iGEM 2024 — Team thessaly


{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)[Introduction](#intro)

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)[Cloning procedure](#clone)

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)[1st
Engineering Cycle](#first)

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)[2nd
Engineering Cycle](#second)

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)[3rd
Engineering Cycle](#third)

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)[Conclusion](#con)

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)[References](#ref)

# ~Engineering~

## Introduction: Pioneering Engineering in Biology

### “Failure is a vital part of all scientific endeavor” -Entrapta, She-Ra, and the Princesses of Power

Synthetic biology is an **interdisciplinary** scientific research field that
utilizes
**engineering principles** to modify and design living organisms and systems. Throughout our iGEM
journey, we consistently applied the **Design-Build-Test-Learn** cycle to **refine**
and **enhance** our project. This involved **characterizing****bioparts** and **creating constructs** with transcriptional units to
establish a thoroughly **characterized**, **measurable**, and **orthogonal
engineering** system. **Trial and error** played a key role in our journey. The setbacks
we encountered helped us **improve** our designs and the way we think as future scientists. Each
challenge taught us valuable lessons, leading to better solutions and a deeper understanding of our work. Our
journey in engineering this system wasn't just about the end product—it was about **embracing the
process** and **learning from every step**.

For engineering **THAELIA** and ensuring optimal outcomes, we divided our system
into four key
components for focused study and experimentation:

• 1st Engineering Cycle: Evaluation of the T7 polymerase system

• 2nd Engineering Cycle: Production of Outer Membrane Vesicles (OMVs)

• 3rd Engineering Cycle: Synthesis of the dsRNA in vitro and assessment of its
silencing
efficacy

This **modular approach** allowed us to focus on each element and**improve** the
final design.

## Cloning procedure:

As iGEM Thessaly 2024, for our cloning experiments, a variety of vectors were used to
accommodate two
different chassis. For *E. coli* strains (DH5α and BL21(DE3)), Golden Braid vectors were utilized,
following the established Golden Braid grammar for modular cloning. The domestication process was carried out
using the Golden Braid Domesticator tool to ensure compatibility and efficient assembly. Additionally, another
cloning method, the Golden Standard method, was employed this year to address the needs of the second chassis,
*Pseudomonas putida.*

By following the steps of the previous iGEM Thessaly teams, we decided to use the
**Golden Braid 2.0**cloning method, that allows the assembly of DNA parts in a standardized and efficient manner by using
Type IIS restriction enzymes, that recognize specific DNA sequences but cleave at a defined distance away from
the recognition site [1, 2].

The first step in GoldenBraid is to create **Level 0** constructs. In this
level, individual DNA
parts (such as promoters, coding sequences, and terminators) are inserted into universal domesticator
plasmids. For these parts to be compatible with the GoldenBraid system, a process called
**domestication** must occur, where internal restriction sites that would interfere with the
assembly process are removed, and specific 4nt overhangs are added to match the GoldenBraid grammar. These
Level 0 constructs then serve as the foundational building blocks for higher-level assemblies in the cloning
process [2].

For creating our Level 0 constructs, we used the **BsmbI** Type IIS enzyme and
the **pUPD2**vector.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng1.png)

### Figure 1: Creating Level 0 constructs by inserting the Basic Parts with the overhangs in pUPD2 vector by using the BsmbI TypeIIS enzyme

To assemble functional devices (transcriptional units), we combined our basic parts using the
Type IIS enzyme
**BsaI**and the **pDGB3 alpha1** and **pDGB3 alpha2**
vectors. In this process, multiple Level 0 parts were assembled into these vectors, resulting in a complete
transcriptional unit, which is referred to as a **Level a construct**.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng2.png)

### Figure 2: Creating Level a constructs by inserting the Basic Parts from pUPD2 vector into pDGB3a1 by using the BsaI TypeIIS enzyme

To move from Level a to Level ω using the Golden Braid cloning system, we utilized the BsmbI
Type IIS
restriction enzyme, similar to how BsaI is used in Level a. In this process, the two Level a constructs were
assembled into the **pDGB3****ω** **vector**, resulting in a complete
Level Ω construct with two transcriptional units.

However, while working with Golden Braid vectors facilitated the straightforward assembly of
DNA constructs,
these vectors utilize the **pBR322** origin of replication, which is incompatible with our
proposed chassis, *P. putida [3]*. So, to successfully implement our
constructs in *P. putida*,
it was necessary to transition from Golden Braid to Golden Standard vectors. The Golden Standard vectors
contain the **pBBR1** origin of replication, which is compatible with *P. putida*,
enabling stable replication and efficient gene expression within this target organism [4]. This transition
ensured that our assembled constructs could function effectively in our chassis.

Read More:Golden Standard Vectors

The Golden Standard assembly system is designed with a highly structured approach to
plasmid
construction,
following a set of rules similar to those found in the **SEVA format**. It organizes plasmid
vectors into three fundamental components: **the origin of replication**, **the
selection
marker**, and **the cargo**. Each of these components is minimized to retain only
the
essential DNA sequences required for functionality, ensuring that plasmid constructs are as efficient as
possible [5].

![](https://static.igem.wiki/teams/5299/league-of-legends/eng3.png)

### **Figure 3: Standard European Vector Architecture****.**

For example, **pSEVA23g19[g1]** is a modular plasmid from the SEVA (Standard
European Vector
Architecture) collection, specifically designed for genetic engineering in Gram-negative bacteria. The
"23"
indicates that it carries a kanamycin resistance gene for selection, while "3g" refers to the pBBR1 origin
of
replication, which supports stable replication across a broad range of bacterial species. The "[g1]" tag
specifies that the plasmid's cargo is 1AI2, associated with level 1 Golden Standard reactions [5].

The structure of pUPD2, despite being a Golden Braid vector, remains compatible with various
cloning methods. By utilizing the BsaI restriction enzyme, Level 0 parts from the pUPD2 vector can be
efficiently integrated into the Level 1 Golden Standard vector pSEVA23g19[g1].

![](https://static.igem.wiki/teams/5299/league-of-legends/eng4.png)

### Figure 4: Assembly of genetic parts in pUPD2 and successful transfer of transcriptional units into pSEVA23g19[g1] Plasmid

## 1st Engineering Cycle : Evaluation of the T7 polymerase system

Our project, THAELIA, is centered on bmRNAi, where dsRNA is produced and delivered to the
fungal pathogen *V. dahliae*. The first critical objective of our system is to generate dsRNA in
sufficient
quantities and efficiently encapsulate it into OMVs for delivery. A key factor in achieving this is the
efficient production of dsRNA, which relies on the T7 polymerase system. Consequently, a major challenge we
needed to address was how to effectively regulate the production of T7 polymerase to ensure optimal dsRNA
synthesis without imposing undue stress on the bacterial chassis.

### First iteration: Identification of the most suitable promoter for T7 polymerase production

**Design**

During our Designing phase, we were full of questions, such as; “*How were we going to
regulate the T7
polymerase production?” ,* And most importantly; “*How could we isolate this function from other
cellular processes?”*

The T7 polymerase is a key component of our engineered bacterial system, driving the
efficient production of
double-stranded RNA (dsRNA). To determine the most suitable promoter for the T7 polymerase production system
we
tested three BG synthetic
promoters (i.e. BG17, BG37, BG42) from the *Zobel et al.* library [6]. We
selected the promoters BG17,
BG42, and BG37 based on findings from *Zobel et al*., which demonstrated their significant activation
during the exponential growth phase. Our objective was to evaluate their temporal activation profiles and
relative strength. We used *E. coli* BL21 (DE3) cells, which are able to express the T7 polymerase
system, so they can serve as a relevant model for our engineered *P. putida* chassis, allowing us to
evaluate our constructs’ effectiveness in a system closely resembling our final design.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng5.png)

### Figure 5: The BG promoters we tested for finding the most suitable promoter for the T7 polymerase production device. We used sfGFP as a reporter gene.

The promoter J23119 served as a positive control because it is a well-characterized
constitutive promoter
from the Anderson family, allowing for direct comparison of promoter strength and consistent activation
throughout all growth phases [7]. Additionally, osmY is a well-characterized
promoter specifically induced
during the stationary phase, making it a valuable positive control for assessing time-dependent activation
and
promoter strength during this phase [8].

![](https://static.igem.wiki/teams/5299/league-of-legends/eng6.png)

### Figure 6: The J231119 and osmY promoters serve as positive controls. We used sfGFP as a reporter gene.

**Build**

We began by cloning the parts we needed (BG37, BG42, BG17, J23119, osmY, ter, sfGFP, T7pol,
T7pro and
T7hyb6ter, which included Golden Braid overhangs, into the pUPD2 vector to create the Level 0 parts. The
promoters were synthesized by IDT with their respective RBSs already integrated. We used *E. coli*
DH5α
cells for the cloning procedure due to their high transformation efficiency.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng7.png)

### **Figure 7. Diagnostic digestion of the generated Level 0 parts** by using EcoRV and NotI. **A.** *in silico* virtual digestion by GelSim Tool from SnapGene. **B**.1 % agarose gel ran at 110 V for 25 min stained with EtBr. The lanes order is as follows: (1) pUPD2 (control), (2) J23119-RBS2, (3) osmY-RBS2, (4)BG37-RBS2, (5) BG42-RBS2, (6) BG17-RBS2, (7) T7 pol.

Next, we transfered the basic parts from the pUPD2 vectors into the Golden Braid vectors:
pDGB3a1 and pDGB3a2
vectors to assemble the level alpha constructs. This step enabled us to generate complete, functional
genetic
modules for further experimentation and analysis.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng8.png)

### **Figure 8. Diagnostic digestion of the generated Level a constructs** by using EcoRV and NotI. **A.** *in silico* virtual digestion by GelSim Tool from SnapGene. **B**.1 % agarose gel ran at 110 V for 25 min stained with EtBr. The lanes order is as follows: 1) pDB3a1, (2) BG37-RBS2-sfGFP-ter, (3) osmY-RBS2 -sfGFP-ter, (4) J23119-RBS1 -sfGFP-ter, (5)BG37-RBS2-T7pol-ter , (6) , (7)P3.1-RBS2-sfGFO-ter , (8) P3.1-RBS1-sfGFP-ter, (9) BG37-RBS1-sfGFP-ter, (10) BG37-RBS1-sfGFP-ter, (11) T7pro-RBS2-sfGFP- T7terhyb6 , (12) BG17-RBS1,-sfGFP-ter (13) BG42-RBS1 -sfGFP-ter

**Test**

After assembling our plasmid constructs, we successfully transformed them into chemical
competent *E. coli*BL21 (DE3). The following day, we prepared 10ml liquid cultures of LB medium, with the proper
antibiotic,
to promote the growth and acclimatization of the transformed bacteria, allowing them to incubate overnight
for
16 hours at 37°C and 210 rpm. On the third day, we centrifuged the cultures and washed the resulting pellets
twice with 0.8% saline solution (NaCl). Subsequently, we performed a 1:100 dilution to measure the optical
density at 600 nm (OD600). To achieve an OD600 of 0.1 with a final volume of 2 mL of M9 culture with the
corresponding carbon source, we applied the dilution equation "Cinitial x Vinitial= C final x V final". M9
medium was selected for this experiment due to the fluorescence interference associated with LB medium. We
then transferred 200 μL of each diluted culture into the wells of a 96-well plate with a clear bottom. Each
construct was tested in five technical repeats. The plate was incubated in a plate reader for 15 hours at
37°C, which is optimal for *E. coli* while shaking at 180 rpm. Measurements were automatically
recorded
every hour, monitoring OD at 600 nm and sfGFP fluorescence at 515 nm (emission peak of sfGFP, R*oberts et
al*. )

![](https://static.igem.wiki/teams/5299/league-of-legends/eng9.png)

### Figure 9: Bacterial growth curve, with M9 medium with 0,2% glucose as a carbon source, based on Optical Density at 600 nm measurements taken every 1 hour. Data points represent the average of five technical replicates, with any values showing a standard deviation greater than 0.2 excluded for clarity. Error bars correspond to standard deviation of n=5 replicates. Blank was subtracted.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng10.png)

### Figure 10: Normalized fluorescence intensity for J23119-RBS2-sfGFP-ter, osmy-RBS2-sfGFP-ter, BG37-RBS2-sfGFP-ter, BG17-RBS2-sfGFP-ter and BG42--RBS2-sfGFP-ter constructs during the time points: 3h, 6h, 10h and 15h. The RFUs (sfGFP measurements at 515 nm) are divided by cell growth (Optical Density at 600nm), in order to normalize all values. *E. coli* BL21 (DE3) cells with pDGB3a1 were used as the (-) control. Error bars correspond to standard deviation of n=5 replicates. Blank was subtracted.

**Learn**

By examining Figure 9 and analyzing the OD600 measurements, we learn that significant
bacterial growth
occurs
between the 1-hour and 8-hour time points for all promoters except BG37. For E. coli cells containing the
BG37
promoter, the growth continues to increase up to the 12-hour mark, with a steady rise in OD600 between the
6-hour and 12-hour time points indicating that the cells are in the exponential phase.

Further insights can be gained from Figure 10, which confirms that the cells remain in
exponential growth
at
the 12-hour time point**. BG37, in particular, demonstrates strong activity during this
period.**
At the 6-hour time point, BG37 shows a notable increase in fluorescence, surpassing that of the other
promoters, highlighting its heightened responsiveness during early exponential growth. By 10 hours, BG37
continues to exhibit superior regulation compared to BG17 and osmy. Even at 15 hours, when the cells are
transitioning out of exponential growth, BG37 remains active, showcasing its sustained effectiveness.

In comparison, while J23119 and BG42 also demonstrate strong performance, BG37
distinguishes
itself with its
early activation at 6 hours, making it particularly suited for systems that require robust expression during
the initial stages of exponential growth. Although BG42 and J23119 provide more consistent expression across
various phases, the results clearly indicate that **BG37 is the optimal choice for targeted regulation
during the exponential phase**. Its early and sustained activity positions it as the most effective
promoter for driving T7 polymerase production in scenarios where rapid and efficient expression is
essential.
**So, by completing this Design-Build-Test-Learn (D-B-T-L) cycle, we successfully integrated the BG37
promoter into our system to facilitate the production of T7 polymerase.**

![](https://static.igem.wiki/teams/5299/league-of-legends/eng11.png)

### Figure 11: Production of T7 polymerase, regulated by BG37 promoter.

### Second iteration: Identification of the most suitable promoter for T7 polymerase production

**Design:Evaluation of T7 polymerase production device**

After identifying BG37 as the most suitable promoter for T7 polymerase production, we aimed
to validate the
functionality of a Level Ω construct designed to efficiently express T7 polymerase under the control of
BG37.
Due to time constraints, we decided to test T7 polymerase production by using a sfGFP reporter under the
control of a T7 promoter. For this reason, the J23119 Anderson and BG37 constitutive promoters were used to
drive T7 polymerase gene expression. The J23119 promoter served as a positive control for continuous T7
production, while *E. coli* cells with the pDGB3omega plasmid were used as a negative control.
Additionally, we aimed to assess the timing of BG37 promoter activation, focusing on whether it activates
specifically during the exponential phase. We used E. coli DH5a cells for this experiment, which are
optimized
for maintaining plasmids, with specific mutations (such as recA1 and endA1) that prevent unwanted
recombination and degradation of foreign DNA and lack the T7 RNA polymerase system [9].

![](https://static.igem.wiki/teams/5299/league-of-legends/eng12.png)

### Figure 12: Level omega constructs lead to T7 polymerase and sfGFP production.

**Build****:**

Once we obtained our level A constructs, as it was described in the previous DBTL, in the
pDGB3a1 and pDGB3a2
vectors, we proceeded to create our level omega constructs (BG37-RBS2-T7polymerase-ter + T7pro-sfGFP-T7ter,
PJ23119-RBS2-T7polymerase-ter + T7pro-sfGFP-T7ter) by cloning them into the pDGB3omega vector.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng13.png)

### Figure 13: Diagnostic digestion of the generated Level ω constructs by using EcoRV and NotI. A. in silico virtual digestion by GelSim Tool from SnapGene. B.1 % agarose gel ran at 110 V for 25 min stained with EtBr. The lanes order is as follows: (1) pDB3omega, (2) BG37-RBS2-T7pol-Ter + T7pro- RBS2-sfGFP-T7ter, (3) J23119-RBS2-T7pol-Ter + T7pro-RBS2-sfGFP-T7ter

**Test**

After assembling our plasmid constructs, we successfully transformed them into chemical
competent *E. coli
DH5a*. The following day, we prepared 10ml liquid cultures of LB medium, with the proper antibiotic,
to
promote the growth and acclimatization of the transformed bacteria, allowing them to incubate overnight for
16
hours at 37°C and 210 rpm. On the third day, we centrifuged the cultures and washed the resulting pellets
twice with 0.8% saline solution (NaCl). Subsequently, we performed a 1:100 dilution to measure the optical
density at 600 nm (OD600). To achieve an OD600 of 0.1 with a final volume of 2 mL of M9 culture with the
corresponding carbon source, we applied the dilution equation "Cinitial x Vinitial= C final x V final". M9
medium was selected for this experiment due to the fluorescence interference associated with LB medium. We
then transferred 200 μL of each diluted culture into the wells of a 96-well plate with a clear bottom. Each
construct was tested in five technical repeats. The plate was incubated in a plate reader for 15 hours at
37°C, which is optimal for *E. coli* while shaking at 180 rpm. Measurements were automatically
recorded
every hour, monitoring OD at 600 nm and sfGFP fluorescence at 515 nm (emission peak of sfGFP, R*oberts**et al*. )

![](https://static.igem.wiki/teams/5299/league-of-legends/eng14.png)

### Figure 14: Bacterial growth curve, with M9 medium with 0,2% glucose as a carbon source, based on Optical Density at 600 nm measurements taken every 1 hour. Data points represent the average of five technical replicates, with any values showing a standard deviation greater than 0.2 excluded for clarity. Error bars correspond to standard deviation of n=5 replicates. Blank was subtracted.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng15.png)

### Figure 15: Normalized fluorescence intensity for J23119-RBS2-T7pol-ter + T7pro-RBS2-sfGFP-T7terhyb6, BG37-RBS2-T7pol-ter + T7pro-RBS2-sfGFP-T7terhyb6 level omega constructs during the time points:1h, 3h, 6h, 10h and 15h. The RFUs (sfGFP measurements at 515 nm) are divided by cell growth (Optical Density at 600nm), in order to normalize all values. *E. coli DH5a* cells with pDGB3omega1 were used as the (-) control. Error bars correspond to standard deviation of n=5 replicates. Blank was subtracted.

**Learn**

In figure 14, we observed that the cells exhibit suboptimal growth, likely due to the
metabolic burden
imposed by high plasmid copy numbers and the potential loss of plasmid over time. This metabolic strain can
reduce overall system performance, as cells must allocate significant resources to plasmid replication and
maintenance. To enhance the efficiency and stability of our system, we propose integrating the T7 polymerase
production device directly into the bacterial chromosome. Chromosomal integration would alleviate the
metabolic load by reducing plasmid-associated demands and ensuring stable, consistent expression of the T7
polymerase, leading to improved overall performance and growth.

Additionally, the BG37 promoter demonstrates strong autoinducible activity during the
exponential phase and
beyond, reaching expression levels comparable to or higher than the constitutive J23119 promoter. This
confirms the utility of BG37 for applications requiring high expression during growth.

### Third iteration: Integration of T7 polymerase into the chromosome

**Design**

Building on insights gained from our previous Design-Build-Test-Learn (DBTL) cycle, we aimed to integrate the
T7 polymerase transcriptional unit directly into the *P. putida* chromosome. This approach was selected
to achieve stable, single-copy expression of the T7 polymerase, eliminating the potential variability and
instability associated with plasmid-based systems[10]. Chromosomal integration
ensures that the gene remains a permanent fixture within the bacterial genome, reducing the risk of loss
during cell division and promoting consistent expression throughout the population [11].

So, to achieve stable expression of the T7 polymerase in *P. putida*, we set out to
integrate the T7 polymerase transcriptional unit directly into the bacterial chromosome. After consulting with
Dr. Esteban Garcia-Martinez, we identified the **mini-Tn7** system as the most suitable tool for
our goal.

The mini-Tn7 system is a broad host-range vector that allows for the stable integration of
single-copy genes into bacterial chromosomes at a neutral site: **attTn7**. This system is
particularly useful for bacteria with single attTn7 sites, such as *P. aeruginosa* and *P.
putida*. The system operates through two key components. The first component is the Tn7-M delivery
vector, contains an R6K replication origin, which makes it a suicide vector; it can replicate only in the
presence of Pir proteins, ensuring that it remains stable and does not persist in the bacterial host after the
desired integration has occurred [12]. Simultaneously, it has a multiple cloning
site (MCS) flanked by Tn7 left (Tn7L) and Tn7 right (Tn7R) sequences.The second component is the
**helper plasmid** (pTNS), which encodes the TnsABCD proteins (Tn7 transposase). These proteins
are responsible for recognizing the Tn7 borders 9 on the delivery vector and facilitating site-specific
integration into the attTn7 site. This precise integration occurs 25 nucleotides downstream of the glmS gene,
ensuring that no essential functions of the host organism are disrupted. Together, these components allow for
accurate and stable insertion of the T7 polymerase gene into the *P. putida* chromosome.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng16.png)

### Figure 16: Components of the Tn7 cloning and integration system

In order to facilitate this process, we needed to construct the pTn-19[g1] plasmid. This
can
be accomplished
by digesting the Golden Standard (GS) plasmid pSEVA23g19[g1] and the vector pTn7-M with the restriction
enzymes PacI and SpeI, as described in *Salgado et al [13].* The
resulting
fragments are then ligated
together to generate the pTn-19[g1] plasmid, which carries both the Level 1 cargo and the necessary Tn7
borders for chromosomal integration.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng17.png)

### Figure 17: Creation of pTN-19[g1]

**Build- Test**

Unfortunately, due to time constraints, we were unable to proceed with the construction of
the pTN-19[g1]
plasmid and the subsequent integration of the T7 polymerase into the chromosome.

## 2nd Engineering Cycle: Production of Outer Membrane Vesicles (OMVs)

The second critical component of developing the THAELIA project involved optimizing the
timing of producing
the Outer Membrane Vesicles (OMVs). In our design, the production of OMVs is initiated by the eTEV protease,
which cleaves the TolB protein at engineered TEV protease recognition sites. We chose to utilize a tolB
mutant
and introduce a tolB variant containing TEV recognition sites because this particular mutant has
demonstrated
superior efficiency in producing Outer Membrane Vesicles (OMVs) in *P. putida* compared to other tol
or
Pal mutants [14]. To achieve this, we aimed for orthogonal production of eTEV
during the
**stationary** phase, allowing for precise control over the timing of protease expression.To
achieve this, we designed the orthogonal production of eTEV protease specifically during the stationary phase,
enabling precise control over the timing of protease expression. This approach ensures that the cleavage of
TolB occurs at the most opportune moment, maximizing OMV production after dsRNA synthesis, allowing the dsRNA
to be efficiently encapsulated within the vesicles. By finely tuning the expression of eTEV, we can enhance
OMV production and
ensure
efficient loading of the dsRNA inside them.

**Design**

**Evaluation of Various RBSs (BBa\_B0030, and BBa\_B0034****)
for** **the P3.1
Stationary Promoter**

During our Designing phase, we hit a brick wall; ‘*How were we going to control the
stationary phase
production of TEV protease?’* And most importantly; ‘*How could we isolate this function from other
cellular processes?’*

The answer to our questions was found through a thorough literature review, where we
encountered the work of
Jaishankar et al. (2020) [15]. In their study, they described a synthetic
auto-inducible promoter active in
the stationary phase, which demonstrated very strong activity. To evaluate this promoter and assess its
suitability for our project, we employed sfGFP as a reporter gene, enabling us to visualize and quantify the
promoter's activity effectively. Furthermore, we designed experiments to test two different Ribosome Binding
Sites (RBSs) to enhance the optimization of the system's performance. We used the promoter J23119 as a
positive control because it is a well-characterized constitutive promoter from the Anderson family, allowing
for direct comparison of promoter strength and consistent activation throughout all growth phases [16].
Additionally, osmY is a well-characterized promoter specifically induced during the stationary phase, making
it a valuable positive control for assessing time-dependent activation and promoter strength during this
phase
[17].

![](https://static.igem.wiki/teams/5299/league-of-legends/eng18.png)

### Figure 18: The constructs we used for P3.1 - RBS\_B0034 examination

![](https://static.igem.wiki/teams/5299/league-of-legends/eng19.png)

### Figure 19:The constructs we used forP3.1- RBS\_B0030 examination

**Build**

We constructed the Level 0 and Level a constructs as described in Engineering Cycle 1.

**Test**

After assembling our plasmid constructs, we successfully transformed them into chemical
competent E. coli
BL21 DE3. The following day, we prepared 10ml liquid cultures of LB medium, with the proper antibiotic, to
promote the growth and acclimatization of the transformed bacteria, allowing them to incubate overnight for
16
hours at 37°C and 210 rpm. On the third day, we centrifuged the cultures and washed the resulting pellets
twice with 0.8% saline solution (NaCl). Subsequently, we performed a 1:100 dilution to measure the optical
density at 600 nm (OD600). To achieve an OD600 of 0.1 with a final volume of 2 mL of M9 culture with the
corresponding carbon source, we applied the dilution equation "Cinitial x Vinitial= C final x V final". M9
medium was selected for this experiment due to the fluorescence interference associated with LB medium. We
then transferred 200 μL of each diluted culture into the wells of a 96-well plate with a clear bottom. Each
construct was tested in five technical repeats. The plate was incubated in a plate reader for 15 hours at
37°C, which is optimal for E. coli while shaking at 180 rpm. Measurements were automatically recorded every
hour, monitoring OD at 600 nm and sfGFP fluorescence at 515 nm (emission peak of sfGFP, Roberts et al. )

**1. Examining RBS1**

![](https://static.igem.wiki/teams/5299/league-of-legends/eng20.png)

### Figure 20: Normalized fluorescence intensity for J23119-RBS1-sfGFP-ter, osmy-RBS1-sfGFP-ter, and P3.1--RBS1-sfGFP-ter constructs during the time points: 3h, 6h, 10h and 15h. The RFUs (sfGFP measurements at 515 nm) are divided by cell growth (Optical Density at 600nm), in order to normalize all values. *E. coli*BL21 (DE3) cells with pDGB3a1 were used as the (-) control. Error bars correspond to standard deviation of n=5 replicates. Blank was subtracted.

**2. Examining RBS2**

![](https://static.igem.wiki/teams/5299/league-of-legends/eng21.png)

### Figure 21: Normalized fluorescence intensity for J23119-RBS2-sfGFP-ter, osmy-RBS2-sfGFP-ter, and P3.1--RBS2-sfGFP-ter constructs during the time points: 3h, 6h, 10h and 15h. The RFUs (sfGFP measurements at 515 nm) are divided by cell growth (Optical Density at 600nm), in order to normalize all values. *E. coli*BL21 (DE3) cells with pDGB3a1 were used as the (-) control. Error bars correspond to standard deviation of n=5 replicates. Blank was subtracted.

**Comparing the performance of P3.1 under the regulation of different RBSs: BBa\_B0030
(RBS1) vs
BBa\_B0034 (RBS2)**

![](https://static.igem.wiki/teams/5299/league-of-legends/eng22.png)

### Figure 22: Bacterial growth curve, with M9 medium with 0,2% glucose as a carbon source, based on Optical Density at 600 nm measurements taken every 1 hour. Data points represent the average of five technical replicates, with any values showing a standard deviation greater than 0.2 excluded for clarity. Error bars correspond to standard deviation of n=5 replicates. Blank was subtracted.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng23.png)

Figure 23: Normalized fluorescence intensity for P3.1-RBS1-sfGFP-ter, and
P3.1--RBS2-sfGFP-ter constructs
during the time points: 3h, 6h, 10h and 15h. The RFUs (sfGFP measurements at 515 nm) are divided by cell
growth (Optical Density at 600nm), in order to normalize all values. Error bars correspond to standard
deviation of n=5 replicates. Blank was subtracted.

**Learn**

By observing Figure 22 and analyzing the OD600 measurements, we notice significant
bacterial
growth between
the 2h and 6h time points. The steady rise in OD600 during this period indicates that the cells are in the
exponential phase.

The results indicate that the **P3.1 promoter** is significantly
**stronger** than
any other promoter tested, exhibiting a continuous increase in expression levels as the bacteria grow.
Notably, after 15 hours of incubation, the fluorescence data demonstrate a marked enhancement in sfGFP
production, underscoring the P3.1 promoter's effectiveness in driving gene expression during the**stationary** phase. It was evident that under the regulation of RBS2, the P3.1 promoter drives a
higher level of expression. Based on our current data, P3.1 performs well even with RBS1, suggesting that a
stronger RBS may not be necessary for optimal system performance. However, further experiments with our
actual
eTEV production system are needed to determine which RBS provides the best regulation.

![](https://static.igem.wiki/teams/5299/myrto/wet-lab-14-1.png)

### Figure: Production of eTEV regulated by P3.1

## 3rd Engineering Cycle: Synthesis of the dsRNA in vitro and assessment of its silencing efficacy

Project Thaelia was developed to safeguard olive trees against *V.**dahliae*,
hence during the
engineering process our main objective was to find the best way to achieve it. It was made clear that one of
the most important aspects of our design is the successful activation of *V.* *dahliae’s* RNAi
mechanism. To achieve that, three conditions should be met:

The dsRNA should be produced in **sufficient quantities** to ensure its
availability for uptake
by the fungal cells

The fungal cells must possess the ability to efficiently **internalize the
OMVs** that carry the
dsRNA molecules

The internalized dsRNA must effectively **induce gene silencing** within the
fungus, leading to
a targeted suppression of essential genes.

While the first condition is multifactorial and difficult to prove, we made the first steps
by examining
basic components of our system. The second condition has already been validated in the literature [18],
leaving the efficiency of gene silencing as the main focus—an exciting and open area for exploration in our
design. We consulted with PhD candidate Afrodite Katsaouni, who has hands-on experience in RNAi experiments,
and Prof. Kalliopi Papadopoulou , expert in plant biotechnology, and they suggested evaluating gene
silencing
*in vitro,* as it is a fast and informative first level of evaluation.

### 1st iteration: Evaluating the effects of dsRNA on V. dahliae

**Design**

Our first task was to identify the ideal target genes for silencing that play a critical
role
in fungal
growth or virulence. Through extensive literature review we documented genes of *V.**dahliae*involved in various essential functions. We engaged with experts PhD
candidate
Afrodite Katsaouni and Dr. Athanasios Dalakouras, both of whom have worked with RNAi and *V.**dahliae**,* and identified three promising genes: Regulator of G signaling 1 (RGS1),
ADP/ATP Carrier (*AAC)* and Thiamine transport protein (*THI20).* To validate our selection we
consulted with Prof. Aliki Tzima and Prof. Kalliopi Papadopoulou, experts in plant and fungal biology.They
confirmed our choice, noting that in literature deletion of these genes exhibit pronounced phenotypic
effects,
suggesting that they influence both growth and virulence. [19,20,21].

**BLAST analysis**

Before finalizing our gene selections, we needed to ensure that targeting them would not
adversely affect
other microorganisms, plants, or humans, nor compromise biodiversity. For this purpose, we performed a blast
analysis, looking for homology between these three fungal genes in *V.* *dahliae* and human,
olive or other microorganisms’ genes.

While some regions of the genes showed homology with genes from other fungal species, no
homology was
detected with olive tree or human genes, suggesting that targeting them wouldn’t pose a human health risk.
At
the beginning of our journey, our main focus was to identify sequences of the AAC, RGS1 and THI20 genes that
lead to efficient silencing and hinder Verticilllium’s growth. We did not prioritize designing dsRNAs that
avoid homology with other organisms but one of our future goals is to improve our system by targeting
sequences with higher species-specificity.

**Target sequences selection**

After selecting our target genes, the next step was to choose what gene regions we would
target. We began by
searching in the literature for gene regions that were successful targets for RNAi. We also talked with our
PI, Prof. Antonis Giakountis, expert in long non-coding mediated transcriptional regulation, who informed us
that depending on the RNAi mechanism of the fungus, different gene regions could be more effective targets.
If
the RNAi mechanism leads to mRNA degradation, a better target would be the 3' UTR, while if it prevents
translation, the 5' UTR would be more effective. Since it’s not clear how *V.* *dahliae*’s
RNAi
mechanism works, we decided to target regions confirmed by literature as successful targets for gene
silencing
[22,23].

Moreover, we consulted with Dr. Athanasios Dalakouras to determine a length that would both
fit within the
OMVs and be effective for RNAi and we concluded that 200 bp dsRNA molecules would be the ideal choice. So,
after taking in consideration the optimal length and the sequences we needed to target, we proceeded on
producing the dsRNA.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng24.png)

### Figure 24: Target gene regions selected for dsRNA sequence design (1:RGS1, 2:AAC, 3:THI20)

**Primer design**

To produce the dsRNAs targeting these regions, we needed to design primers with T7 promoter
overhangs, as we
would be using T7 polymerase for their *in vitro* synthesis. In our design our bacteria will produce
dsRNA 200 bp long, so we tailored our primers to amplify a region of this size. Additionally, we designed
primers for RT-qPCR to assess gene expression levels. We designed our primers using the Geneious software
with
the guidance of PhD candidate Dimitris Rallis who provided us with useful tips on how to design primer pairs
suitable for our experiments.

**Build**

**Synthesis of dsRNA Sequences**

To evaluate whether we can induce gene silencing in *Verticillium* *dahliae*
with our designed
dsRNAs, we needed to apply these molecules to developing fungal hyphae. The first task was to generate the
DNA
template required for *in vitro* transcription of the dsRNA sequences.

To achieve this, we first cultivated V. dahliae on Potato Dextrose Agar (PDA) to obtain
hyphal tissue. From
the harvested tissue, we extracted total RNA, which was used to synthesize cDNA, providing the essential
starting material for the following steps.

As illustrated in Figure 25, we used primers containing T7 promoter overhangs to amplify
the
target gene
sequences (Fig.25-1) and incorporate the T7 promoter sequence necessary for *in vitro* transcription
(Fig.25-2). The initial PCR product was then used as a template for a second round of PCR amplification
(Re-PCR) to increase the yield of the desired DNA fragments (Fig.25-3).

After confirming the correct PCR amplification through gel electrophoresis, the DNA bands
corresponding to
our target sequences were extracted from the gel (Fig.25-4). With the purified DNA template in hand, we
proceeded to *in vitro* transcription using T7 RNA polymerase to synthesize the dsRNA molecules
(Fig.25-5,6). This dsRNA was ultimately used for gene silencing experiments in *V.* *dahliae*.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng25a.png)
![](https://static.igem.wiki/teams/5299/league-of-legends/end25b.png)

### Figure 25: Steps for dsRNA production. 1-2) PCR with T7 promoter overhang primers to incorporate T7 sequence into the sequence of interest, 3) Re-PCR to increase yield, 4) Gel electrophoresis and extraction, 5-6) In vitro transcription for dsRNA synthesis.

**Test**

**Monitoring fungal growth**

Next, we proceeded to examine whether these dsRNA molecules effectively silenced these key
genes, operating
under the hypothesis that successful gene silencing could have a visible effect on fungal growth and gene
expression. We cultivated *V.* *dahliae* in Potato Dextrose Broth (PDB) and isolated its
conidia, which are asexual reproductive structures that generate new hyphae [24].
Then, we prepared a 96-well
plate, as illustrated in Figure 26, by adding PDB, fungal conidia and dsRNA to each well. The plate was
incubated for 10 days, during which we measured OD595 every 24 hours to monitor the growth of *V.**dahliae*.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng26.png)

### Figure 26: 96 well plate layout. Row A serves as the blank control, containing only PDB. Row B contains PDB and conidia, acting as our experimental control. Rows C, D, and E are designated for the dsRNA treatment, each containing PDB, conidia, and dsRNA targeting a specific gene.

**Analysis of Gene Expression Levels**

In order to have a more holistic view of the effects of the dsRNAs to the fungus we
performed
an RT-qPCR to
assess expression levels of the target genes. We expected that the dsRNA would activate the fungal RNAi
machinery leading to the silencing of the targeted genes and thus causing reduced expression levels.

We pooled the hyphae from six wells in each row into a single tube, resulting in two
technical replicates
for each treatment. We then extracted RNA from these samples and synthesized cDNA. Finally, we performed
RT-qPCR on one of the technical replicates from each row to assess expression levels of RGS1, AAC and THI20.

**Learn**

The OD595 measurements (Fig. 27,28) indicated that there was little variation in fungal
growth across all
conditions. This limited response could be attributed to several factors:

• Given the small volume of dsRNA used, inaccuracies in pipetting may have resulted in
insufficient dsRNA being
added

• It may be necessary to optimize the dsRNA concentration to achieve a more pronounced effect

• The dsRNA sequences may not have been ideal for activating the RNAi mechanism of the fungus

• These gene regions may not be ideal targets for impacting fungal growth

• Silencing these genes may have no effect in fungal growth but in virulence

• This experiment may not be suitable to observe the effects of gene silencing in the fungus.
In this case we
would also need *in planta* experiments to acquire more prominent results.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng27.png)

### Figure 27: Monitoring of fungal growth in the span of 10 days. The graph illustrates fungal growth in every condition.

![](https://static.igem.wiki/teams/5299/league-of-legends/eng28.png)

### Figure 28: Growth curves for *V.* *dahliae*in each treatment. From the slope we can see that in all conditions the fungus develops normally.

To have a glimpse of what’s happening inside the fungus, we attempted to assess gene
expression with RT-qPCR.
Unfortunately, due to our inexperience and the sensitivity of the technique we weren’t able to produce
reliable data, while the limited time prevented us from repeating the experiment. We observed variability in
Cq values between our technical replicates, which could be the result from pipetting errors or even from
degradation of RNA before cDNA synthesis. In the future we want to repeat the experiment to obtain more
accurate results.

To be able to draw conclusions about the effect of these dsRNA sequences we would need to
repeat both the
plate reader assay and the RT-qPCR. Due to limited time we couldn’t repeat these experiments but we hope
future iGEM teams will further develop this project.

### Proposed redesign

While we couldn’t see the genes’ expression levels due to unreliable data, we also didn’t
see
any significant
difference in fungal growth. To address this in the future we will utilize bioinformatic tools to predict
siRNAs with a high likelihood of effective gene silencing. We will design 200 bp dsRNA sequences that
incorporate these predicted siRNAs to increase the chances of successful gene silencing. Such tools are
siDirect and BLOCK-iT™ RNAi Designer from Thermo Fisher Scientific. Both tools are suitable to design siRNAs
[25] for gene silencing, since they use algorithms to check for knockdown
effectiveness and thermodynamic
stability. For the efficacy they use empirically based rules that rely on the fact that the knockdown
efficiencies of siRNAs are dependent on their sequences [26]. Using tools like
that will allow us to test
sequences that have theoretically better chances for inducing gene silencing. We will also be able to
compare
our results taking us a step closer to understanding the RNAi mechanism of *V.**dahliae**.*

**Additional planned DBTLs**

The first cycle didn’t result in conclusive data, however, we identified flaws in our
design
and generated
new questions that necessitated further investigation.To address them, we discussed with Dr. Athanasios
Dalakouras, who made us realize the need to enhance our experimental approach. He recommended that we target
multiple genes simultaneously to determine whether a multifaceted attack on the fungus would have a more
pronounced effect on fungal growth and gene expression. Accordingly, we devised a plan for two additional
DBTL
cycles, which will progressively provide us with insights into the effects of dsRNA on *V.**dahliae*.

### 2nd iteration: Evaluating silencing when targeting two genes at a time

In this cycle we’ll examine the effect that dsRNA has on the fungus when targeting two
genes
at a time. Will
fungal growth be more affected than when targeting one gene? Would we observe any change in gene expression?

We will apply dsRNA to fungal conidia, targeting two genes simultaneously and by measuring
OD595 and
performing RT-qPCR we would evaluate its effects in hyphal growth and gene expression. Then, we will compare
the results between the two cycles to determine whether the effects of dsRNA treatment are enhanced with
this
approach.

### 3rd iteration: Evaluating silencing when targeting all three genes at the same time

In this cycle, we will investigate the effect of dsRNA on the fungus by targeting all three
genes
simultaneously. Our objective again is to evaluate whether applying greater pressure to the fungus through
dsRNA treatment leads to a more significant impact on its growth and gene expression.

We will apply dsRNA to fungal conidia, targeting two genes simultaneously and by measuring
OD595 and
performing RT-qPCR we would evaluate its effects in hyphal growth and gene expression. Then, we will compare
the results with the two previous cycles to determine whether the effects of dsRNA treatment are enhanced
with
this approach.

## Conclusion

In conclusion, utilizing the D-B-T-L cycle throughout various aspects of our project has
proven to be highly
beneficial, allowing us to systematically explore and enhance each component. We gained valuable insights
into
the evaluation of the T7 polymerase system, the synthesis of dsRNA in vitro and its subsequent assessment
for
silencing efficacy. This
modular approach not only facilitated our focus on each individual element but also significantly improved
the
overall design and functionality of our final construct.

## References

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[24] Naito, Y., & Ui-Tei, K. (2012). SiRNA design software for
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*Frontiers in Genetics*, *3*, 102. doi:10.3389/fgene.2012.00102

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)
[Our Experimental Design](#design)

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)
[Experiments](#exp)

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)
[Materials](#mat)

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)
[Protocols](#protocols)

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)
[Lab Book](#lab)

![](https://static.igem.wiki/teams/5299/sidebar-icon.png)
[References](#ref)

## ~Our Experimental Design~

In our quest to provide a solution for V. dahliae and create THAELIA, our primary goal was to assess all the
different parts of the design experimentally, separately, in order to demonstrate the overall feasibility of
our design. We approached our system step by step to gain insights and improve our approach. While our
experiments provided insights into different aspects of our design, more experimental and biological repeats
are needed to fully characterize the system and ensure the reliability of our data. A larger number of repeats
would reduce variability and provide a clearer picture of how our system behaves under various conditions.
However, within the time constraints, we tried to gather as much data as possible. Our work provides a
starting point for **future studies to expand upon.**

| Experiment | Short Description |
| --- | --- |
| Experiment 1: Characterization of regulatory components of T7 polymerase production system and eTEV production device | We conducted plate reader assay using E. coli BL21 DE3 cells containing the T7 expression system, which share similar traits with P. putida cells (our Project Design chassis). Our aim was: 1) to identify the most suitable promoter for the T7 polymerase production system, 2) to evaluate various RBSs for the P3.1 stationary promoter. We measured sfGFP levels and OD600. |
| Experiment 2: Further characterization of BG37 promoter and evaluation of T7 polymerase system | We conducted experiments with E. coli DH5a cells, which do not contain the T7 polymerase system. Using these cells, we aimed to better characterize our BG37 promoter by testing different sources of carbon and backbones and measuring the differences observed. We also aimed to determine if our T7 polymerase production device functions as a level 2 construct. We measured sfGFP levels and OD600 |
| Experiment 3: Further characterization of BG37 promoter in our proposed chassis | We conducted experiments with P. putida KT2440, which is our Project Design chassis. Our goal was to further characterize the BG37 promoter by measuring the sfGFP levels and OD600 |
| Experiment 4: Evaluation of gene silencing in *Verticillium dahliae* | We conducted experiments in order to determine efficiency of our gene silencing system targeting *V. dahliae.* We produced dsRNA in vitro to treat fungal cultures. We measured OD595 and used RT-qPCR to compare fungal growth and gene expression levels. |

## ~Experiments~

### Experiment 1: Characterization of regulatory components of T7 polymerase production system and eTEV production device

Our system is built on two main functions: 1) dsRNA production via T7 polymerase, and 2) OMV generation
through TEV protease activity. To ensure optimal performance, our first objective was to test and identify the
most suitable *promoters* for regulating T7 polymerase and TEV production. Given that our design
requires
distinct regulation across growth phases, we aimed to *find the best promoters*  to drive T7 polymerase
expression during the exponential phase and eTEV production in the stationary phase.

![](https://static.igem.wiki/teams/5299/wetter1.png)

### Figure 1: The regulation of our system: 1) T7 polymerase is produced during the exponential phase, leading to dsRNA production, 2) eTEV is produced during the stationary phase, leading to OMVs production

Read More:Experiment 1

### Experiment 2: Further characterization of BG37 promoter and evaluation of T7 polymerase system

Our next goal was to further characterize the BG37 promoter to ensure that it is activated specifically
during the exponential phase. In our design, the BG37 promoter regulates T7 production, which drives the
production of dsRNA. To evaluate its functionality, we first tested the BG37 promoter in a Level omega
construct, where it controls T7 production, with an sfGFP reporter gene positioned downstream of the T7
promoter. This step was important, as testing in a Level 2 construct is as close to the proposed design as
possible for a first evaluation of the functionality of the promoter.

![](https://static.igem.wiki/teams/5299/wetters3.png)

### Figure 2: BG37 promoter is responsible for T7 polymerase production

Additionally, understanding that various growth conditions can significantly influence promoter activity, we
focused on testing the BG37 promoter under different carbon sources. The choice of carbon source can affect
cellular metabolism, growth rates, and energy availability, all of which may impact the promoter’s
functionality [2]. To evaluate this, we used an sfGFP reporter gene positioned
downstream of the BG37 promoter
to assess how different carbon sources, specifically citrate and glucose, influence its performance [1].
Moreover, since various plasmid backbones have different characteristics (e.g. origin of replication) that are
needed to work in different chassis, we compared the pSEVA23g19g1 vector with the Golden Braid pDGB3a1 vector,
starting with E. coli DH5α as an intermediate step before transitioning to P. putida. Simultaneously, we
validated that its function is consistent across different genetic backbones, which reduces the likelihood of
vector-specific behavior or artifacts [2].

Read More:Experiment 2

### Experiment 3: Further characterization of BG37 in our design chassis: P. putida

We chose to further characterize the BG37 promoter in P. putida, our proposed chassis, to verify its
autoinducible properties during the exponential phase. By using sfGFP as a reporter gene and comparing BG37
with well-characterized promoters: J23119 (Anderson constitutive promoter) and OsmY (stationary phase-specific
promoter) , we aimed to assess its strength and activation across different growth stages in P. putida. This
allows us to determine how effectively BG37 performs in our system during key phases of cell growth [3].

Read More:Experiment 3

### Experiment 4: Evaluation of gene silencing in vitro

While designing our project and reading about RNAi our interest was heightened. Could we actually observe the
impact of dsRNA on the fungus? Fascinated by this possibility, we designed this experiment with the aim to
assess the effect that dsRNA has on the fungus. To be able to do that though we first have to create the DNA
template needed for the in vitro transcription for the production of dsRNA and cultivate V. dahliae. To obtain
our results we measured OD595 and performed an RT-qPCR reaction. In the Read More button below you can see in
more detail the process of this experiment and our results. All protocols and photos are also provided on this
page.

The other part of our system that we wanted to test was the effect of dsRNA on V. dahliae. We designed this
experiment to investigate how dsRNA influences both fungal growth and expression levels of the target genes.
We chose genes that according to literature are necessary for the correct development and virulence of the
fungus. To determine whether dsRNA targeting these genes impacts fungal growth, we measured OD595, while gene
expression levels were assessed using RT-qPCR.

Read More:Experiment 4

## ~Materials~

Read More: Bacterial Strains

| Strains | Genotype |
| --- | --- |
| E. coli |  |
| E. coli  DH5a | Δ(argF-lac)169, φ80dlacZ58(M15), ΔphoA8, glnX44(AS), deoR481, rfbC1, gyrA96(NalR), recA1, endA1, thiE1, hsdR17 |
| E. coli  BL21 DE3 | F-, ompT, gal, dcm. Ion, hsdSB(rB-mB-), λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]), [malB+]K-12(λS), pLysS[T7p20 orip15A](CmR) |
| E. coli  HB101 | F–mcrB mrr hsdS20(rB–mB–) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) gln V44 λ– |
| E. coli  DH5aλpir | λpir phage lysogen of DH5α |
| E. coli  PIR2 | F-Δlac169 rpoS(Am) robA1 creC510 hsdR514 endA recA1 uidA(ΔMlui)::pir |
| P. putida |  |
| P. putida KT2440 | Wild type strain derived from P. putida  mt-2 cured of the pWW0 plasmid |
| P. putida  Δrnc | Δrnc |

Read More: Vectors

| Vectors | Description |
| --- | --- |
| pTnS-2 | ApR, ori R6K, TnSABC+D operon |
| pRK600 | CmR, ori ColE1, tra+mob+ of RK2 |
| pTn7-M | KmR, GmR, ori R6K, Tn7L and Tn7R extremes, standard multiple cloning site |
| pUPD2 | CamR, ori pMB1, lacZ gene |
| pDGB3alpha1 | KanR, ori pBR322, lacZ gene |
| pDGB3alpha2 | KanR, ori pBR322, lacZ gene |
| pDGB3omega1 | SpecR, ori pBR322, lacZ gene |
| pSEVA23g19[g1] | KanR, ori pBRR1, lacZ gene |

Read More:Reagents, Antiobiotics and Media

|  |  |
| --- | --- |
| Antibiotics | Kanamycin, Chloramphenicol, Ampicillin, Gentamycin, Spectinomycin. |
| Reagents | Reagents for isolation of plasmid DNA from bacteria: “NucleoSpin® plasmid” kit from Macherey-Nagel |
| Nutrient Media for Bacterial Cultures | LB BROTH Luria Broth (LB) Broth was used in liquid form, dissolved in dH2O and sterilized, for liquid bacterial cultures. The desired antibiotic should also be added. LB AGAR Luria Broth (LB) Agar was dissolved in dH2O, sterilized and added in Petri dishes for bacterial cultures. The desired antibiotic should also be added before its solidification. M9 MEDIUM Prepared according to the recipe provided in this page. |
| Enzymes | For the Restriction Enzyme Digestion and Ligation cloning experiments, the following enzymes were used: BsaI-HF, BsmBI, and T4 Ligase. For the Polymerase Chain Reaction, KAPA HiFi DNA Polymerase and Kapa SYBR Green 2x Mix from Kapa Biosystems were used according to manufacturers’ instructions For the diagnostic digestion experiments, the following enzymes were used: NotI, EcoRV-HF, BaeGI, XbaI For DNase I treatment, DNase I from ThermoscientificTM and rDNase I from TakaraTM were used according to manufacturers’ instructions For in vitro dsRNA production Thermo fisherTM T7 polymerase was used according to manufacturer's instructions |
| DNA electrophoresis in agarose gel | TAE 50X ELECTROPHORESIS BUFFER The DNA electrophoresis buffer with a final volume of 1L contains 100 ml EDTA (0.5M), 242g Tris Base, 57.1 liquid acetic acid and ddH2O to final volume. The working solution is diluted from 50x to 1x. NEB Gel Loading Dye, Purple 6X NEB 1kb Plus DNA ladder molecular weight control Aqueous solution of ethidium bromide (10mg/ml) |
| Kits | “SuperscriptTM II RT kit” was used for cDNA synthesis “NucleoTrap Kit” from Macherey-NagelTM was used for gel extraction |

## ~Protocols~

### General Recipes

Read More : LB (Lysogeny broth) Medium Recipe

Read More : M9 Medium Recipe

Read More : Glycerol Stock

Read More : Working concentrations and stock
solutions

Read More : X- Gal preparation

### General Protocols

Read More : Plasmid isolation- Minipreps

Read More : One-pot Dig-Lig reaction

Read More : Restriction Digestion of DNA

Read More : 96 well plate preparation

Read More : Plasmid recovery from filtered paper

### Working with E. coli

Read More : Heat-shock Transformation Protocol for
E.coli

Read More : Inoue Chemical Competent Cells for E.coli

### Working with P. putida

Read More : Electrocompetent Cells for P. putida

### Evaluation of gene silencing

Read More : RNA Extraction

Read More : Phenol-Chloroform extraction

Read More : V. dahliae
conidia isolation + measurement

## ~Lab Book~

Throughout our journey, we made sure to keep a detailed lab book to reference our past
experiments and tweak any steps we deemed important. When conducting a protocol, each member of the
Experimental Wetlab Team took note of their actions. This facilitated the improvement of our experiments and
ensured cohesion in our practices, as each member could reference the notes of another member at any time.

Read More:Lab Book

## References

[1] Blázquez, B., et all. (2023). Golden Standard: a
complete standard, portable,
and interoperative MoClo
tool for model and non-model proteobacteria. In Nucleic Acids Research (Vol. 51, Issue 19, pp. e98–e98).
Oxford University Press (OUP). <https://doi.org/10.1093/nar/gkad758>

[2] Zobel, S., Benedetti, I., Eisenbach, L., de
Lorenzo, V., Wierckx, N., &
Blank, L. M. (2015). Tn7-Based
Device for Calibrated Heterologous Gene Expression in Pseudomonas putida. In ACS Synthetic Biology (Vol. 4,
Issue 12, pp. 1341–1351). American Chemical Society (ACS). <https://doi.org/10.1021/acssynbio.5b00058>

[3] Esteban Martínez-García, Angel Goñi-Moreno, Bryan
Bartley, James McLaughlin,
Lucas Sánchez-Sampedro,
Héctor Pascual del Pozo, Clara Prieto Hernández, Ada Serena Marletta, Davide De Lucrezia, Guzmán
Sánchez-Fernández, Sofía Fraile, Víctor de Lorenzo, SEVA 3.0: an update of the Standard European Vector
Architecture for enabling portability of genetic constructs among diverse bacterial hosts, Nucleic Acids
Research, Volume 48, Issue D1, 08 January 2020, Pages D1164–D1170,<https://doi.org/10.1093/nar/gkz1024>

[4] Pédelacq, J.-D., Cabantous, S., Tran, T.,
Terwilliger, T. C., & Waldo, G. S.
(2006). Engineering and
characterization of a superfolder green fluorescent protein. Nature Biotechnology, 24(1), 79–88.
doi:10.1038/nbt1172

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