion vessels.

**Methods**

* Our genetic parts were digested using different pairs of restriction enzymes. The restriction enzymes were used in specific amounts with various elements, ensuring that each part has different "sticky ends" on each side for directional cloning.
* The vector was cut with the same pair of restriction enzymes used for a specific genetic part to make it compatible with each Part.
* We ran the digested vectors and PCR products on PCR, specifically the digestion protocol, which consists of 2 stages

The vector was cut with the same pair of restriction enzymes used for a specific genetic part to make it compatible with each Part.

#### Incubation and inactivation

+ Stage (1) Heat activation step to provide the optimal temperature (37 °C for 1 hr) for most restriction enzymes to cleave DNA efficiently.
+ Stage (2) Heat-inactivation step to denature and kill the restriction enzymes at 85 °C, preventing them from cutting the DNA during the next ligation step.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/23.webp)

Purpose

Materials

Methods

◆ ◆ ◆

# Ligation

Press the button on the right side

**Purpose**
To ligate the excised genetic insert into its corresponding vector, forming a novel recombinant plasmid in a final reaction volume of 20 μL

**Materials**

* T4 DNA Ligase.
* Ligation Buffer (10X).
* Nuclease-free water.
* Vector DNA.
* Insert DNA.

**Methods**

* We added 5 μL of the digested PCR product to 10 μL of the digested corresponding vector with 2 μL of the buffer ligase, 1 μL ligase enzyme, and 1 μL ATP.
* The final component ratio was 2:1 of vector to insert volume (10 µL vector, 5 µL insert).
* Finally, this mixture was incubated at room temperature for 1 hour, which is the optimal temperature to form phosphodiester bonds, sealing the nicks in the DNA backbone.

Purpose

Materials

Methods

◆ ◆ ◆

# Transformation

Press the button on the right side

**Purpose**
To introduce the ligated plasmids into JM109 competent cells to assess the viability of the recombinant constructs, following the established protocol.

**Materials**

* DNA vector.
* Competent cells.
* Culture media.
* Ampicillin.
* Ice.
* Distilled water.
* Falcon tube.
* Transformation tubes.
* LB media.
* Shaker.

**Methods**

* 5 μL of the ligated plasmids was added to Jm109 competent cells.
* Then, the heat shock protocol was performed to increase the efficiency of DNA uptake by bacterial cells as follows:

+ The mixture was left on the ice for 10 min, as the cold temperature stabilizes the cell membranes, making them more receptive to the plasmid DNA.
+ Then, the mixture was exposed to heat shock by being transferred to a hot bath for 45 seconds. This creates a thermal imbalance across the cell membrane, allowing the DNA to enter.
+ Then, they were turned back to the ice for 2 minutes, allowing for the recovery and stabilization of the bacterial membranes.

* 250 μL of LB media was added to each of the 13 tubes, as it provides nutrients for the cells to recover and begin expressing the genes on the newly acquired plasmid.
* Then, they were left on the shaker for 1 hour, ensuring proper aeration for the cells as they recover and begin expressing the antibiotic resistance gene on the plasmid.
* We melted the solid media.
* Then, 100 μL of Ampicillin and 50 μLof X-gal were added to the media, as the Ampicillin selects the bacteria that successfully took the plasmid, while the X-gal is used to differentiate between recombinant and non-recombinant plasmids.
* The media was poured on the plates.
* Then, 100 μLof Jm109 competent cells were added to the plates containing the selection media.
* We spread the cells using glass beads.
* Finally, the plates were incubated at 37 °C overnight, allowing the transformed bacteria to grow into colonies.

Purpose

Materials

Methods

◆ ◆ ◆

# PCR

Press the button on the right side

**Purpose**
To isolated the level 1 ligated plasmids from JM109 competent cells to assess the viability of the recombinant constructs, following its transformation.

**Materials**

* DNA fragments.
* Distilled water.
* Pipette tips.
* PCR tubes.
* 96-well plate.
* Eppendorf tube.

**Methods**

* The lyophilized primers were resuspended by adding the following amount of distilled water to the lyophilized primers:
* Then, we prepared the working solution of each of the level 1 ligated parts by adding 10 µL of its stock solution to 90 µL of distilled water for a total of 100 µL for each primer. Then we mixed it properly.
* Aiming for a final pure volume of 50 µL, we added 25 µL of Master mix (2x concentration) for 3 PCR tubes.
* Then, we added 2 µL of the working solution of the forward and reverse primers for each fragment to the master mix. Subsequently, we mixed them properly.
* Finally, we added 0.5 µL of the template fragment.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/8.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/90.webp)

Purpose

Materials

Methods

◆ ◆ ◆

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

![Hero Section Image](https://static.igem.wiki/teams/5545/hero/section-hero-recovered-07.webp)

1 Introduction

2 Dry lab

3 Wet lab

![](https://static.igem.wiki/teams/5545/img/right-side-lung.svg)

# Introduction

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).](https://2025.igem.wiki/afcm-egypt/safety-and-security)

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).](https://2025.igem.wiki/afcm-egypt/proof-of-concept)

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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/soft/comp.webp)

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.

![Complex structure visualization](https://static.igem.wiki/teams/5545/soft/cd1la7e.webp)

![One copy RMSD L7Ae](https://static.igem.wiki/teams/5545/softt/protein-rmsd-1.webp)

RMSD graph of L7Ae protein with one copy C/D box.

![One copy RMSD C/D box](https://static.igem.wiki/teams/5545/softt/rna-rmsd.webp)

RMSD graph of C/D box RNA with L7Ae.

### 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).

![Complex structure visualization](https://static.igem.wiki/teams/5545/model2025/m2gif1alphafold-ezgif-com-gif-to-webp-converter.webp)

GIF shows the 3D structure of CO-BERA-C/D box with the protein complex.

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

![One copy RMSD L7Ae](https://static.igem.wiki/teams/5545/softt/protein-rmsd-1.webp)

RMSD graph of L7Ae protein with one copy C/D box.

![One copy RMSD C/D box](https://static.igem.wiki/teams/5545/softt/rna-rmsd.webp)

RMSD graph of C/D box RNA with L7Ae.

### 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.

![One copy RMSD C/D box](https://static.igem.wiki/teams/5545/softt/5ns.webp)

![One copy RMSD C/D box](https://static.igem.wiki/teams/5545/softt/rna-rmsd.webp)

Fig shows RMSD graph of Argonaute protein with 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.

![Final system setup](https://static.igem.wiki/teams/5545/softt/rna1f.webp)

Fig shows RMSF graph of Guide siRNA per RNA nucleotide.

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.

![Final system setup](https://static.igem.wiki/teams/5545/softt/proteind.webp)

Fig shows RMSF of Argonaute protein per residue.

# 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:

| Part name | Code |
| --- | --- |
| Terminator | 1-A |
| RBS | 1-B |
| CMV promoter | 1-C |
| The mammalian vector. | 1-D |

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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/4.webp)

## 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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/7.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/6.webp)

## 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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/9.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/0000.webp)

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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/tasksrashidy/last-aly.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/1.webp)

Figure: Gel electrophoresis showing the size verification of fragments 1,2 and 3.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/pmge36e-plasmid.webp)

Figure: Gel electrophoresis showing the size verification of pMG36E plasmid.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/newgel/newgel/dna-ladder-5.avif)

Figure: Gel electrophoresis showing the size verification of the parts isolated from he distribution kits.

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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/123.webp)

## 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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/newgel/newgel/agar.avif)

Culture media showing the bacterial transformation of our fragments.

## 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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/4.webp)

Gel electrophoresis showing the size verification of fragments 1,2 and 3

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/newgel/image-7.avif)

Gel electrophoresis showing the size verification of the parts amplified by M13 primers

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/15.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/16.webp)

## 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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/19.webp)

## 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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/22.webp)

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/experiment/23.webp)

## 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.

| Part name | Theoretical length | Code |
| --- | --- | --- |
| P11 promotor | 60 | 1-A |
| Antitoxin promoter | 92 | 1-B |
| SV40 Poly A signal | 122 | 1-C |
| Trrn B terminator | 72 | 1-D |
| RBS | 11 | 1-E |
| T7 promoter | 18 | 1-F |
| P-kat A promoter | 66 | 1-G |
| P32 promoter + lac R operon | 188 | 2-A |
| Pkat A + Rep repressor | 83 | 2-B |
| Pkat A + operator | 86 | 3-A |
| T7 + SVTR kozak | 38 | 3-B |
| P170-Cp promoter | 326 | 3-C |
| P32 promoter with lac operon | 188 | 3-D |

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/tasksrashidy/fragments-dna-horizon.webp)

#### 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.

| Part name | Theoretical length | Code |
| --- | --- | --- |
| T2A + His tag + TSLP | 566 | 1-A |
| secE + L7Ae | 552 | 1-B |
| GFP | 717 | 1-C |
| CO-BERA expression non conditioned | 908 | 1-D |
| Conditioned CO-BERA + CL-operator | 450 | 1-E |
| Conditioned CO-BERA no operator | 430 | 1-F |
| Duf4811-L7Ae | 867 | 2-A |
| CO-BERA circuit + Rep operator | 508 | 2-B |

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/dna-ladder-1.webp)

#### 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.

| Part name | Theoretical length | Code |
| --- | --- | --- |
| TSLP | 494 | 1-A |
| Toxin anti-toxin | 615 | 1-B |
| Lac repressor | 1083 | 1-C |
| Rep repressor | 660 | 1-D |
| LLO | 1587 | 1-E |
| Foldase and L7Ae | 1293 | 1-F |

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/dna-ladder.webp)

#### 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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/tasksrashidy/img-24.webp)

## 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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/newgel/newgel/agar2.avif)

## 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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/gel/unnamed.webp)

## 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.

![Figure: Pulsatile vs. continuous release](https://static.igem.wiki/teams/5545/newgel/newgel/agar3.avif)

## 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.

◆ ◆ ◆

{% endblock %}