iGEM 2023 — Team wageningenur

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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![cherry image](https://static.igem.wiki/teams/4663/wiki/icons-logos/cherry-1.png)
![hero bacteria](https://static.igem.wiki/teams/4663/wiki/icons-logos/bacterium-hero.png)

## May

Week 1 - 15th of May to 21st of May

* Defined subprojects, got introduced to labs.

  Week 2 - 22nd of May to 28th of May

Wet Lab

* Amplified fragments of promoters believed to be effective in cold temperatures from *Pseudomonas fluorescens* SBW25.
* Obtained the *Pseudomonas fluorescens* etHAn strain with a functional type III secretion system from the Department of Plant and Environmental sciences of the University of Copenhagen in Denmark.

Dry Lab

* Learned how to code in NetLogo for the agent-based model.
* First trial of generating output of florigen protein change from the plant model neural network.

## June

Week 3 - 29th of May to 4th of June

Wet Lab

* Linearized pSEVA 64 plasmids and ordered a GFP fragment and type III secretion signal tag.
* Root exudates and plasmids containing root exudate sensitive parts were ordered.
* A list of nine interesting antiflorigen candidates was generated.

Dry Lab

* An ordinary differential equation (ODE) model of *Escherichia coli* was converted from MATLAB to Python and ran successfully for the first time.

  Week 4 - 5th of June to 11th of June

Wet Lab

* Performed Golden-Gate assemblies of the cold-responding promoters into pSEVA 23 sfGFP plasmid backbone.
* Performed Golden-Gate assemblies of the pSEVA 64 plasmid with a GFP insert. One with and one without a type III secretion signal.
* Primers for putting root exudate-sensitive parts in pSEVA plasmids were designed and ordered. Stock solutions of root exudates were made.
* The first attempt to transform *P. fluorescens* SBW25 with a plasmid.
* Designed primers and antiflorigen inserts arrived and were put into pET-16b plasmids using restriction digestion and ligation. The resulting plasmids were transformed into *E. coli* Dh5α strains.

Dry Lab

* Validated the Python ODE model with the MATLAB ODE model.

  Week 5 - 12th of June to 18th of June

Wet Lab

* Improve *P. fluorescens* etHAn transformants with a spacer between the ribosome binding site and the start codon of sfGFP for enhanced protein production.
* Recieved the plasmids containing root exudate-sensitive parts from which individual gene fragments were amplified and purified.
* The first successful colony PCR of an *E. coli* Dh5α strain with a constructed plasmid containing an antiflorigen gene was obtained.

  Week 6 - 19th of June to 25th of June

Wet Lab

* Amplification of homology arms for the barcode construct from the *P. fluorescens* SBW25 genome.
* First plant growth experiment with *Arabidopsis thaliana* Col-0 on different growth media.
* Performed growth and toxicity assays for *P. fluorescens SBW25* on rhamnose and cuminic acid.

Dry Lab

* Added a temperature response to the Python ODE model.
* Added a temperature dependency for florigen production to the plant model.

  Week 7 - 26th of June to 2nd of July

Wet Lab

* Multiple transformation methods were attempted from which one that works was found for *P. fluorescens* SBW25.
* Transformed *P. fluorescens* SBW25 with ‘cold’-promoter plasmids.
* Transformed *P. fluorescens* etHAn with GFP and type III secretion signal containing plasmids.
* Isolation of the pCym promoter fragment from its host plasmid.
* Performed a Golden-Gate assembly of the *dapB* overexpression gene in a pSEVAb23 plasmid.

Dry Lab

* The first version of the agent-based model (ABM) for microbial communities was built and tested.
* The influence of different temperatures on simulated florigen concentrations was tested and the temperature dependency was tuned to reflect results from literature.

## July

Week 8 - 3rd of July to 9th of July

Wet Lab

* Plant growth experiments of *A. thaliana* Col-0 in Fahrraeus slides.
* All root exudate fragments were rearranged and inserted in their respective pSB1C3 plasmids compatible with BioBrick assembly. These plasmids were transformed in *E. coli* Dh5α and sequenced.

Dry Lab

* Redesigned and debugged the ABM for microbial communities.

  Week 9 - 10th of July to 16th of July

Wet Lab

* The transformed *P. fluorescens* SBW25 with the ‘cold’-promoter plasmids were confirmed via sequencing.
* First colonisation experiment of *P. fluorescens* SBW25 on *A. thaliana* Col-0 roots.
* Most of the root exudate sensing modules that were cloned with a GFP-reporter gene in SEVA plasmids and transformed in *E. coli* Dh5α were confirmed via sequencing.
* Seven of the nine antiflorigen plasmids now showed successful colony PCR results

Dry Lab

* Obtained the weather data of 2015-2022 from a weather station in Wageningen.

  Week 10 - 17th of July to 23rd of July

Wet Lab

* Performed a Gibson assembly on the barcode fragments.
* Performed a plate reader experiment to get data on the secretion of sfGFP from the *P. fluorescens* etHAn strain.
* Most of the roots exudate sensing plasmids transformed into *P. fluorescens* SBW25 were confirmed via sequencing.
* The salicylic acid sensing module in *P. fluorescens* SBW25 was characterized and proven responsive.
* Seven of the nine antiflorigen plasmids were confirmed via sequencing and the plans to keep on trying to create the two remaining plasmids were scrapped.
* Transformed the first *E. coli* BL21 production strains with the antiflorigen plasmids.

Dry Lab

* Ribosome concentration for different temperatures validated between the ODE Python model and in vivo measurements.

  Week 11 - 24th of July to 30th of July

Wet Lab

* Characterized colonisation behaviour of *P. fluorescens* SBW25 under the fluorescence microscope.
* The arabinose sensing module in *P. fluorescens* SBW25 was characterised and proven responsive.
* The creation of seven different antiflorigen producing *E. coli* BL21 strains were confirmed via colony PCR and the first antiflorigen production and purification run was tried.

Dry Lab

* The ccdA-ccdB toxin-antitoxin system of the ODE Python model validated against the measurements of the previous Wageningen iGEM team Colourectal.

## August

Week 12 - 31st of July to 6th of August

Wet Lab

* Transformed the barcode construct into *E. coli* Dh5α.
* Creation of *P. fluorescens* etHAn double transformation with the sfGFP secretable and RFP non-secretable counterstaining.
* Transformation of *P. fluorescens* SBW25 with the pGNW plasmid.
* The cuminic acid sensing module in *P. fluorescens* SBW25 was characterised and proven responsive.
* Performed a Golden-Gate assembly of the rhamnose inducible system into a pSEVAb23 plasmid.

Dry Lab

* Tested the ABM and started to collect data for data analysis.

  Week 13 - 7th of August to 13th of August

Wet Lab

* The barcode construct assembly was confirmed via sequencing.
* First try of working with a vibratome to visualise sfGFP injection into *A. thaliana* Col-0 roots.
* The naringenin sensing module in *P. fluorescens* SBW25 was characterised and proven responsive.
* Introduction of the previously characterised RNA toehold switch in *P. fluorescens* SBW25 and proven to be functional.
* Produced and purified the TFL1 and ATC/CEN antiflorigens in *E. coli* BL21 production cultures.

Wet Lab

* Included growth rate and substrate consumption in the ODE Python model.

  Week 14 - 14th of August to 20th of August

Wet Lab

* Cross-sectional detection of sfGFP inside *A. thaliana* Col-0 using a vibratome.
* Produced and purified the CsAFT and PsTFL1 antiflorigens in *E. coli* BL21 production cultures.

Dry Lab

* Added real life temperature and day length in the plant model to simulate daily florigen concentrations and flowering time.

  Week 15 - 21st of August to 27th of August

Wet Lab

* Performed a tri-parental conjugation of the barcode construct into *P. fluorescens SBW25*.
* First try of sfGFP production induction by *A. thaliana* root exudates.
* Produced and purified the BvFT1 and CEN-like 1 antiflorigens in *E. coli* BL21 production cultures.

Dry Lab

* The plant model is able to generate florigen concentrations and the flowering time for the 2000 days between 1st of January 2015 and 31st of December 2022.

## September

Week 16 - 28th of August to 3rd of September

Wet Lab

* Performed a buffer exchange to get the antiflorigens from their elution buffer into PBS buffer.
* Plate reader assay of incubation of ‘cold’-promoter-GFP bearing *P. fluorescens* SBW25 strains in different temperatures.

Dry Lab

* Introduced the rhamnose and cuminic acid sensing systems in the ODE Python model for different temperatures.
* Constructed a neural network to predict daily florigen concentrations, flowering time and minimum temperature.

  Week 17 - 4th of September to 10th of September

Wet Lab

* Confirmation of recombination of the barcode construct in *P. fluorescens* SBW25.
* The quorum sensing module from *Vibrio fischeri* responsive to AHL was introduced in *P. fluorescens* SBW25.

Dry Lab

* Built and tested the cell level ABM.
* Tested multiple data transformation techniques for the plant model to predict future florigen concentrations, flowering time and minimum temperature.

  Week 18 - 11th of September to 17th of September

Wet Lab

* The introduced quorum sensing module was characterised in *P. fluorescens* SBW25 for a wide range of AHL concentrations.

Dry Lab

* Data collecting and analysis of the cell level ABM.

  Week 19 - 18th of September to 24th of September

Wet Lab

* Assessed the primer sets for the LAMP assay.
* Plasmids containing the RNA toehold switch combination of induction by the quorum sensing module and the salicylic acid sensing module were introduced in *P. fluorescens* SBW25.

Dry Lab

* Collected data and performed data analysis on the interaction of the microbial community ABM model and the cell level ABM model.

  Week 20 - 25th of September to 1st of October

Wet Lab

* Started the in vivo *A. thaliana* Col-0 experiment to assess the efficacy of the heterologously produced antiflorigens on flowering delay.
* Characterisation of the introduced RNA toehold switch induced by AHL and salicylic acid in *P. fluorescens*.

Dry Lab

* Constructed a separate neural network to predict future temperature from current temperature time series.

## October

Week 21 - 2nd of October to 7th of October

Dry Lab

* Combined the temperature prediction neural network with the plant model neural network.

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{% block title %}Engineering{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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![safety header](https://static.igem.wiki/teams/4663/wiki/headers-of-pages/engineering-new.png)
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Index

[##### **Index**](#top)
[![](https://static.igem.wiki/teams/4663/wiki/icons-logos/engineering/dapb-knock-out-db-to.png)](#DBTL1)
[![](https://static.igem.wiki/teams/4663/wiki/icons-logos/engineering/microscopy.png)](#DBTL2)
[![](https://static.igem.wiki/teams/4663/wiki/icons-logos/background/references.png)](#references)

**Introduction**

For nearly half a year, we engaged in research to explore the capacity of Pseudomonas fluorescens in delivering antiflorigens to plant roots to regulate plant flowering. Our team collectively advanced our individual subprojects by undergoing multiple cycles of **Design-Build-Test-Learn** (DBTL). In the following section, we highlight two DBTL cycles we were particularly fond of. One highlights the DBTL cycle of **a fluorescence microscopy experiment**, while the other highlights the DBTL cycle of our ***dapB* knockout procedure** which is connected to the biosafety part of our project.

“Properly speaking, such work is never finished; one must declare it so when, according to time and circumstances, one has done one’s best.” — Johann Wolfgang von Goethe

![dbtl image](https://static.igem.wiki/teams/4663/wiki/icons-logos/dbtl-cycle.png)

## Fluorescence microscopy

Our project aims to inject antiflorigens into the roots of fruit trees using *Pseudomonas fluroescens*. For this reason, one of the tasks of our project was to investigate the capability of *P. fluorescens* to attach to the roots of the model plant *Arabidopsis thaliana*, and release a superfolder Green Fluorescent Protein (GFP) equipped with a Type-III Secretion System-specific secretion signal into the roots.

**Round 1: Preliminary studies on colonisation behaviour of *P. fluorescens***

**Design 1:**

Our experimental design includes the following steps:

1. Growth of *A. thaliana* in Fahraeus slides
2. Addition of GFP-secreting *P. fluorescens* to the plants.
3. Fluorescence microscopy on living plants and cross-sectioned roots.

**Build 1:**

We want to study the potential of *P. fluorescens* to **inject GFP into roots** of *A. thaliana*. To this end, GFP with a T3SS specific secretion signal was introduced to pSEVA64 vector via Golden Gate cloning and transformed into *P. fluorescens* to create the plasmid shown in Figure 1. RFP is expressed as a second protein under the control of the same promoter, however without a secretion signal. pSEVA64 was shown effective in *Pseudomonas putida*, a close relative to *P. fluorescens*, and was therefore a promising candidate for integration in *P. fluorescens* [1]. The secretion sequence of the T3SS was adopted from as Jansen et al. (2022) [2].

1. Negative control: GFP is produced but cannot be secreted due to the lack of a T3SS secretion signal.
2. Positive control: *gfp* was placed under the control of a functional transcriptional promoter + RBS combination sourced from a previously published [3].
3. Test sample: *gfp* is expressed under the control of a strong promoter (Part: BBa\_J23100), in the expectation that this leads to high protein secretion into plants.

![pseupomona package image](https://static.igem.wiki/teams/4663/wiki/dbtl/dbtl1.png)

**Figure 1**: Plasmid map of pSEVA64 for integration into*P. fluorescens* (test sample). GFP is coupled T3SS-secretion signal under the control of the strong constitutive promoter BBa:J23100. RFP is expressed to counterstain the bacteria, however, RFP has no secretion signal and can therefore not be secreted.

**Test 1:**

*A. thaliana* was grown on Fahrraeus slides in 1/2 MS medium (see [plant growth protocol](https://2023.igem.wiki/wageningenur/protocols)). After 6 days, the roots were approximately 2 cm long and had developed their main structures, including the differentiation and elongation zones, as well as lateral roots and root hairs, which was enough to study the basic attachment behaviour of our bacterium.

We conducted a 7-day experiment to **investigate bacterial attachment** on ten different roots. Inoculation was performed by adding the bacteria samples to the roots, incubating for 1 hour, and then washing the roots to remove unattached bacteria. **On the initial day**, following inoculation, the **bacteria were uniformly distributed across the entire root** (Figure 1B).

However, **in the subsequent days**, it became evident that they exhibited a higher propensity for **colonisation of the basal zone of the root**, which corresponds to the differentiation zone near the point where the stem initiates, with only a minimal presence of bacteria in the apical region of the root (Figure 1C-D). An overview of different root regions can be found in Figure 1A.

The analysis of the cross-section was hindered by the inability of the vibratome to section the unfixed plant tissue. The tissue was too soft, and bent away from the knife, making the sectioning impossible.

![pseupomona package image](https://static.igem.wiki/teams/4663/wiki/dbtl/dbtl2.png)

**Figure 2:** Overview of tissues and root regions in the root system ofA. thaliana and microscopic pictures ofP. fluroescens attached to root ofA. thaliana (100x magnification). A. Overview picture; B:*P. fluorescens* is evenly attached to the root of A. thaliana (day 0); C: Fluorescence microscopic and merged channel picture of*P. fluorescens* attached to the elongation zone ofA. thaliana (day 2); D: Increasing colonialisation towards root-basal region, with a decreased number of bacteria at apical site (day 1).

**Learn 1:**

First, we gained the knowledge that **bacteria mainly colonise the basal zone of the roots** where the roots are mature and closer to the surface. This observation cross validates our [agent-based model](https://2023.igem.wiki/wageningenur/model) which posits that most bacteria colonise just beneath the soil surface.

Second, we learned that we could not detect GFP secretion into the plant by just looking on the surface of the plant. To get insights of how GFP is potentially secreted into the plant, **cross-sections were needed** as described below in the Round 2.

Third, from discussions with the plant microscopy expert [Norbert de Ruijter](https://2023.igem.wiki/wageningenur/attributions), we learned that fixation of the plant material might lead to the fluorescence quenching of GFP. However, we learned that is inevitable to make proper cross-sections of the plant roots with the vibratome.

**Round 2: Protein secretion detection on root-cross sections**

**Design 2:**

In this iteration, we **improved our design** with a fixation step and implemented learnings from round 1. Roots were **fixated** for 1 h at room temperature in 4% paraformaldehyde, making the root stiffer and allowing proper cross-sectioning of the roots. Assuming that all bacteria secrete similar amounts of GFP into the plant, we anticipated that in densely colonised parts of the root more GFP is secreted into the roots compared to rarely colonised parts such as the root tip.

**Build 2:**

**No further bacteria needed to be built** for this round. The used bacteria include the above mentioned GFP-producing negative, positive, and test samples.

**Test 2:**

**After fixation**, roots were cut at the beginning and end of the differentiation zone (Figure 3). Cut roots were embedded in the improved embedding medium (6% agarose gel instead of 4%). The embedded roots were directly sectioned on the vibratome (25-50 µm thick sections) and analysed on a **fluorescence microscope**.

This time, **utilizable cross-sections** of the root could be made. Under the fluorescence microscope, we detected that the thickness of 50 µm was slightly too thick, which complicated the detection of internal root structures. **Reduction to 25 µm** allowed an optimal visualisation of main structures.

Finally, we could **visualise attached bacteria** and investigate whether they could secrete visible amounts of GFP into the plant. As it can be observed in Figure 4B, GFP-producing bacteria can be visualised attaching to the epidermis. Further, green fluorescent structures are visible in the core of the plant. No evident accumulation of green fluorescent particles is observed in the epidermis. The negative control shows a cross section of an A. thaliana root that was not grown in the presence of bacteria. The negative control shows similar patterns of green fluorescence in the cell wall of the epidermis and the stele (Figure 4D).

![pseupomona package image](https://static.igem.wiki/teams/4663/wiki/dbtl/dbtl31.png)

**Figure 3:** Tissues and regions ofA. thaliana root system. For vibratome sectioning, roots were cut below the shoot and above the elongation zone, because most bacteria were detected in the intermediate zone.

![pseupomona package image](https://static.igem.wiki/teams/4663/wiki/dbtl/dbtl41.png)

**Figure 4:** Phase contrast-(left) and fluorescence microscopy (right) picture ofA. thaliana root cross sections (thickness: 25 µm) A+C: Phase contrast pictures, 20x magnification. Main structures of the root are visible such as root hairs, epidermis, cortex and the slightly brown stele. B: Fluorescence microscopy picture 1, bacteria are visible as green dots surrounding the epidermis, no major accumulation of GFP is visible inside the epidermis. Green fluorescent structures are visible in the stele. D: Fluorescence microscopy picture 2, autofluroescent structures in epidermis and stele.

**Learn 2:**

The findings of this experiment provide first insights into the **injection of proteins from *P. fluorescens* into roots**. However, it has to be critically questioned how GFP might have accumulated in the core of the root while no GFP is detected at the epidermis. From literature we know that oxidative stress can lead to the production of secondary metabolites in plants, which can result in autofluorescence at similar wavelengths to GFP [4, 5]. Therefore, we refrain from making the statement that the fluorescence at the stele is GFP-derived.

In a **future** experiment, one might consider coapplying **a reducing agent** like DTT to the fixation procedure to lower the quantity of oxidative metabolites to obtain results that leave less scope for interpretation. Furthermore, one could increase the sensitivity of GFP detection by using GFP-specific antibodies (immunostaining). These modifications would not only enhance the accuracy of the findings but also serve as a validation of the nature of observed fluorescent structures, such as those seen in the root's stele (Figure 4), confirming whether they are GFP or of plant origin.

**Conclusion:**

Within two iterative cycles of the DBTL, we improved the methodology for detecting GFP originating from *Pseudomonas fluorescens* in plant roots. First, we had a broad overview of where bacteria attach and tried to already detect injected GFP in the epidermis and root hairs.

Next, we **enhanced the procedure for detecting intracellular GFP** by analysing cross-sectional samples from root regions where we estimated a high bacterial presence, particularly in the basal portions of the roots.

In the final phase of optimisation, we **refined the cross-sectioning procedure** for our specific research objectives. This refinement involved the introduction of a root-fixation step and an increase in the concentration of agarose within the embedding medium. These improvements were essential to increase the precision and reliability of our detection method.

## *dapB* knockout

As described in the Results section, **our biocontainment strategy** is based on **knocking out an essential gene** from the genome of *P. fluorescens* and introducing it to the cell on a plasmid under the control of an inducible expression system. The envisioned knockout procedure consists of two main parts, the **integration of a suicide plasmid**, and the subsequent **deletion of the gene of interest**. Both parts were subjected to DBTL cycles which are presented below. To find an [Overview](https://2023.igem.wiki/wageningenur/description) of this method together with the methods used visit our [Results](https://2023.igem.wiki/wageningenur/wetlab-biosafety) page.

**Part I - integration of a suicide plasmid into the genome**

**Round 1: Electroporation**

**Design 1:**

Based on literature research, we chose *dapB* as suited candidate to knock out in our *P. fluorescens* host for our biocontainment strategy. *dapB* is an essential gene encoding for dihydrodipicolinate reductase, an enzyme that plays a key role in the biosynthesis of lysine and diaminopimelate (DAP) which is an essential building block of peptidoglycan of all gram-negative bacteria [6]. Strains devoid of *dapB* are therefore auxotrophic for both compounds (DAP and lysine). This gene has been knocked out before by Gal et al. (2003) using SOE-PCR and two-step allelic exchange [7]. However, in this project, we chose a slightly different approach which was proposed by Wirth et al. (2020) as a method to modify *Pseudomonas putida*, a close relative of *P. fluorescens* [8].

**Build 1:**

We started by constructing **a suicide plasmid** based on the pGNW backbone and containing homology arms needed for a first recombination event to happen. The plasmid was assembled via Golden Gate and transformed into *E. coli* DH5α λpir via chemical transformation. The correctly assembled plasmids were purified and used to **transform *P. fluorescens*** via electroporation.

![](https://static.igem.wiki/teams/4663/wiki/dbtl/dbtl51.jpg)

**Figure 5:***E. coli* DH5α λpir containing pGNW-dapB-KO. Fluorescence of the cells, resulting from the expression of agfp transcriptional unit present on pGNW, suggests correct plasmid assembly.

**Test 1:**

The bacteria were plated on LB with kanamycin, however, despite many tries, no colonies were observed.

**Learn 1:**

The most likely reason explaining why no transformants were obtained was the failure of electroporation to deliver the suicide plasmid to *P. fluorescens*. Therefore, we decided to **try a different method of transformation**

**Round 2: Tri-parental mating**

**Design 2:**

We chose another approach proposed by Wirth et al. (2020) [8] which is based on **tri-parental mating**, a form of **conjugation** in which a helper strain facilitates the transfer of a plasmid between two other bacteria. This method uses *E. coli* DH5α λpir containing the pGNW-based plasmid as a donor, *P. fluorescens* as an acceptor, and *E. coli* HB101 as a helper.

**Build 2:**

We followed the procedure of tri-parental mating according to the [protocol](https://2023.igem.wiki/wageningenur/protocols).

**Test 2:**

We managed to obtain fluorescent *P. fluorescens* colonies indicating **successful transformation**. This was further confirmed by colony PCR and subsequent electrophoresis which showed bands of the expected size indicating integration into the genome

![pseupomona package image](https://static.igem.wiki/teams/4663/wiki/dbtl/dbtl7.jpg)

**Figure 6:** A:*P. fluorescens* colonies after transformation with the pGNW-based plasmid showing fluorescence. B: Agarose gel visualising the results of colony PCR of*P. fluorescens* after transformation with pGNW. Two bands of the expected size proved that the plasmid was successfully incorporated into the genome.

**Learn 2:**

**Tri-parental mating** turned out to be a **successful** method of delivery of a suicide plasmid to *P. fluorescens* SBW25. A single recombination event resulted in the incorporation of pGNW into the genome.

**Part II - creating the knockout**

**Round 1: DAP lysine supplementation from the medium**

**Design 1:**

In order to **knock out the gene of interest**, *P. fluorescens* containing pGNW must be transformed with a pQURE6-H plasmid encoding **I-SceI endonuclease**. The enzyme recognises I-SceI sites flanking the homology arms introduced with the suicide pGNW plasmid and cuts the DNA. These double-strand breaks can be fixed by the cell by a second recombination event which can either lead to restoration of the wild-type genotype or **a knock-out**.

**Build 1:**

We delivered pQURE6-H via **chemical transformation**, a method with which other teammates had successfully introduced different plasmids into *P. fluorescens* in the meantime. The transformants were plated on LB with the addition of DAP and lysine in the concentrations determined by Gal et al., as well as 2 mM toluic acid to induce the expression of the meganuclease I-SceI.

**Learn 1:**

Red colonies appearing on the plates indicated that chemical transformation **successfully delivered pQURE6-H to the cell** because a red reporter unit is cloned into this plasmid’s backbone (Figure 7). However, **colony PCR** proved that all the tested colonies had a wild-type genotype (Figure 8). Despite several trials, the result was always **unsuccessful**.

![pseupomona package image](https://static.igem.wiki/teams/4663/wiki/dbtl/dbtl8.jpg)

**Figure 7:** Red colonies of*P. fluorescens* after transformation with pQURE6-H.

![pseupomona package image](https://static.igem.wiki/teams/4663/wiki/dbtl/dbtl9.png)

**Figure 8:** Agarose gel visualising the results of a colony PCR after transformation of*P. fluorescens* with pGNW incorporated into the genome with pQURE6-H. The bands correspond to the length of 1850 bp, which means that the gene of interest was not deleted.

**Learn 1:**

The inability to obtain a knockout **despite the successful transformation** with pQURE6-H was **intriguing**. After analysing all the steps, we concluded that the most likely reason was the fact that the **cells with successful deletion of *dapB* could not survive** in the medium used due to insufficient concentrations of DAP and lysine.

**Round 2: overexpression of *dapB***

**Design 2:**

Therefore, we **redesigned our approach**. Rather than supplying DAP and lysine from the medium, we decided to **transform the cells with the overexpression plasmid first**, and then carry out the knockout procedure. Since before the transformation with pQURE6-H, the cells would already contain the overexpression plasmid, we decided to change the method of pQURE6-H delivery. Instead of using chemical transformation, we chose to deliver it via **tri-parental mating**, which had proven to be more efficient.

**Build 2:**

We **constructed the overexpression plasmid** based on the pSEVAb33 backbone with *dapB* placed under the control of the strong constitutive promoter J23100 (BBa\_J23100). After confirmation of the correct assembly, the plasmid was delivered to *P. fluorescens* containing pGNW via chemical transformation. Afterwards, the cells were transformed with pQURE6-H via **tri-parental mating** using *P. fluorescens* with pGNW incorporated into the genome as an acceptor, *E. coli* DH10β containing pQURE6-H as a donor, and *E. coli* HB101 as a helper strain.

**Test 2:**

The cells were **successfully transformed** with the overexpression plasmid, which was checked by colony PCR. Transformation with pQURE6-H was successful. However, after induction, the **colony PCR** once again indicated that **all the bacteria had a wild-type genotype**.

**Learn 2 and final conclusions:**

In the end, we did not manage to knock out the *dapB* gene. One possible reason is that the **I-SceI meganuclease** was not activated properly or was **inactive**. In that case, however, the genome would not be cut at all, and the suicide plasmid would still be in the genome. This was however not the case. The fact that we could not obtain a knockout even when introducing *dapB* on a plasmid suggests that the genomic copy remained essential for the survival of the cell. One possible explanation is that the **high levels of enzyme** resulting from overexpression were **too burdensome** for the cells. This could force the plasmid to mutate making it unable to sustain the growth of cells with a knockout.

Therefore, the next step of this cycle would be to **sequence the overexpression plasmid**. If the results indeed showed the presence of **mutations affecting *dapB* expression**, we would repeat the procedure described in Part II Round 2 but using a promoter and RBS that give lower expression levels.

References

[1] Damalas, S. G., Batianis, C., Martin‐Pascual, M., de Lorenzo, V., & Martins dos Santos, V. A. (2020). SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards. Microbial biotechnology, 13(6), 1793-1806.

[2] Jensen, C., Korolev, A., Corredor-Moreno, P., Minter, F., Dodds, P. N., & Saunders, D. G. (2022). Caveats of Using Bacterial Type Three Secretion Assays for Validating Fungal Avirulence Effectors in Wheat. Molecular Plant-Microbe Interactions, 35(12), 1061-1066.

[3] Upadhyaya, N. M., Mago, R., Staskawicz, B. J., Ayliffe, M. A., Ellis, J. G., & Dodds, P. N. (2014). A bacterial type III secretion assay for delivery of fungal effector proteins into wheat. Molecular Plant-Microbe Interactions, 27(3), 255-264.

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