iGEM 2024 — Team uzurich

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☰ Timeline

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## Timeline

Week 0

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

This page provides an overview of our progress in the wetlab. For a more detailed insight, please visit the  [Experiments page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).
While everyone was involved in plasmid assembly using the Golden Gate cloning system, Jessi, Seraina and Vanessa initially focused on testing different DGCs and knocking down the PDE using the CRISPRi system. Mattia was also involved in the PDE system and then concentrated on developing the kill switch with Ekaterina and Linus. However, there was significant collaboration and overlap among all team members throughout the project.
For further details visit the [Attributions page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D).

### Week 0

#### Prior to the lab

In the month prior to starting in the lab, our wet lab instructor Gabriel introduced us to the Golden Gate Cloning system.
During this time, we designed plasmids for the different levels of the cloning process, ordered our gene fragments and supplies for the lab, and planned our experiments accordingly.
Our instructor, Zaira, guided us through the process of designing an sgRNA for the knockdown of the PDE gene.
This preparation laid the foundation for the upcoming lab work.

### Week 1

#### 8.7 – 12.7

##### Golden Gate Level 0

We made competent cells from *E. coli* SY327 and transformed level 0 parts from the iGEM kit. Heat shock transformation was also performed with the pBP plasmid, which served as the level 0 backbone for our ordered gene fragments.
Successful transformations were stored in glycerol, while unsuccessful assemblies were repeated.

##### CRISPRi

We miniprepped *E. coli* pSCB2 to extract the pSCB2 plasmid, which was then used to insert our sgRNA via CRISPRi inverse PCR.
After confirming the amplification by gel electrophoresis, we treated the samples with DpnI to remove the native methylated DNA.
This ensured that only our unmethylated plasmid containing the sgRNA was present in the sample.
Following ligation, the pSCB2-sgRNA plasmid was transformed into *E. coli* SY327.

### Week 2

#### 15.7 – 19.7

##### Golden Gate Level 0

After two unsuccessful attempts to transform certain iGEM kit parts, our supervisor suggested that we switch to the *E. coli* DH5α strain.
We were able to successfully transform the parts with this strain, which is known to improve efficiency with challenging plasmids.
We performed a level 0 Golden Gate assembly for our ordered gene fragments, using the mini prepped pBP as the backbone. We transformed our level 0 constructs along with the backbones from the iGEM kit.
Colony PCR was done for multiple level 0 colonies and those with the correct fragment length were sent for sequencing, most of which were successful.

##### CRISPRi

Additionally, we did a colony PCR to verify whether last week's transformations were successful and whether the *E. coli* colonies contained the plasmid with our sgRNA. We purified the DNA from these colonies and sent it for sequencing, which confirmed that two of them had the correct insert.
In addition we performed a triparental conjugation to transfer the pSCB2-sgRNA plasmid from *E. coli* to *P. sp.* IsoF dCas9.

![Week 8](https://static.igem.wiki/teams/5250/project/notebook/img-w2-min.jpg)

### Week 3

#### 22.7 – 26.7

##### Golden Gate Level 0 & Level 1

The failed level 0s were reassembled, though some were still unsuccessful.
Successful level 0s were miniprepped and used to assemble our level 1 testing and final construct plasmids.
White colonies from the level 1 testing constructs were sent for sequencing directly (as our level 1 backbones contain GFP as a reporter gene), while the final constructs were incubated over the weekend.

##### Biofilm Staining

We performed a colony PCR with colonies from the triparental conjugation, which showed no difference to the control (which lacked the helper strain), suggesting the helper strain may not be necessary.
We prepared overnight cultures for the polysaccharide staining assay, incubating our *P. sp.* IsoF dCas9 pSCB2-sgRNA as well as the negative control (IsoF dCas9) both with and without rhamnose. The rhamnose-treated cultures were expected to show increased polysaccharide production, which could be seen by eye already the next day.
The overnight cultures were then plated on Congo red derivative plates and incubated over the weekend.

### Week 4

#### 29.7 – 2.8

##### Golden Gate Level 1

After receiving the sequences of the level 1 plasmids the unsuccessful ones were reassembled using an overnight protocol for difficult assemblies.
Unfortunately, sequencing revealed that none of the reassembled level 1 final construct plasmids had worked, so we decided to postpone further attempts to a later stage after troubleshooting.

##### Biofilm Staining & c-di-GMP Assay

The polysaccharide staining plates were viewed under the microscope and analyzed using ImageJ.
We performed an initial c-di-GMP assay comparing several DGCs, which included: WspR wt, a WspR mutation, DGC IsoF wt and two DGC IsoF mutations.
The different mutations are expected to increase the c-di-GMP production compared to the wildtype, however the results of this assay were inconclusive.

![Week 4](https://static.igem.wiki/teams/5250/project/notebook/img-w4.jpg)

### Week 5

#### 5.8 – 9.8

##### Golden Gate Level 1

After further research we decided to redo the level 1 plasmids using higher concentrations of gene fragments and backbones and adding a buffer specific to our assembly enzyme, BsaI.

##### c-di-GMP Assay

We conducted a preliminary c-di-GMP assay to optimize the conditions for measuring c-di-GMP production, as the results of last week’s assay were unsuccessful.
Cells were either unwashed or washed with saline solution and adjusted at different values of OD600.
The pSCB2-sgRNA plasmid was used as a negative control, while a strain with an upregulated DGC was used as a positive control.
Despite these adjustments the assay still did not reflect the expected difference in c-di-GMP production.

### Week 6

#### 12.8 – 16.8

##### Golden Gate Level 1

We repeated the level 1 plasmid assembly, doubling the concentrations of the backbone and gene fragments.
Despite this, the number of white colonies did not increase, indicating that the efficiency of assembly did not improve.
Sequencing of the white colonies revealed that the cloning was unsuccessful.

##### C-di-GMP Assay

Following our instructor's advice, we repeated the preliminary measurement, adjusting to lower values at OD600 and used saline solution instead of LB to reduce background noise.
We focused on controls to optimize conditions instead of testing our DGC constructs.
Negative controls included an upregulated PDE in *P. sp.* IsoF (pBBR1MCS5 PA5295) and an empty plasmid (pBBR1MCS5) in *P. sp.* IsoF, while an upregulated DGC in *P. sp.* IsoF served as a positive control (pBBR1MCS5 yedQ).
The results reflected the expected c-di-GMP levels, particularly at low OD600 values.

Testing our DGCs in *E. coli* under the optimized conditions showed inconclusive results.
We believe this is because the DGCs are not in their native strain, leading to the absence of other essential metabolic steps required for c-di-GMP upregulation.
We decided to conjugate our DGCs into the *P. sp.* IsoF strain.

##### Toxin Testing

We conducted a preliminary toxin assay using rhamnose induced overnight cultures. We measured OD600 over a 16-hour period expecting that the toxin-induced cultures would exhibit reduced growth.
However, the rhamnose-treated cultures displayed a higher OD600, leading us to question whether the OD is a good indicator of cell death, whether the cells were using rhamnose as an extra source of energy and whether our rhamnose promoter might be the issue.

![Week 8](https://static.igem.wiki/teams/5250/project/notebook/img-w6-min.jpg)

### Week 7

#### 19.8 – 23.8

##### Adapting Plasmid Backbones

We attempted triparental conjugation to transfer our different testing DGC constructs (pT21Rhyz01 to pT21Rhyz05) from *E. coli* SY327 to *P. sp.* IsoF dCas9, but the conjugation was unsuccessful.
Upon further research, we discovered that our plasmid is not replicative in *P. sp.* IsoF.
Instead of recloning them into a replicative plasmid using Golden Gate assembly, we opted for digestion-ligation into the pBBRMCS5 plasmid, which is commonly used for gene expression in *P. sp.* IsoF.
We amplified the transcription units on the above-mentioned plasmids with recognition sites for specific restriction enzymes (both in the MCS of the pBBRMCS5 plasmid) and then performed digestion-ligation into pBBRMCS5.
The first attempt was unsuccessful, probably due to insufficient digestion, so we repeated it the next week.

Since none of our previously used backbones are replicative in *P. sp.* IsoF we had to adapt our plasmids.
We identified three backbones in the iGEM distribution kit that are replicative in *P. sp.* IsoF, namely pJUM23-1A, pJUMP43-2A and pJUMP45-2A and transformed them into *E. coli* SY327.

##### Toxin Testing

We decided to test the functionality of our rhamnose promoter, to ensure that this is not the problem with our toxin.
We did so using a plasmid that included RFP as a reporter gene.
We performed a preliminary test which, after an overnight incubation with rhamnose, exhibited a red color, confirming that the rhamnose promoter was active.
To quantify the promoter’s activity at different rhamnose concentrations and the potentially toxic effect of rhamnose on the cell, we performed an overnight growth assay, measuring OD600 and fluorescence.
This showed that the promoter’s maximum activity with the minimum negative effects of rhamnose was at 1%.

### Week 8

#### 26.8 – 30.8

##### Golden Gate Level 1

After encountering issues with our initial Golden Gate cloning attempts, we had to reclone many of our level 1 plasmids.
We used the pJUMP23-1A backbone, as this one is replicative in *P. sp.* IsoF.
We ordered our transcription units as a single unit to improve cloning efficiency.
After talking to our Wet Lab advisor, we adjusted our protocol to a 3:1 ratio of gene fragment to backbone (instead of a 1:1 ratio) and transformed less of the Golden Gate assembly.
Most of the colonies were white, indicating the Golden Gate assembly had a much higher efficiency than the previous attempts.
Sequencing the colonies confirmed that 17 out of 20 plasmids were cloned successfully.

##### Toxin Testing

Given that the three toxin testing plasmids (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03), shared the same non-replicative backbone as the DGC plasmids, we applied the same amplification and digestion-ligation strategy with pBBRMCS5.
However, this process failed and was repeated the following week. Our toxin test failed to show the expected effect in both *E. coli* SY327 and *E. coli* DH5alpha, due to a gyrase mutation conferring resistance to CcdB.
After further research we identified *E. coli* HB101 as suitable and transformed our plasmids into this strain.

##### Digestion-Ligation of DGC Testing Plasmids

Since the digestion-ligation of the DGC Transcription Units failed last week, we repeated the experiment.
Extending the digestion period allowed us to successfully clone the testing plasmids with the five different DGCs into the pBBRMCS5 plasmid.

![Week 8](https://static.igem.wiki/teams/5250/project/notebook/img-w8.jpg)

### Week 9

#### 2.9 – 6.9

##### Biofilm Staining & c-di-GMP Assay

This week we conducted further testing on different DGC constructs, including two of our selfmade DGC mutations.
We first conjugated our plasmids into *P. sp.* IsoF via triparental conjugation.
We then set up two experiments to measure the c-di-GMP expression of our different DGCs (WspR wt, a WspR mutation, DGC IsoF wt and two DGC IsoF mutations), our knocked down PDE as well as our controls (*P. sp.* IsoF dCas9, *P. sp.* IsoF with an empty plasmid, the upregulated DGC yedQ).

We first measured the c-di-GMP levels through fluorescence intensity.
For this we inoculated the colonies with rhamnose once overnight and once for 5 hours only, which produced more promising results than the overnights.
For our second experiment, we stained the biofilms by plating these strains on plates with a Congo red-derived dye and inoculating them over the weekend. We analyzed the results of the staining the following week.

##### Golden Gate Cloning

Following previous challenges with Golden Gate, we repeated the cloning of level one and two plasmids.
These included testing plasmids for sensing, toxins and a strain with our mutated DGC to inoculate our plants with our final construct plasmids.
According to the cPCR gel of the level 2 cloning, it failed.
After troubleshooting, we decided to redo the cPCR the following week using Q5 Polymerase instead of GoTaq, as the Q5 is more suited for amplification of sequences as long as our level 2 plasmids.

##### Sensing

In preparation for the sensing experiment next week, we did a triparental conjugation of our plasmid into *P. sp.* IsoF.

##### Toxin Tests

After difficulties with our toxin tests in the previous weeks, we chose *E. coli* HB101 as a new ccdB sensitive strain to test our toxin on.
We performed a triparental conjugation of our plasmid into *E. coli* HB101. Unfortunately, the cells were not affected by the toxin.
After some trouble shooting we decided to try the toxin test in two further strains, *E. coli* MC1061 and *E. coli* CSH50.

### Week 10

#### 9.9 – 13.9

##### Golden Gate Cloning

The colony PCR of last week’s Golden Gate assembly indicated that the cloning didn’t work.
To ensure the cPCR wasn't the issue we repeated it with more colonies, again using Q5 polymerase instead of GoTaq for better long-sequence amplification.
This second cPCR also came back negative.
After troubleshooting, we decided to increase the insert to vector ratio to 5:1 and repeated the assembly of the remaining level 1 and level 2 plasmids.

##### Biofilm Staining

A second biofilm staining assay of the strains containing the different DGCs and the sgRNA to knockdown the PDE was conducted, since there was a mistake in the controls of the last assay.
This time we included a negative control: a strain containing an upregulated PDE.

##### Toxin Testing

Both *E. coli* CSH50 and *E. coli* MC1061 showed no significant response to the toxin, as the cells continued growing.
For the sake of being thorough and due to time constraints, we conjugated and tested our toxin constructs in *P. sp.* IsoF once more with no significant effect.

##### Sensing

Colony PCR confirmed that the cloning of our sensing plasmid was successful.
After the first assay failed, further research revealed that the xylose-XutR complex binds not to the Pxut itself, but to an operator sequence located after it, which was missing in our construct.
To address this, we planned to add the missing sequence via digestion-ligation or PCR-amplification and blunt-end ligation, and designed primers accordingly.

##### Gibson Assembly

We used Gibson assembly to replace spectinomycin resistance with kanamycin resistance on a backbone, as both backbones initially had the same resistance, and this change was necessary to select for both of our final plasmids in the same bacterium.
After amplifying the level 2 backbone and kanamycin resistance from a level 1 backbone with specific primers, we ran a gradient PCR to improve efficiency and assembled them into a new level 2 backbone.
We sent one promising colony for sequencing and will get the results next week.

##### Plant Experiments

We wanted to test the effect of our c-di-GMP regulating constructs on the growth of plants in wet and dry conditions.
Unfortunately, as the cloning of our own DGC system with a constitutive promoter didn’t work in time, we used *P. sp.* IsoF pBBR1MCS5 yedQ as a comparable construct to represent how a DGC overexpression can still have an effect on plant growth.
Therefore, we inoculated MicroTom plants (*Solanum lycopersicum*) with *P. sp.* IsoF pBBR1MCS5 yedQ, a PDE overexpression (*P. sp.* IsoF pBBR PDE), our strain with the empty plasmid (*P. sp.* IsoF wt pBBR1MCS5) and LB as a negative control.
We collected data from 4 plants as a T0 measurement.

![Week 10](https://static.igem.wiki/teams/5250/project/notebook/img-w10.jpg)

### Week 11

#### 16.9 – 20.9

##### Golden Gate Level 2

We conducted a Golden Gate assembly of our feedback loop plasmids (pT32Rhyz01-pT32Rhyz03) and our final construct plasmids (p21Rhyz04 and p22Rhyz04), which contained all our target constructs.
To prevent self-ligation of the backbone, we pre-digested it and treated it with FastAP phosphatase.
However, the colony PCR results were inconclusive, after sending them for sequencing.

##### c-di-GMP Assay

We repeated the c-di-GMP assay using the same strains as in previous experiments.
Our *P. sp.* IsoF DGC mutant, R196A, showed the highest levels of c-di-GMP.

##### Biofilm Staining

We examined the biofilm staining plates under a microscope and captured images for analysis.
Results indicated that the WspR mutation exhibited the highest biofilm formation, while the controls (*P. sp.* IsoF pBBR1MCS5 empty plasmid and *P. sp.* IsoF wt) showed the least.

##### Sensing

We performed the PCR amplification and blunt end ligation to introduce the operator sequence into our sensing plasmid.
After transformation into *E. coli* SY327 we sent some colonies for sequencing, which unfortunately came back as incorrect as the primer bound to the RBS on the second transcription unit.

##### Plant Experiments

We did a colony-forming unit (CFU) count on the dilution series plates with countable colonies (between 30-300).
Additionally, we measured the dry weight of the plants that had been drying in the oven over the weekend.
The CFU count showed some unexpected inconsistency between the different samples, indicating that future results should be interpreted with caution.
The dry weight of the aerial part of the plants matched our expectation from the previous wet weight measurements.

### Week 12

#### 23.9 – 27.9

##### C-di-GMP Assay

We conducted our final c-di-GMP assay, once again using various strains to compare the c-di-GMP levels across our DGC and PDE constructs.
Consistent with previous results, the DGC R196A variant exhibited the higher c-di-GMP levels than the wildtype sequence.

##### Toxin Testing

We modified the toxin test by plating adjusted overnights containing our toxin construct, induced by the rhamnose promoter, onto rhamnose plates with varying concentrations (0%, 1%, and 1.5%).
This experiment was conducted for both *E. coli* and *P. sp.* IsoF, using IsoF with an empty plasmid (pBBR1MCS5) and *E. coli* with a plasmid containing GFP as controls.

##### Sensing

As the sequencing of our sensing plasmid, in which we tried to implement the operator sequence with PCR amplification and blunt-end ligation returned as incorrect, we went forward with the oligo annealing as a second option to introduce the missing operator sequence.
After the annealing reaction, ligation and transformation we sent for sequencing, the results of which we got the following week.

##### Swimming Motility Assay

We assessed the motility of bacteria carrying our different DGC constructs, using the same strains from our c-di-GMP assays, as reduced motility supports biofilm formation.
As anticipated, the DGC constructs exhibited lower motility compared to the control strains.
The motility assay was conducted twice.

##### Plant Experiments

We collected data from the remaining plants inoculated in Week 10, which involved measuring the aerial mass and plating a dilution series of bacteria from the rhizosphere.
The dry weight of the plants was recorded the following week.

![Week 10](https://static.igem.wiki/teams/5250/project/notebook/img-w12-min.jpg)

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{% set sources = [
{"names": "[1] Chandran Sathesh-Prabu, Rameshwar Tiwari, Doyun Kim & Sung Kuk Lee. (2021).", "title": "Inducible and tunable gene expression systems for Pseudomonas putida KT2440. Nature Scientific reports, (11:18079).", "link": "https://doi.org/10.1038/s41598-021-97550-7"},
{"names": "[2] Hailing Nie, Yujie Xiao, Jinzhi He, Huizhong Liu, Liang Nie, Wenli Chen and Qiaoyun Huang. (2020).", "title": "Phenotypic-genotypic analysis of GGDEF/EAL/HD-GYP domain-encoding genes in Pseudomonas putida. Environmental Microbiology Reports, (12(1), 38-48).", "link": "https://doi.org/10.1111/1758-2229.12808"},
{"names": "[3] Beat Christen, Matthias Christen, Ralf Paul, Franziska Schmid, Marc Folcher, Paul Jenoes, Markus Meuwly and Urs Jenal. (2006).", "title": "Allosteric Control of Cyclic di-GMP Signaling. THE JOURNAL OF BIOLOGICAL CHEMISTRY, (281(42), pp. 32015-32024).", "link": "https://doi.org/10.1016/S0021-9258(19)84115-7"},
{"names": "[4] Nabanita De, Michelle Pirruccello, Petya Violinoca Krasteva, Narae Bae, Rahul Veera Raghavan, Holger Sondermann. (2008).", "title": "Phosphorylation-Independent Regulation of the Diguanylate Cyclase WspR. PLoS Biology, (6(3):e67).", "link": "https://doi.org/10.1371/journal.pbio.0060067"},
{"names": "[5] B J Lugtenberg, L V Kravchenko, M Simons. (1999).", "title": "Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization. Environmental Microbiology, (1(5), 439-446).", "link": " https://doi.org/10.1046/j.1462-2920.1999.00054.x"},
{"names": "[6] Matthew Ferenc. (2017).", "title": "Plasmids 101: Common Lab E. coli Strains.", "link": "https://blog.addgene.org/plasmids-101-common-lab-e-coli-strains."},
{"names": "[7] Soyoung Park and Karin Sauer. (2022).", "title": "Controlling biofilm development through cyclic di-GMP signaling. Adv Exp Med Biol., (1386, 69-94).", "link": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9891824/"},
{"names": "[8] Eike H. Junkermeier and Regine Hengge. (2023).", "title": "Local signaling enhances output specificity of bacterial c-di-GMP signaling networks. microLife, (4, 1-11)", "link": "https://doi.org/10.1093/femsml/uqad026"},
{"names": "[9] Victor G. Tagua, Maria Antonia Molina\_henares, Maria L. Travieso, Rafael Nisa-Marttinez, José Miguel Quesada, Manuel Espinosa-Urgel and Maria Isabel Ramos-Gonzalez. (2022).", "title": "C-di-GMP and biofilm are regulated in Pseudomonas putida by the CfcA/CfcR two-component system in response to salts. Environmental Microbiology, (24 (1)).", "link": "https://doi.org/10.1111/1462-2920.15891"},
{"names": "[10] He Zhang, Shuan Tao, Huimin Chen, Yewei Fang, Yao Xu, Luyan Chen, Fang Ma, Wei Liang. (2024).", "title": "The biologican function of the type II toxin-antitoxin system ccdAB in recurrent urinary tract infections. Frontierts Microbiology, (vol 15).", "link": "https://doi.org/10.3389/fmicb.2024.1379625"}
] %}
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## Construct

---

Our goal is to engineer a genetic circuit within *Pseudomonas sp.* IsoF that allows the overproduction of biofilm only when the bacteria are in proximity to plant roots.
To achieve this targeted biofilm production we integrated a sensing system that reacts to the plant exudate xylose.
To prevent the spread of our engineered bacteria we incorporated a kill switch using a toxin-antitoxin system in our construct.
Additionally, we built a positive feedback loop which further enhances the biofilm production. This feedback loop also serves as a regulatory switch that activates the kill switch as soon as the bacteria sense xylose. This way, our positive feedback loop is connected to both biofilm overproduction and the activation of the kill switch, ensuring the controlled bacterial activity.
Thus, our construct consists of four main components and, accordingly, we have divided the engineering description into those four parts:

* Sensing
* Enhancement of Biofilm Production
* Feedback Loop and
* Kill Switch

We chose Golden Gate Assembly for our cloning method.
The iGEM distribution kit supplied us with basic parts, such as constitutive promoters, different ribosome binding sites (RBS), GFP and a terminator ([see Parts for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)).
We also utilized several of the provided backbones: pJUMP28-1A, pJUMP23-1A,pJUMP28-1B, pJUMP29-1C, pJUMP29-1D, pJUMP43-2A and pJUMP45-2A.

Additionally we received a level 0 backbone, the pBP, from this year’s iDEC team Evolution Suisse from the University of Zurich and ETH Zurich, for which we are very grateful.
We researched all the gene sequences, attached the appropriate adaptors and ordered them through Twist.

![Construct](https://static.igem.wiki/teams/5250/project/engineering/picture-1-updated-min.png)

**Figure 1:** This illustration represents our full construct to enhance biofilm formation only when the bacteria are in proximity to plant roots. Created with BioRender.com

##### Two state system: Connection of kill switch to feedback loop

By connecting the kill switch, feedback loop and the xylose-sensing module, we created a two-state system, in which the feedback loop acts as a switch.
In the first state, before the plant gets inoculated with our engineered bacteria, neither biofilm production is increased nor the kill switch is active.

When the bacteria encounters plant roots, for example after we inoculate the plant with our engineered bacteria, xylose triggers the feedback loop.
This initiates the overproduction of biofilm components and simultaneously the production of toxin.

Due to the self-activation and the self-amplifying characteristic of the feedback loop, it enhances biofilm production even further and also leads to a constant production of the toxin.
This means that the feedback loop acts as a switch from the first state to the second state.
When the feedback loop and therefore the second state is activated, the toxin production is sustained.
However, the antitoxin is produced only in the presence of xylose and therefore only when the bacteria are close to the root.
If the bacteria move away from the root and xylose is no longer sensed, toxin production continues without the neutralizing antitoxin, leading to the death of the bacteria.

## Sensing

---

Since we aim for targeted biofilm production, our sensing system should be based on an interaction between plants and bacteria.
We need a molecule, which is secreted by plants, recognized by bacteria and can simultaneously activate their gene expression. We decided on using a plant exudate from tomato plants as an inducer.
After a lot of research we found a sensing mechanism, originally developed for *Pseudomonas putida* KT2440, which relies on the detection of xylose [1](#ref1).
Xylose is a sugar commonly exuded by tomato plants, as well as the roots of other plant species [5](#ref5).
Given the prevalence of xylose in root exudates, our sensing system and therefore our entire construct is broadly applicable across a wide range of plants.

In the presence of xylose, the XutR (Xylose-binding protein) binds to the sugar and forms a complex.
This complex then binds to the XutR operator, which then activates the xylose-induced promoter PXut [1](#ref1).

### Cycle 1

#### Design and Build

Due to a misunderstanding when reading the paper, we thought the XutR complex would bind to the PXut directly.
To test the functionality of the PXut, we therefore assembled the level 2 testing plasmid pT12Rhyz01 ([see Parts for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)) without the operator sequence after the PXut.
pT12Rhyz01 consists of pT11Rhyz01, pT11Rhyz02 and two linker transcription units.
The xylose-binding protein XutR is constitutively expressed using the promoter BBa\_J23119 on the first transcription unit, since we don’t want the promoter’s activation to be limited due to the insufficient expression of XutR but rather due to the presence or absence of xylose.
The second transcription unit encodes the xylose-induced promoter (PXut) and a GFP (without the operator sequence).
We introduced the pT12Rhyz01 plasmid into *E. coli* SY327 and then transferred pT12Rhyz01 into *Pseudomonas sp.* IsoF using Triparental Conjugation ([see Experiments for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)).

#### Test

Since the xylose-induced promoter is specifically designed for *P. putida* KT2440, we decided to test the system exclusively in *P. sp.* IsoF without testing it first in *E. coli* SY327.
We performed a xylose sensing assay to determine whether PXut can be induced by XutR in the presence of xylose, and to identify the concentration of xylose required to trigger promoter activation.
The following xylose concentrations were tested: 0.01mM, 0.1mM, 0.2mM, 0.5mM, 1mM, 2mM, 5mM, 10mM and 20mM.

Overnight cultures were adjusted to an OD of 0.01 and 180µl of each adjusted culture were added to a 96-well-plate.
They were induced with the different xylose concentrations and kept for 8 hours at room temperature.
Each strain was tested in triplicate.
The GFP expression was then measured every hour for a total of 15 hours.
This sensing assay ([see Experiments for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)) failed, there was no increase in GFP when higher xylose concentrations were present.

#### Learn

After re-reading the paper[1](#ref1) and re-evaluating Robert M. Q. Shanks (Co-Author of the paper) input, given when we contacted him in spring regarding his paper, we realized that the Xylose-XutR complex doesn’t bind directly to the PXut.
Instead, it binds to a XutR operator sequence located between PXut and the ribosome binding site.
Therefore we were missing a sequence between our xylose induced promoter and RBS.

### Cycle 2

#### Design and Build

![Xylose Illustration](https://static.igem.wiki/teams/5250/project/engineering/picture-18-xylose-illustration.webp)

**Figure 2:** XutR and xylose bind the operator and induce gene expression. Created with BioRender.com

To address the issue, we decided to perform a PCR-amplification followed by blunt-end ligation with the said operator sequence.
We therefore ordered a primer including the operator sequence along with a region complementary to the ribosome binding site (RBS) on the vector.
We then amplified the vector of the sensing plasmid pT12Rhyz01 using the primer, aiming to insert the operator sequence between the PXut promoter and the RBS.
The sensing plasmid pT12Rhyz01 consists of two transcription units: one consists of a constitutive promoter controlling XutR and the second consists of xylose inducible promoter pXut that regulates GFP.
Since the plasmid contains two transcription units, there are two RBS sites present.
The primer had to be designed to bind the RBS between PXut and GFP, as the operator sequence needed to be inserted between the PXut and the RBS.
However, due to the presence of two RBS sites, we expected nonspecific binding of the primer to the second RBS.
To solve this issue, we gel-purified the correct DNA fragment and proceeded with the gel purified vector.
After DpnI treatment, we performed a ligation and transformed the newly cloned plasmid into *E. coli* SY327.

Unfortunately the PCR-amplification and blunt-end ligation were unsuccessful in the tested colonies.
As a result, we opted to implement the operator sequence using oligo annealing followed by digestion and ligation.
We ordered the operator sequence as two oligonucleotides, with SalI and SphI recognition sites as overhangs.
The sequences were annealed according to the oligo annealing protocol.
Next, we designed primers specific to the sensing plasmid lacking the operator sequence (pT12Rhyz01).
The forward primer was designed to overlap with the RBS on the plasmid, while also incorporating the SphI recognition site.
The reverse primer overlapped with the PXut promoter on the plasmid and included the SalI recognition site.
After amplifying the plasmid backbone using these primers, we performed digestion and ligation with the annealed oligos and the amplified backbone.

The newly assembled plasmid was transformed into *E. coli* SY327 and then introduced into *P. sp.* IsoF using Triparental Conjugation.

![plasmid map testing sensing plasmid](https://static.igem.wiki/teams/5250/project/engineering/picture-2-pm-testing-sensing-plasmid-pt12rhyz01-1.png)

**Figure 3:** Plasmid map of p12Rhyz01

#### Test

Unfortunately the oligo annealing and digestion ligation failed, due to unspecific binding of the primer.
For future experiments we will therefore repeat this step and then conduct the second sensing assay.

## Enhancing Biofilm: Targeting DGCs and PDEs

---

![DGC Illustration](https://static.igem.wiki/teams/5250/project/engineering/picture-19-dgc-illustration.webp)

**Figure 4:** DGC Illustration. Created with BioRender.com

![PDE Illustration](https://static.igem.wiki/teams/5250/project/engineering/picture-20-pde-illustration.webp)

**Figure 5:** PDE Illustration. Created with BioRender.com

The core component of our construct is the overexpression of the biofilm produced by *Pseudomonas sp.* IsoF.
Research has shown that higher c-di-GMP levels lead to enhanced production of biofilm components [7](#ref7),[2](#ref2).
Thus, our objective is to maximize the c-di-GMP concentration in the cell.
The c-di-GMP concentration is regulated by two enzyme families: diguanylate cyclases (DGCs) and c-di-GMP specific phosphodiesterases (PDEs)[9](#ref9).

DGCs are responsible for synthesizing c-di-GMP, whereas the PDEs degrade it [9](#ref9).
We targeted both enzyme families to increase the intracellular concentration of c-di-GMP as much as possible.
First, we implemented a modified DGC to boost the production of c-di-GMP.
Additionally, using a CRISPRi system, we knocked down a PDE on the chromosome of *P. sp.* IsoF to further increase c-di-GMP levels and thereby promote biofilm formation.

### Diguanylate cyclase

#### Design and Build

We targeted the diguanylate cyclases (DGCs) by introducing a modified DGC into *P. sp.* IsoF.
DGCs are either single-domain or multi-domain proteins that catalyze the production of c-di-GMP by cyclization of two guanosine triphosphate (GTP) molecules to one c-di-GMP molecule[9](#ref9).
Various DGCs contain a conserved GG(D)EF domain and within this domain there is a so-called I-site (Inhibitory site) [8](#ref8).

The I-site is an allosteric binding site specific for c-di-GMP.
When c-di-GMP binds there, it reduces the enzyme’s activity and thereby prevents further production of c-di-GMP.
C-di-GMP thus acts as a non-competitive inhibitor for the DGC.
This negative feedback mechanism serves as a self-regulatory process of the intracellular c-di-GMP concentration.
Specifically targeting this inhibitory site, or rather preventing c-di-GMP from binding to the I-site, may result in an increase in the enzyme’s activity even when c-di-GMP concentrations are already high and therefore a further increase in the c-di-GMP levels within the cells.
Altering the inhibitory site and thus preventing c-di-GMP from binding to it has been successfully done in the DGC from *Pseudomonas aeruginosa*, WspR[4](#ref4).

Nabanita De. et al.[4](#ref4) discovered that c-di-GMP binds to two arginine residues (R242 and R198) within the GGDEF domain of the WspR diguanylate cyclase.
They created two site-directed mutants of the enzyme by substituting one of the arginine residues by alanine.
The purified enzymes showed higher activity and no detectable c-di-GMP was bound to the I-site, indicating that the binding of c-di-GMP and therefore inhibition of the enzyme was disrupted in both mutations [4](#ref4).
Since both mutated DGCs showed an increased activity we decided to focus on testing one of these mutations, specifically the R242A variant.
To compare it to the wildtype sequence, we ordered the WspR DGC in two variants: one retaining the wildtype sequence and the other modified, so that the arginine at position 242 is replaced with alanine.

Although this strategy has been applied to DGCs in other bacterial species, such as the PleD DGC of *Caulobacter crescentus*[3](#ref3), it has never been tried on DGCs native to *Pseudomonas sp.* IsoF.
Therefore we decided to explore this approach in one of the DGCs naturally occurring in *P. sp.* IsoF.
Hailing Nie et al. conducted a comparative analysis of various DGCs and PDEs from *Pseudomonas putida* KT2440 with the main aim of understanding their role in biofilm formation and swimming motility[2](#ref2).
P. putida KT2440, a well-established model organism with similarities to *P. sp.* IsoF, served as a reference point for our project.
In their research, the DGC PP\_1494 in *P. putida* KT2440 showed the highest increase in biofilm formation when overexpressed in a mutant.
After examining a gene bank for *P. sp.* IsoF, we identified its homologous DNA sequence, PisoF\_00565 and chose to focus on this DGC.
As mentioned, this I-site is typically conserved across different DGCs with GGDEF domains and thus we assume this is also true for the PisoF\_00565 DGC of *P. sp.* IsoF.
Despite the conservation of the GGDEF domain and the I-site[3](#ref3) across different DGCs, the exact sequence and structure of these regions vary, which presented us with a challenge, especially since there is not a lot of data on *P. sp.* IsoF.

Nabanita De. et al. performed a sequence alignment of the GGDEF domains of WspR proteins across various Pseudomonas species to identify the locations of the I-site within these sequences.
In both the WspR and PleD diguanylate cyclases, c-di-GMP binds to an arginine residue. When this arginine is substituted with alanine, it disrupts the binding site of c-di-GMP[3](#ref3).

Building on this approach we created two mutants of the PisoF\_00565 DGC sequence.
Alpha Fold was used to identify the GGDEF domain in the sequence of PP\_1494 and therefore also in PisoF\_00565.
We then performed our own sequence alignment using Benchling to identify the I-site within the GGDEF domain of our target DGC PisoF\_00565. After identifying the binding site, we also mutated the corresponding arginine with an alanine.
In the first mutant, we altered the codon corresponding to an arginine at position 196 and replaced it with a codon for alanine. In the second mutant, the arginine residue at position 240 was substituted with an alanine.
Both those sequences and the wildtype sequence of PisoF\_00565 were ordered.

![plasmid map testing sensing plasmid](https://static.igem.wiki/teams/5250/project/engineering/picture-26-min.png)

**Figure 6:** Sequence Aligment. Created with BioRender.com

| DGC | host organism | mutated amino acid |
| --- | --- | --- |
| DGC PisoF wt | *Pseudomonas sp.* IsoF | *none* |
| DGC PisoF R196A | *Pseudomonas sp.* IsoF | arginine at position 196 was substituted with an alanine |
| DGC PisoF R240A | *Pseudomonas sp.* IsoF | arginine at position 240 was substituted with an alanine |
| DGC WspR wt | *Pseudomonas aeruginosa* | none |
| DGC WspR R242A | *Pseudomonas aeruginosa* | arginine at position 242 was substituted with an alanine |

**Table 1:** DGCs used

As a result, we have five different diguanylate cyclases:
the wild-type WspR of *P. aeruginosa*, the WspR mutant R242A, the wild-type PisoF\_00565 of *P. sp. IsoF* and our genetically engineered mutants PisoF\_00565 R196A and the PisoF\_00565 R240A.
All those different sequences, with the right adaptors, were ordered from Twist.
The aim now is to test, which of the different DGCs produce the most c-di-GMP and therefore lead to the highest formation of biofilm components.
Five different testing plasmids, carrying the five different DGCs, were assembled and transformed into *E. coli* SY327: pT21Rhyz01, pT21Rhyz02, pT21Rhyz03, pT21Rhyz03, pT21Rhyz04 and pT21Rhyz05 ([see Parts List for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)).
Each of those plasmids contain an inducible Rhamnose promoter and one of the five DGC sequences.
Additionally we performed Triparental conjugation ([see Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)) and the five plasmids were transformed into *P. sp.* IsoF using the helper strain *E. coli* SY327 pRK2013.

The initial conjugation attempt was unsuccessful and after thorough research, we discovered that our plasmids are not replicative in *P. sp.* IsoF due to an incompatible origin of replication.
Instead of re-cloning them into a replicative plasmid using Golden Gate assembly, we chose to perform a digestion ligation into the pBBRMCS5 plasmid, which was provided by the lab.
The pBBRMCS5 plasmid is commonly used for gene expression in *P. sp.* IsoF in the lab and therefore replicative in *P. sp.* IsoF.

To begin, we designed primers that would partially bind to our transcription units on the plasmids while also incorporating SacI and SpeI recognition sites on each end.
We then carried out a PCR to amplify the transcription unit from the level 1 plasmid and simultaneously extended it with the SacI and SpeI sites for later implementation into the multiple cloning site of the pBBRMCS5 plasmid, which contains the same recognition sites in their multiple cloning site. We confirmed the amplification by checking the amplified sequence length on a gel.
After DNA purification, we proceeded with a digestion of both the backbone and transcription units at 37 °C for one hour, followed by ligation. Alongside the five ligation reactions we included two controls where we replaced the T4 DNA ligase with water.
After transformation, the controls showed numerous colonies, suggesting that our digestion was inefficient.
A colony PCR on three colonies per construct revealed that the transcription unit had not been successfully inserted.
We repeated the experiment, extending the digestion by 30 minutes.
The controls still showed more colonies than expected but colony PCR on 16 colonies per construct confirmed that the DGC transcription unit had been successfully integrated into the pBBRMCS5 plasmid in at least one of the tested colonies per construct.

These five newly cloned plasmids pBBR pT21Rhyz01, pBBR pT21Rhyz02,pBBR pT21Rhyz03,pBBR pT21Rhyz04 and pBBR pT21Rhyz05 were then conjugated into *P. sp.* IsoF using triparental conjugation.

![DGC Ligation PCR](https://static.igem.wiki/teams/5250/project/engineering/picture-23-dgc-ligation-pcr.webp)

**Figure 7:** Colony PCR revealed that the digestion ligation into the pBBR1MCS5 backbone of the transcription units containing the different DGCs was successful.

#### Test

To compare the different enzymes and their mutants, we measured the c-di-GMP concentration of the five different *P. sp.* IsoF strains containing the respective testing plasmid, using the c-di-GMP assay kit from Lucerna Technologies.

Preliminary tests were conducted to identify the optimal conditions for the assay.
After adjusting the protocol based on the preliminary results, the c-di-GMP assay was performed three times ([see Experiments for details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)).

The overnight cultures were adjusted to an OD of 0.03, then induced by adding 125ul of 40% Rhamnose to achieve a final concentration of 1% in a 5ml tube.
This led to the expression of the enzyme.
The cultures were incubated for 5 hours and then washed with NaCl solution to eliminate background interference from the LB medium.
The absorbance of each overnight culture was measured with a spectrophotometer and adjusted to an optical density (OD) of 0.25.
A black 96-well plate was set up according to the c-di-GMP assay protocol.
As negative controls, we used *P. sp.* IsoF dCas9 (contains no plasmid but dCas9 is implemented on genome), *P. sp.* IsoF pBBR1MCS (*P. sp.* IsoF containing the empty plasmid) and as a positive control we used *P. sp.* IsoF pBBR1MCS YedQ (constitutively overexpressed DGC).
Into each well, 50ul of each of the adjusted and diluted cultures, were added. Each strain was tested in triplicate (3 replicates per strain).
The plate was incubated in the dark for 14 hours.
Then the fluorescence was measured with a plate reader, with the first excitation set at 469nm and emission at 501nm, followed by a second measurement with excitation set at 482nm and emission at 505nm.

After the c-di-GMP assay, we performed a biofilm staining assay ([see Experiments for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)).
A square plate containing 50ml LB medium with agar and 1.25ml of a Congo-Red derived dye (2mg/ml) was prepared and dried.
This dye stains polysaccharides and therefore the biofilm produced by our *P. sp.* IsoF strains.
The cultures were adjusted to an OD of 1 and 10ul of each adjusted strain were plated onto the prepared plate.
The plate was incubated at 30°C for two days.

Additionally we conducted a swimming motility assay, to assess whether the introduction of a DGC would affect the bacterial motility.
Elevated levels of c-di-GMP are associated with reduced motility, which in turn promotes biofilm formation, since a lower motility is beneficial for forming a robust biofilm[2](#ref2).

![dgc-pisof-00565](https://static.igem.wiki/teams/5250/project/engineering/picture-3-dgc-pisof-00565-1.png)

**Figure 8:**Plasmid map of pBBR pT21Rhyz01

![dgc-pisof-00565-r196](https://static.igem.wiki/teams/5250/project/engineering/picture-4-dgc-pisof-00565-r196-1.png)

**Figure 9:** Plasmid map of pBBR pT21Rhyz02

![dgc-pisof-00565-r240](https://static.igem.wiki/teams/5250/project/engineering/picture-5-dgc-pisof-00565-r240-1.png)

**Figure 10:** Plasmid map of pBBR pT21Rhyz03

![dgc-p-aerug-xutr](https://static.igem.wiki/teams/5250/project/engineering/picture-6-dgc-p-aerug-xutr-1.png)

**Figure 11:** Plasmid map of pBBR pT21Rhyz04

![dgc-p-aerug-xutr-r242](https://static.igem.wiki/teams/5250/project/engineering/picture-7-dgc-p-aerug-xutr-r242-1.png)

**Figure 12:** Plasmid map of pBBR pT21Rhyz05

Previous

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#### Learn

The c-di-GMP assay, the biofilm staining assay and the motility assay show that introducing certain diguanylate cyclases (DGCs) can significantly increase intracellular c-di-GMP levels, promote polysaccharide production and reduce bacterial motility.
These three results all indicate that the different *P. sp.* IsoF strains containing different DGCs do indeed produce and form a stronger biofilm.

Our mutated DGC PisoF\_00565 R196A resulted in a significantly higher concentration of c-di-GMP compared the wildtype sequence of PisoF\_00565. This indicates that we successfully modified the enzyme’s I-site, which usually reduces enzyme activity when c-di-GMP binds.
By altering the sequence of the DGC we prevented c-di-GMP from binding to the negative allosteric site, which effectively enhanced the enzyme’s activity. This has never been done and showed before for any DGC native to *P. sp.* IsoF.
For further details on the results, please see the [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

Given that the mutated WspR diguanylate cyclase (DGC WspR R242A), native to *P. aeruginosa*, resulted in the greatest increase in c-di-GMP levels and polysaccharide production, we chose to incorporate this mutated WspR DGC into our final plasmid construct.

Additionally, we discovered that the origins of replication OriV9 (pBBR322), p15A and pUC are not replicative in *P. sp.* IsoF.
This insight needs to be taken into consideration when designing and introducing other plasmids into *P. sp.* IsoF.

### Phosphodiesterases

#### Design and Build

![CRISPRi llustration](https://static.igem.wiki/teams/5250/project/engineering/img-new-sgrna.png)

**Figure 13:** CRISPRi Illustration. Created with BioRender.com

Phosphodiesterases, which contain either an EAL or HD-GYP domain, are the second enzyme family that controls the c-di-GMP concentration in the cell[9](#ref9).
They degrade c-di-GMP by either linearizing it into a pGpG or converting it into two molecules of GMP [2](#ref2).
Our aim is to inhibit the activity of these enzymes to increase the c-di-GMP concentration.
Therefore, we aim to target and knock down one of the PDEs encoded on the chromosome of *P. sp.* IsoF using a CRISPR interference (CRISPRi) system.
CRISPRi is a modified version of the CRISPR-Cas9 gene-editing technology and was designed to repress gene expression without cutting the DNA.

Unlike the standard Cas9 protein, which cleaves DNA, the CRISPRi system utilizes a catalytically inactive variant known as dead Cas9 (dCas9).
DCas9 can still be guided to a specific DNA sequence by a guide RNA, but it can no longer cut the DNA.
Instead, it binds to the target sequence and physically blocks the RNA polymerase and therefore prevents the transcription of the targeted gene.
This system thus allows knockdown of a gene encoding for a Phosphodiesterase.

We again utilized the comparative analyses of Hailing Nie et al. to identify a suitable PDE for knockdown.
Among the *P. putida* KT2440 mutants they created, the one lacking PDE\_0914 showed the highest increase in biofilm formation.
After examining the gene bank for *Pseudomonas sp.* IsoF, we identified the homologous gene in *P. sp.* IsoF, PisoF\_02645.
We chose to target this PDE for our knockdown.

Since the research group we were working with already has a well-established functioning CRISPRi system that is not based on Golden Gate assembly but on PCR amplification and blunt-end ligation, we decided to initially test the knockdown of our selected PDE using their cloning method.
After testing, we will then clone it into our final plasmid using Golden Gate assembly.

The CRISPRi system requires two key components: the modified dCas9 protein and a guide RNA (sgRNA).
We were provided with a *Pseudomonas sp.* IsoF strain, which already contains the gene encoding dCas9 integrated on its chromosome.
We will refer to this strain as *P. sp.* IsoF dCas9.
The dCas9 gene sequence is under the control of a rhamnose-inducible promoter.
The CRISPRi system is therefore rhamnose inducible, which allows us to control the expression of the dCas9 and our knockdown.

The sgRNA consists of two main parts: a tracr-cr sequence and a guide sequence.
The tracr-cr sequence is 35 base pairs long and forms a hairpin structure, which is essential for the dCas9 to remain bound to the target DNA sequence and stops the DNA polymerase to function.
The sequence for this tracr-cr is: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC.

For an efficient knock-down the non-template strand should be targeted, meaning that the guide RNA should have the same sequence as the template strand.
The guide RNA itself should be 20-22 nucleotides long and again consists of two parts: the first 10 nucleotides should be unique within the *Pseudomonas sp.* IsoF genome to avoid off-target effects.
The remaining sequence (10-12 nucleotides) does not necessarily need to be unique. Additionally, those 10 unique nucleotides must be located adjacent to a PAM motif (Protospacer Adjacent Motif), which in our system is CCN on the non-template strand.
The PAM sequence is essential because it is recognized by the Cas nucleases and dictates where the enzyme binds.

Our next step was to design our sgRNA.
So the sgRNA was designed by first identifying a PAM motif close to the promoter.
This leads to a high efficient knockdown since the DNA polymerase can’t transcribe any nucleotides when it is already inhibited at the promoter.
Then we ensured that the 10 adjacent nucleotides were unique across *P. sp.* IsoF genome, in both orientations forward and reverse, by examining the *P. sp.* IsoF gene bank.
And lastly, we extended the guide sequence by an additional 12 nucleotides.

The final sequence of the sgRNA, excluding the PAM sequence, is:
5’-CAATGAAACCAATACACTAT
*GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC*-3’.

In this sequence the underlined part represents the guide RNA, while the italicized part represents the tracr-cr RNA.

![sgRNA](https://static.igem.wiki/teams/5250/project/engineering/img-new.png)

**Figure 14:** Our sgRNA to knockdown PDE PisoF\_02645. Created with BioRender.com

This sequence was then synthesized as a primer by Microsynth.
Following the CRISPR interference protocol ([see Experiments detailed protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)), the sgRNA was cloned into the plasmid using PCR amplification and blunt-end ligation.
We performed PCR amplification to insert our sgRNA into the pSCB2-sgRNA-Gm plasmid.
This plasmid already contains the tracr-crRNA sequence, which allows the tracr-crRNA part of our sgRNA to anneal directly to the template plasmid.
The guide RNA sequence, however, is not yet present on the plasmid. Since the guide RNA is physically linked to the tracr-crRNA, the DNA polymerase extends the sequence by adding nucleotides during the PCR reaction, resulting in a plasmid that includes both the guide RNA and the tracr-crRNA.
To eliminate the original template plasmid (pSCB2-sgRNA-Gm) that does not contain our guide RNA, we treated the reaction mixture with DpnI. DpnI is a restriction enzyme that specifically digests GATC motifs where adenine residues are methylated.
Since only the template plasmid is methylated, DpnI selectively digests it, resulting in a product which only contains our desired plasmid, called p1DRhyz04.

After purifying the PCR product, we proceeded with ligation and transformed the p1DRhyz04 plasmid into *E. coli* SY327. We then performed colony PCR and sent the amplified sequence for sequencing to ensure that the correct plasmid was introduced into our *E. coli* strain.
Finally, we performed a triparental conjugation to introduce p1DRhyz04 into *P. sp.* IsoF dCas9.

![pscb2-p1drhyz04-sgrna](https://static.igem.wiki/teams/5250/project/engineering/picture-8-pscb2-p1drhyz04-sgrna-1.png)

**Figure 15:** Plasmid map of p1DRhyz04

#### Test

Since the gene knockdown is only effective in *P. sp.* IsoF, we were only able to test our system in *P. sp.* IsoF dCas9 and could not perform parallel experiments in *E. coli* SY327.
To evaluate our gene knockdown, we initially conducted a c-di-GMP assay, following the same procedure as the assay for our different DGCs and also using the same controls.
We induced the overnight cultures with 125ul of 40% rhamnose after adjusting them to an OD of 0.02.
Then we washed the cells, let them grow and adjusted them again to an OD of 0.25.
A black 96-well plate was set up according to the c-di-GMP assay protocol.
We added 50ul of the adjusted cultures to each well and each strain was tested in triplicate.
The plate was then incubated in the dark for one hour. Then the fluorescence was measured using a plate reader and the first measurement was taken with excitation at 469nm and emission at 501nm, the second measurement with excitation set at 482nm and emission at 505nm.

After the c-di-GMP assay we conducted a biofilm staining assay ([see Experiments for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)), again following the same procedure as the biofilm staining assay for our different DGCs.
The overnight culture of each strain was induced with rhamnose and adjusted to an OD of one. 10ul of each adjusted strain was plated onto the prepared dyed plate.
The plate was incubated at 30°C for two days.
The absorbance of each strain was then measured using a fluorescence microscope.

Additionally we conducted a swimming motility assay, to assess whether the knockdown of a PDE would affect the bacterial motility. Elevated levels of c-di-GMP are associated with reduced motility, which in turn promotes biofilm formation, since a lower motility is beneficial for forming a robust biofilm[2](#ref2).

#### Learn

The biofilm staining assay confirms that PisoF\_02645 is knocked down when implementing our sgRNA and dCas9 into *P. sp.* IsoF.
This knockdown then leads to a higher c-di-GMP concentration in the cell and therefore an increased production of polysaccharides.
The motility of the *P. sp.* IsoF strain with the knocked-down PDE was also reduced.
These results all indicate that this strain produces a stronger biofilm.

Since our CRISPRi system works as intended we proceeded to order the transcription unit of our sgRNA (T7 promoter and sgRNA) with the appropriate overhangs.
We then cloned it directly into one of our level 2 plasmids.

## Feedback loop

---

### Design and Build

![Construct](https://static.igem.wiki/teams/5250/project/engineering/picture-1-updated-min.png)

**Figure 16:** This illustration represents our full construct to enhance biofilm formation only when the bacteria are in proximity to plant roots. Created with BioRender.com

Our positive feedback aims at enhancing biofilm production even further with its self-amplifying characteristic and acts as a regulatory switch that activates both our kill switch and biofilm overproduction.
Finding an appropriate signaling molecule was challenging.
It needed to be a peptide that is not naturally produced by our bacteria to avoid leaky activation of the system, which could prematurely kill the bacteria.
After a lot of research, we chose T7 RNA Polymerase (T7 RNAP) as our molecule.
In our genetic circuit, the XutR-Xylose complex triggers the transcription of T7 RNAP.
Once produced, T7 RNAP binds to its specific promoter, the T7-like promoter, initiating the transcription of itself.
This amplifies its own expression, which creates a self-amplifying loop.
The T7 RNAP also activates the diguanylate cyclase and sgRNA, since both are under the control of a T7-like promoter, leading to an enhanced biofilm formation.
Simultaneously, the kill switch is activated by induction of toxin production.

To verify the functionality of this feedback loop and confirm that higher concentrations of T7 RNAP lead to increased gene expression, we designed three level 2 testing plasmids: pT32Rhyz01, pT32Rhyz02 and pT32Rhyz03 ([see "Parts" for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)) using the Golden Gate assembly.

The pT32Rhyz01 contains two transcription units (and two linker transcription units). The first one is a constitutive promoter and a T7 RNAP (pT32Rhyz01). The second one is a T7-like promoter and a GFP (pT31Rhyz02).
The aim of this plasmid is to test whether the T7-like promoter is effectively activated by the T7 RNAP.

The pT32Rhyz02 is designed to test the impact of T7 RNAP without a feedback loop.
It includes three transcription units: a constitutive promoter drives the expression of T7 RNAP (pT31Rhyz01), a T7-like promoter controls the expression of GFP (pT31Rhyz02).
Additionally, a separate T7 promoter controls the expression of RFP, a gene unrelated to the feedback loop (pT31Rhyz03).

The pT32Rhyz03 simulates the feedback loop.
It also contains three transcription units: a constitutive promoter drives T7 RNAP (pT31Rhyz01), a T7-like promoter leads to the expression of an additional T7 RNAP and a T7-like promoter controlling GFP.
The aim is to compare the GFP expression of pT32Rhyz02 and pT32Rhyz03 to identify whether the RNA polymerase is suitable as a self-amplifying feedback molecule.

![pt32rhyz01](https://static.igem.wiki/teams/5250/project/engineering/picture-9-pt32rhyz01-1.png)

**Figure 17:** Plasmid map of pT32Rhyz01

![pt32rhyz02](https://static.igem.wiki/teams/5250/project/engineering/picture-10-pt32rhyz02-1.png)

**Figure 18:** Plasmid map of pT32Rhyz02

![pt32rhyz03](https://static.igem.wiki/teams/5250/project/engineering/picture-11-pt32rhyz03-1.png)

**Figure 19:** Plasmid map of pT32Rhyz03

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### Test

We encountered some challenges during the cloning of our level 2 plasmids.
Initially, all the colonies we obtained were green, suggesting that the GFP either hadn’t been properly excised or had re-ligated with the backbone.
To address this issue, we increased the insert-to-backbone ratio to 5:1 (instead of 3:1).
However, after another unsuccessful attempt, we decided to pre-digest the backbone and perform gel extraction to remove the GFP before adding the inserts.
This adjustment resulted in all white colonies, but due to time constraints, we were unable to test the feedback loop unfortunately.

## Kill Switch

---

### Design and Build

To ensure that the bacteria die when they move away from the plant roots, we incorporated a kill switch into our construct using the gyrase-inhibiting Toxin/Antitoxin system CcdB/CcdA.
The ccdB gene encodes the toxic protein CcdB, which inhibits the DNA gyrase and therefore leads to cell death.
The ccdA gene encodes the antitoxin protein CcdA that neutralizes the toxin by forming a CcdA-CcdB complex and therefore protecting the cell[10](#ref10).

Our first goal was to determine the amount of CcdB required to effectively kill the cell.
Therefore we designed three testing plasmids pT41Rhyz01, pT41Rhyz02 and pT41Rhyz03 ([see Parts for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)) with Golden Gate Assembly and transformed into *E. coli* SY327.
Each of these plasmids contains the toxin gene but with a different ribosome binding site (RBS) strength.
pT41Rhyz01 has the weakest RBS leading to little toxin production, while pT41Rhyz03 has the strongest RBS leading to high toxin production.
The expression of the toxin is controlled by an rhamnose promoter to ensure the toxin is produced only when we induce it with rhamnose.

The first toxin test ([see Experiments for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)) in *E. coli* SY327 showed that the bacteria does not get killed by the toxin, indicating that *E. coli* SY327 is resistant to CcdB.
Those three plasmids were then transformed into *E. coli* DH5alpha and the same toxin test was conducted. Again, the experiment failed and the bacteria weren’t killed.
After in-depth research we found that *E. coli* SY327 as well as E. coli DH5alpha had a mutation in their gyrase and were therefore resistant to our toxin [6](#ref6).

We identified three suitable *E. coli* strains[6](#ref6): E. coli HB101, *E. coli* MC1061 and *E. coli* CSH50.
We prepared competent cells following the protocol ([see Experiments protocol for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)) and transformed our plasmids into this new strain.

Since these three testing plasmids also contain the pJUMP28-1A backbone, we assumed that they would also not replicate in *P. sp.* IsoF.
We therefore performed the same digestion ligation to insert them into the pBBRMCS5 plasmid, as we did with the DGC testing plasmids described earlier.
The initial ligation attempt was unsuccessful but after prolonging the digestion the second attempt was successful.

Additionally we constructed pT41Rhyz04 with Golden Gate Assembly, which contains a rhamnose inducible promoter and the antitoxin gene ccdA.
Level 2 testing plasmid pT42Rhyz01 was then assembled with pT41Rhyz04 and one of the three toxin testing plasmids to test whether the antitoxin CcdA is potent enough to rescue the cell and transformed into *E. coli*.
The assembly was not successful.
The toxin assays failed.

Since the bacterial growth was not inhibited, there is no reason for testing whether the antitoxin can block the toxin. Therefore, we have decided not to proceed with recloning.

### Test

We performed the same toxin assay 1 ([see Experiments for more details](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)) for all different *E. coli* strains.
The toxin assay consists of an OD measurement overnight.
The overnight cultures were adjusted to an OD of 0.01 and induced with different rhamnose concentrations (0%, 1%, 1.5%).
Then they were incubated for four hours.
195 ul of bacterial culture was added to a 96-well plate and the OD600 was measured overnight.

The same toxin test was done on *P. sp.* IsoF containing the three toxin testing plasmids on the pBBRMCS5 backbone.
Additionally we performed toxin assay 2 on two strains: *E. coli* CSH50 and *P. sp.* IsoF.

##### plasmids with backbone pJUMP28-1A (replicative in *E. coli* SY327)

![pt41rhyz01](https://static.igem.wiki/teams/5250/project/engineering/picture-12-pt41rhyz01-1.png)

**Figure 20:** Plasmid map of pT41Rhyz01

![pt32rhyz02](https://static.igem.wiki/teams/5250/project/engineering/picture-13-pt41rhyz02-1.png)

**Figure 21:** Plasmid map of pT41Rhyz02

![pt32rhyz03](https://static.igem.wiki/teams/5250/project/engineering/picture-14-pt41rhyz03-1.png)

**Figure 22:** Plasmid map of pT41Rhyz03

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##### plasmids with backbone pBBR1 MCS5 (replicative in *P. sp.* IsoF)

![pbbr1-pt41rhyz01](https://static.igem.wiki/teams/5250/project/engineering/picture-15-pbbr1-pt41rhyz01-1.png)

**Figure 23:** Plasmid map of pBBR pT41Rhyz01

![pbbr1-pt41rhyz02](https://static.igem.wiki/teams/5250/project/engineering/picture-16-pbbr1-pt41rhyz02-1.png)

**Figure 24:** Plasmid map of pBBR pT41Rhyz02

![pbbr1-pt41rhyz03](https://static.igem.wiki/teams/5250/project/engineering/picture-17-pbbr1-pt41rhyz03-1.png)

**Figure 25:** Plasmid map of pBBR pT41Rhyz03

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### Learn

For *E. coli* CSH50 we see a significant decrease in growth when inducing it with more rhamnose.
This is not the case for *P. sp.* IsoF, the CFU remains consistent across all tested rhamnose concentrations.
We can thus conclude that the toxin CcdB does inhibit the growth of *E. coli* CSH50 in this second toxin assay but it does not inhibit the growth of *P. sp.* IsoF.
We don’t know why the first toxin test didn’t lead to the same result as the second toxin test for *E. coli* strains.
Since our aim is to implement this Kill Switch into *P. sp.* IsoF, our main focus was on determining whether this toxin was effective in *P. sp.* IsoF.
For this bacterial species, the result is clear.
In both experiments there is no inhibition in growth for *P. sp.* IsoF. We can thus conclude that the toxin CcdB doesn’t effect the growth of *P. sp.* IsoF significantly.
For the future, we would therefore research more on toxin antitoxin systems specifically designed for *P. sp.* IsoF and implement one of those into our genetic circuits.

## Design of final construct plasmids

---

Based on our test results, we designed two final construct plasmids p21Rhyz02 and p22Rhyz02 with eight transcription units in total.
We chose to implement the sgRNA, to knockdown phosphodiesterase PisoF\_02645, and the diguanylate cyclase of *Pseudomonas aeruginosa* that is mutated at position 242 (WspR DGC R242A), since this mutation resulted in the highest intracellular c-di-GMP levels and highest polysaccharide production.
The toxin assay indicated that a sufficient level of toxin is required to impact bacterial growth.
Therefore, we incorporated the toxin with a strong RBS into our final construct.

Instead of implementing a linker transcription units to fill the second level 2 plasmid, we introduced a second DGC transcription unit (T7 promoter and WspR DGC R242A).
This even further enhances biofilm formation when the feedback loop is induced.

To clone the two level 2 plasmids p21Rhyz02 and p22Rhyz02, we used pJUMP43-2A(sfGFP) and pJUMP45-2A backbones from the iGEM distribution kit, as they are replicative in *P. sp.* IsoF.
Since we need all eight transcription units for our construct to work, we needed to introduce both plasmids into *P. sp.* IsoF.
However both backbones carry a spectinomycin resistance, which posed a challenge in selecting bacteria that carried both plasmids, rather than just one.
Therefore we performed Gibson Assembly to change the resistance marker on the pJUMP45-2A(sfGFP).
We amplified the kanamycin resistance of the pJUMP29-1B(sfGFP) with primers overlapping our backbone and simultaneously amplified the backbone without the spectinomycin resistance with primers overlapping the kanamycin resistance gene.
One of the plasmids that showed GFP expression and grew on kanamycin plates was sent for sequencing, but unfortunately, the sequence was incorrect.
The sequence we received matched that of pJUMP29-1B, indicating that the template had been transformed into *E. coli* SY327.
Due to time constraints, we did not repeat the Gibson Assembly and decided to focus on working on individual components rather than repeating the Gibson assembly to integrate our final level 2 plasmids into *P. sp.* IsoF.
Had we chosen to repeat the Gibson assembly, we could have performed a DpnI treatment after amplification to eliminate the template and potentially improve the efficiency of the cloning.

We also encountered challenges while cloning the level 2 plasmids and were ultimately only able to clone one, p21Rhyz02. Unfortunately, due to time limitations we could neither introduce the final level 2 plasmids into P. isoF nor conduct any testing.

##### plasmids with backbone pBBR1 MCS5 (replicative in *P. sp.* IsoF)

![p21rhyz02](https://static.igem.wiki/teams/5250/project/engineering/picture-24-p21rhyz02-1.png)

**Figure 26:** Plasmid map of p21Rhyz02

![p22rhyz02](https://static.igem.wiki/teams/5250/project/engineering/picture-25-p22rhyz02-1.png)

**Figure 27:** Plasmid map of p22Rhyz02

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## Conclusions

---

Our goal was to engineer a genetic circuit within *Pseudomonas sp.* IsoF that allows the overproduction of biofilm only when the bacteria are in proximity to plant roots.
Our construct consists of four components and we were able to successfully clone and implement following parts of the construct:

1. **Enhancing Biofilm:** We successfully enhanced biofilm formation of *P. sp.* IsoF by targeting both enzyme families responsible for regulating c-di-GMP levels in the cells.

1. We were able to knockdown a phosphodiesterase in the genome of *P. sp.* IsoF using CRISPRi.
   We increased intracellular c-di-GMP level, which in turn led to increased polysaccharide production and reduced motility.
   All of those are key factors for a robust biofilm formation.
2. We were also able to increase c-di-GMP level by introducing different diguanylate cyclases into the bacteria.
   This again led to an increased production of polysaccharides and inhibition of motility and therefore to a stronger biofilm formation.
3. We successfully mutated the DGC PisoF\_00565, removing its negative regulation through c-di-GMP.
   Our mutated DGC PisoF\_00565 R196A resulted in higher c-di-GMP concentration compared to the wildtype sequence. This indicates that we successfully modified the enzyme’s I-site, which usually reduces enzyme activity when c-di-GMP binds. By altering the sequence of the DGC we prevented c-di-GMP from binding to the negative allosteric site, which effectively enhanced the enzyme’s activity.
   This has never been done and showed before for any DGC native to *P. sp.* IsoF.

2. **Sensing system:** We successfully assembled our xylose-sensing system, in which xylose binds to a xylose-binding protein to start gene expression.
   This system was introduced into *P. sp.* IsoF.
   Unfortunately the testing of the sensing system was not yet successful.

3. **Kill Switch:** We successfully assembled and tested plasmids containing the CcdB toxin with varying RBS strengths.
   We successfully introduced and tested these transcription units into *P. sp.* IsoF.
   Unfortunately the toxin failed to inhibit bacterial growth.

4. **Feedback loop:** We designed a positive feedback loop that simultaneously functions as a regulatory switch in vitro.
   However, due to cloning issues and time constraint, we were unable to clone or test the plasmids required for this system.

5. **Final construct plasmids:** We successfully cloned, next to many level 0 and level 1 plasmids, one out of two final construct level 2 plasmids, the p21Rhyz02 plasmid. Unfortunately we were unable to conjugate it into *P. sp.* IsoF and test it.

## References

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[{{ source.names }} *{{ source.title }}*](%7B%7B%20source.link%20%7D%7D)

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{% set sources = [
{"names": "[1] Jake D. Callaghan,Nicholas A. Stella,Kara M. Lehner,Benjamin R. Treat,b Kimberly M. Brothers, Anthony J. St. Leger,and Robert M. Q. Shanks (2020).", "title": "Xylose-Inducible Promoter Tools for Pseudomonas Species and Their Use in Implicating a Role for the Type II Secretion System Protein XcpQ in the Inhibition of Corneal Epithelial Wound Closure. Environmental Microbiology Reports.", "link": "https://doi.org/10.1128/AEM.00250-20"},
{"names": "[2] Nie, H., Xiao, Y., He, J., Liu, H., Nie, L., Chen, W. & Huang, Q. (2019).", "title": "Phenotypic–genotypic analysis of GGDEF/EAL/HD‐GYP domain‐encoding genes in Pseudomonas putida. Environmental Microbiology Reports, 12(1), 38–48.", "link": "https://doi.org/10.1111/1758-2229.12808"}
] %}
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{% block title %}{% endblock %}
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## 1. Characterization of the Xylose promoter

---

We aimed for our bacteria to sense xylose from the root exudate of plants.
The experimental approach was to test whether the presence of xylose would induce the expression of GFP.
To achieve this, we used a plasmid containing two transcriptional units (pT12Rhyz01).
The first unit included a constitutive promoter (pJ23119) inducing the expression of the XutR protein.
When xylose is present, XutR binds it, which then turn activates the second transcription unit, driven by the Pxut promoter, leading to the expression of GFP.
Additionally to pT12Rhyz01, we included a negative control with an empty plasmid, a wild-type *Pseudomonas sp.* IsoF strain, and a positive control in our assay.
The positive control entails a plasmid that constitutively expresses GFP.

Before starting the actual experiment we adjusted the cultures to OD 0.01 and into each well of a 96-well plate we added 20 μl of xylose and 180 μl of our adjusted culture.
In order to measure the fluorescence we used a plate reader.
We tested different xylose concentrations to find the optimum and to determine the amount of xylose the plant would need to exude in order to activate our promoter: 20mM, 10mM, 5mM, 2mM, 1mM, 0.5 mM, 0.2 mM, 0.1 mM, 0.01 mM.
We used a 24-hours program to measure OD and fluorescence every 30 minutes.
It is expected that it will take up to 6 hours until first fluorescence will be detected and up to 25 hours until maximal intensity is reached.[1](#ref1)

## 2. Biofilm quantification

---

Our goal is to enhance biofilm formation. Quantifying biofilm production poses a challenge since it has a complex structure.
A biofilm is a community of bacteria that adhere to each other and produce a matrix of extracellular polysaccharides.

To measure biofilm formation, we decided to focus on these three key factors:

1. Quantifying the intracellular concentration of c-di-GMP
2. Measuring the production of polysaccharides
3. Analyzing the motility of the bacteria

### 2.1 c-di-GMP assay

Quantifying the intracellular c-di-GMP concentration is our first method to quantify biofilm formation, since we decided to increase biofilm formation by increasing the c-di-GMP concentration in the cell.
For this purpose, we decided to use the cyclic di-GMP Assay Kit from Lucerna Technologies, which is based on a fluorescence signal generated by c-di-GMP.
The c-di-GMP sensor used in this assay consists of a c-di-GMP riboswitch and a Spinach aptamer.
When c-di-GMP binds to the riboswitch, it stabilizes the Spinach aptamer.
This allows the fluorophore DFHBI-1T to bind and emit a fluorescent signal.
The fluorescence is then measured using a fluorescence plate reader.

Before proceeding with the actual c-di-GMP experiments, we needed to optimize the protocol provided with the kit to establish the ideal conditions.
Therefore, we first preliminary experiments.

#### Preliminary experiment

In the preliminary phase, we tested different conditions to optimize the assay.
We selected four control strains for this: *P. sp.* IsoF wt, *P. sp.* IsoF pBBR1MCS5, *P. sp.* IsoF pBBR1MCS5 PA5295 and *P. sp.* IsoF pBBR1MCS5 YedQ.

*P. sp.* IsoF pBBR1MCS5 PA5295 served as a negative control, because it contains an upregulated phosphodiesterase gene, PA5295, which reduces intracellular c-di-GMP levels.
*P. sp.* IsoF pBBR1MCS5 YedQ served as a positive control, since it carries a constitutively overexpressed diguanylate cyclase gene, YedQ, which increased the c-di-GMP concentration.
*P. sp.* IsoF pBBR1MCS5 and *P. sp.* IsoF served as additional controls, one containing the empty plasmid and the other not containing any plasmid.

We tested the assay by preparing the cells in two conditions: washed and unwashed.
For the washed condition, we washed the cells with NaCl solution to eliminate background interference from the LB medium.
This was done by centrifuging 1 ml of cell culture at 5000 rpm for 5 minutes, discarding the supernatant and resuspending the pellet with 1 ml of NaCl solution.
For the unwashed condition, the cell culture remained in LB medium.

Additionally we adjusted the washed and unwashed cells to different optical densities (OD600 of 0.25, 0.5, 1, 2) using a spectrophotometer, to determine the optimal cell density to measure the c-di-GMP concentration.

We quickly observed that leaving the cells in LB medium results in significant background noise, which compromises data reliability.
Washing the cells provided more consistent and accurate results.
Of the different optical densities tested, an OD600 of 0.25 lead to the most reliable outcome with a clear progression from the negative control to the wildtype and the positive control.

As indicated in the assay protocol, we measured the fluorescence at two different wavelength settings.
The first measurement was taken with an excitation wavelength set at 482 nm and emission wavelength at 505 nm.
For the second measurement the excitation was set at 469 nm and the emission at 501 nm. Both wavelength sets provided similar results.
Based on these findings, we decided that for future experiments we will always wash the cells with NaCl solution and adjust the cell culture to an OD600 of 0.25.

#### Final c-di-GMP assay

After determining the optimal condition and optical density, we could proceed to measure the c-di-GMP concentration in our strains with different upregulated DGCs and downregulated PDE.
As negative controls, we used *P. sp.* IsoF wt, *P. sp.* IsoF dCas9, *P. sp.* IsoF pBBR1MCS5 (which contains the empty plasmid) and *P. sp.* IsoF pBBR1MCS5 PA5295 (constitutively overexpressed PDE).
For the positive control we used *P. sp.* IsoF pBBR1MCS5 YedQ (constitutively overexpressed DGC).

We first adjusted our overnight cultures of each strain to an OD600 of 0.02 and prepared two tubes for each culture.
In one set, we induced expression by adding 125 µl of 40% rhamnose to achieve a final concentration of 1% in 5 ml volume.
Since our DGCs and PDEs are regulated by a rhamnose-inducible promoter, this induction was needed to express or inhibit the enzymes.
The second set of cultures, without rhamnose, served as a control to test whether the rhamnose itself influenced intracellular c-di-GMP concentration.

The cultures were grown for four hours. After that, we washed the cells with NaCl solution, by centrifuging 1 ml of cell culture for 5 min at 5000 rpm, discarding the supernatant and resuspending the pellet in 1 ml of NaCl solution.
We measured the absorbance with a spectrophotometer and adjusted the cultures to an OD600 of 0.25. Following the protocol we further diluted the cell cultures 1:10 with RNase-Free water.
Each strain was measured in triplicate.

50µl of the diluted culture was pipetted into a black 96-well plate and filled up to 70 µl with RNase-Free water.
To each well, 50µl of the 4X c-di-GMP assay (CA) buffer and 50µl of 4X Bacterial Compatibility (BC) Reagent were added.
A fresh 10X Fluorophore stock was prepared for each assay by diluting the 1000X Fluorophore Stock 1:100 with RNase-free water.
Then, 10µl of the 20X c-di-GMP sensor was added to each well.

To generate a standard curve for c-di-GMP concentration in order to calculate the c-di-GMP concentration, we prepared a dilution series of seven standards.
Standard 1 was created by mixing 75 µl of c-di-GMP stock with 75 µl of RNase-free water, leading to a c-di-GMP concentration of 500 nM.
For standard sample 2-6, 70µl of each preceding dilution was mixed with 70ul of RNase-free water.
Standard sample 7 contained only 70µl of RNase-Free water.
20µl of each standard was added to the plate with 5µl of NaCl solution.
The same reagents used for the bacterial samples were added to the wells.

All samples were mixed thoroughly and the plate was incubated in the dark at room temperature for 14 hours.
Fluorescence was then measured using a plate reader with gain at 60 and two wavelength settings: the first measurement with an excitation at 482 nm and emission at 505 nm and the second measurement with an excitation set at 469 nm and emission at 501 nm.

### 2.2. Biofilm staining assay

Since a biofilm, among other things, consists of a matrix of different polysaccharides, we decided to measure the production of polysaccharides as our second method to quantify biofilm formation.
We decided to stain the polysaccharides produced by the different *P. sp.* IsoF strains, using a Congo Red derived dye.
This dye stains different polysaccharides and it was kindly provided by the lab.

For the biofilm staining assay, we prepared two square plates, each containing 50mL of LB medium with agar and 1.25 ml of the Congo Red derivative (2mg/mL).
In one of the plates, we added 1.25 ml of 40% rhamnose, while the other plate remained without rhamnose.
Alongside the *P. sp.* IsoF strains carrying different DGC genes and the sgRNA to downregulate the PDE, we used the following strains as negative controls: *P. sp.* IsoF wt, *P. sp.* IsoF dCas9, *P. sp.* IsoF pBBR1MCS5 (containing the empty plasmid) and *P. sp.* IsoF pBBR1MCS5 PA5295 (constitutively overexpressed PDE).
*P. sp.* IsoF pBBR1MCS5 YedQ (constitutively overexpressed DGC) served as a positive control.

We prepared two sets of overnight cultures. In one set, we added 1% rhamnose to each overnight to induce the expression or inhibition of the enzymes.
The second set of cultures, without rhamnose, served as a control to test whether the rhamnose itself influenced intracellular c-di-GMP concentration.
We adjusted the cultures to an optical density of 1 and plated 10ul of each adjusted strain onto their respective plate, either to the one containing rhamnose or the one without.

The plate was incubated at 30°C for three days.
After incubation, the fluorescence, indicating the level of stained polysaccharides, was measured using a fluorescence microscope.
The images were processed and analyzed using imageJ.

### 2.3. Swimming Motility Assay

We measured the motility of our bacteria, as reduced motility has been linked to increased biofilm formation.[2](#ref2)
The assay was conducted using 0.3% semisolid agar plates.
The strains tested included the *P. sp.* IsoF wt DGC and its two mutations, R196A and R240A.
Additionally, we evaluated *P. aeruginosa* WspR wt, its mutation R242A and also the strain containing the sgRNA to knockdown a PDE.
Our goal was to determine whether these mutations resulted in decreased motility, which is beneficial for the biofilm.
Controls included *P. sp.* IsoF wt dCas9 (no plasmid) and *P. sp.* IsoF wt with the empty plasmid pBBR1MCS5.
*P. sp.* IsoF pBBR1MCS5 YedQ, containing a constitutively expressed DGC, served as a positive control while *P. sp.* IsoF pBBR PDE served as a negative control.

We adjusted the cultures to an OD600 of 0.25 and plated 10 µl of the sample. After incubating the plates overnight we measured the largest swimming zone diameter of each plate.

## 3. Plant Experiment

---

Our aim was to measure the effectiveness of our bacterial treatment in boosting plant growth under desertified conditions.
We inoculated the soil of the plants with four different treatments, each treatment condition consisting of 3-4 plants.
The first set of plants received sterile LB medium (negative control), the second received LB medium + *Pseudomonas sp.* IsoF wt. with an empty backbone (negative control), the third received LB medium + *P. sp.* PA5295, an upregulated PDE (negative control), the last set of plants received *P. sp.* YedQ, an overexpressed DGC (positive control).

The initial idea of the plant experiment was to test our final construct, but unfortunately we were not able to clone it in time for this experiment.
Therefore, we decided to use the plant experiment to test the general hypothesis of the project: does an enhanced biofilm have a beneficial effect on the plant?

To see if the bacteria does have an influence, we measured the state of the plant before and after two weeks of our bacterial treatment.
We decided to measure the weight of the rhizosphere, the weight of the aerial part of the plant (wet and dry) and the CFUs within the rhizosphere.

To weigh the rhizosphere, we uprooted the plant from the soil, carefully removed the soil that was still attached and placed the composite in a small plastic bag and weighed it. We subtracted the weight of the individual plastic bag from each sample.

We then proceeded with measuring the colony forming units (CFU) in our extracted rhizosphere.
For this, we added 3 ml of NaCl solution (0.9%) to the bag, sealed it and gently agitated the bags for 3 minutes to ensure the solution made contact with the roots and rhizosphere. We transferred 1 ml of this saline solution + rhizosphere composition into an Eppendorf tube.
We then performed a dilution series with 6 x 1:10 dilutions.
After this we plated each with the right antibiotic (our bacteria was kanamycin resistant) and the antifungal Cycloheximide.

We weighed the aerial part of each plant by putting it in a falcon tube (wet weight).
The samples were dried for 3 days in the oven at 55°C and weighed them again (dry weight).
We subtracted the weight of the individual falcon tube from each sample.

The first set of measurements was taken immediately after the treatment.
This consists of one plant per bacterial treatment.
The second set of measurements was taken two weeks later, ecording data from the rest of the plants (3 plants per condition and strain inoculated).
During these two weeks we set up two different conditions for the plant: dry and wet.
The first group received 25 mL of water every 4 days, mimicking drought conditions, whereas the second group received 50 mL of water each day, which mimics normal wet condition.
We did this to determine whether an overproduction of biofilm would help a plant experiencing drought, compared to a plant under normal wet conditions.

## 4. Toxin Assay

---

### Toxin assay 1

#### Overview

In order to have a functional kill switch, all parts of it needed to be fine tuned to each other.
First, the toxin expression has to be optimized.
The aim is to get a high enough expression to kill the cell, but low enough for the antitoxin production to override the toxin’s effect.
To alter the toxin expression we decided to test different RBS strengths: a weak, medium and a strong one.
The idea of our final construct is to have the toxin production regulated by the feedback loop, while the antitoxin production is linked directly to the xylose sensing system.
This effectively binds our bacteria to the plant roots, preventing the unwanted spread of our bacteria.

#### Experiment

Cells are grown in liquid culture, adjusted to an OD600 of 0.01 and grown again for 4 hours to get the cells from their resting into a metabolically active phase.
Since in this first experiment the toxin expression is induced by rhamnose each strain is then inoculated with 0%, 1% and 1.5% rhamnose, the latter two inducing the rhamnose promoter.
Triplicates of 1 ml of each are plated in a 96-well plate and their OD600 is measured periodically overnight (shaking, 37°C) using a plate reader.
Although measuring cell growth via OD600 is not ideal—since cell death is detected only when the growth curve flattens, making it difficult to distinguish between bacterial killing and the natural plateau phase—it should be still sufficient to detect the effectiveness of our toxin.
A growth curve with an earlier and lower plateau would suggest growth inhibition in the bacterial cells, indicating the toxin's impact.

The toxin/antitoxin system CcdB/CcdA was designed for *E. coli* and we therefore decided to test its functionality first in *E. coli* before implementing it into *P. sp.* IsoF.
For those experiments, a constitutively expressing GFP plasmid (pJUMP29-1D(sfGFP)) was included as a negative control.
To ensure we could see growth inhibition by measuring the OD600, we induced the same strain with a sufficient amount of Kanamycin to inhibit growth.
For the three strengths of toxin expression, plasmids pT41Rhyz01, pT41Rhyz02 and pT41Rhyz03 were transformed into the respective *E. coli* strain tested.

The same experiment was done in *P. sp.* IsoF.
A strain with an empty plasmid (pBBR1MCS5) and the wild type IsoF strain with no plasmid was used as negative controls.
The positive control was *P. sp.* wildtype strain again induced with enough Kanamycin to observe growth inhibition.
For the three strengths of the toxin expression, the plasmids pBBR pT41Rhyz01, pBBR pT41Rhyz02 and pBBR pT41Rhyz03 were used.
The toxin test in *P. sp.* IsoF was also modified, as its optimal growing conditions are different.
The cells were grown and measured under 30°C and the time allocated to adjusting the cultures to a metabolically active phase had to be extended as the average replication time of *P. sp.* IsoF is ~1.5 - 2hrs.

Over the course of the project, we adjusted the protocol to only grow the cells for another 30 - 60min after they were adjusted to an OD600 of 0.01, as this proved to be a more efficient way to reach the same result.
This adjustment was specific to the growth rate of our cells and would have to be adjusted individually to the application if this protocol were used.

### Toxin assay 2

After several failed attempts with the toxin assay described above, we realized that measuring OD values was not the optimal method for assessing the toxin's effect.
Further research revealed that previous iGEM teams had conducted similar toxin experiments using inductive plates.
Based on this, we decided to adopt the same approach.

We plated the toxin-expressing strains with different ribosome binding site (RBS) strengths (weak, medium, strong) on agar plates.
We added rhamnose at concentrations of 1% and 1.5% to induce toxin expression.
Additionally, we plated the strains on agar without rhamnose as a negative control.

After incubating the plates overnight at the appropriate temperatures (37°C for *E. coli* and 30°C for *P. sp.* IsoF), we performed a classical colony-forming unit (CFU) count.
The fewer colonies observed compared to the controls indicated a higher likelihood that the toxin was effectively killing the bacteria.

### Next Steps in the Toxin Experiment

The next phase of the experiment would have been to test the balance between the toxin and antitoxin.
We planned to include the antitoxin in the construct and observe if its expression, alongside the toxin, would "rescue" the bacteria.
If the bacteria survived while both the toxin and antitoxin were being expressed, it would suggest that the expression levels of both proteins were balanced.
However, we did not have sufficient time execute this experiment.

Up to this point, both the toxin and antitoxin were expressed under the control of the rhamnose promoter, rather than the promoters intended for the final construct.
In the final version, the toxin was meant to be regulated by the T7 promoter, while the antitoxin would be controlled by the xylose-inducing system.
To make the kill switch functional in the final construct, it would have been necessary to design additional experiments to fine-tune the expression of both proteins under this different regulatory system.

## 5. Feedbackloop Assay

---

Due to lack of time, we did not carry out the following experiments which would have been conducted to test the functionality of our feedback loop.
First, we wanted to test whether the T7 RNA polymerase would activate the T7 promoter.
We constructed a level 2 plasmid so that if it did, GFP would be expressed.
We planned on using a plate reader to measure the fluorescence every hour for 8 hours (with excitation at 485 nm and emission at 535 nm). The fluorescence intensity would then be adjusted to the OD600 value of the culture at the time.

We would have used a constant GFP expressing strain as a positive control (*E. coli* SY327 pJUMP28-1A(sfGFP)) and an empty backbone as a negative control (*E. coli* SY327 pBP) to remove background fluorescence.

The goal of the second part would then be to determine whether the feedback loop in the construct introduced a positive feedback mechanism.
Specifically, we wanted to see if activation of the loop would progressively increase RNAP expression rather than maintaining it at a constant level.

To test this, we compared two Level 2 plasmids.
Both contained the RNAP and T7 promoter systems and expressed GFP as a readout for measuring and comparing expression levels.
One plasmid mimics the feedback loop since contained two RNAPs and two T7 promoter, while the other doesn't mimic the feedback loop as it only contains one RNAP.
If the plasmid with the feedback loop produced more GFP, it would suggest that the feedback loop enhances the final expression of the gene controlled by the T7 promoter, confirming its role as a positive feedback loop.
The fluorescence would have been measured the same way as described above.

The third part of the experiment would have involved testing whether this feedback loop was also autoinductive—meaning that, once activated, it could remain active without an external source.
However, due to time constraints, we were unable to conduct any tests related to the feedback loop experiment.
Consequently, we did not think and develop a specific experiment to test this final requirement.

## Protocol

---

Show Protocol

[Download Protocol](https://static.igem.wiki/teams/5250/project/experiments/updated-experiments-protocols.pdf)

## References

{% for source in sources %}

[{{ source.names }} *{{ source.title }}*](%7B%7B%20source.link%20%7D%7D)

{% endfor %}

{% endblock %}

{% set sources = [
{"names": "[1] Hailing Nie, Yujie Xiao, Jinzhi He, Huizhong Liu, Liang Nie, Wenli Chen and Qiaoyun Huang. (2020).",
"title": "Phenotypic-genotypic analysis of GGDEF/EAL/HD-GYP domain-encoding genes in Pseudomonas putida. Environmental Microbiology Reports, (12(1), 38-48).",
"link": "https://doi.org/10.1111/1758-2229.12808"},
{"names": "[2] Minh-Hoa Dao-Thi, Laurence Van Melderen, Erwin De Genst, Hassan Afif, Lieven Buts, Lode Wyns, Remy Loris. (2005).",
"title": "Molecular Basis of Gyrase Poisoning by the Addiction Toxin CcdB, Journal of Molecular Biology (V. 348, I. 5, pg 1091-1102).",
"link": "https://doi.org/10.1016/j.jmb.2005.03.049"},
{"names": "[3] Le Roux FBinesse J, Saulnier D, Mazel D. (2007).",
"title": "Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector. Appl Environ Microbiol73",
"link": "https://doi.org/10.1128/AEM.02147-06"},
{"names": "[4] Ferenc, M. (2024).",
"title": "Plasmids 101: Common Lab E. coli Strains.",
"link": "https://blog.addgene.org/plasmids-101-common-lab-e-coli-strains"}
] %}
{% extends "layout\_TOC.html" %}
{% set header\_background\_url = "https://static.igem.wiki/teams/5250/project/results/headpage-41.jpg" %}
{% block title %}{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

## Achievements

---

* We demonstrated that knocking down a native phosphodiesterase in *P. sp.* IsoF significantly enhances biofilm formation.
* We demonstrated that introducing certain diguanylate cyclases (DGCs) can significantly increase c-di-GMP levels, promote polysaccharide production and therefore enhance biofilm formation.
  -> We were able to successfully target both enzyme families involved in c-di-GMP production to increase its intracellular concentration.
* We demonstrated that targeting the I-site of a DGC can lead to an increased activity of an enzyme.
* We successfully modified the enzyme's I-site to stop c-di-GMP from binding to the negative allosteric binding site which effectively enhanced the enzyme's activity. This has never been done before. One of our mutated DGCs, DGC PisoF R196A, led to the higher c-di-GMP levels compared to the wildtype DGC PisoF sequence in some experiments.
* We demonstrated that *P. sp.* IsoF with enhanced biofilm-forming ability can promote the growth of Micro Tom Tomato.

## Enhancing Biofilm Production

---

### 1. Phosphodiesterases

**Aim:** Our aim was to knockdown PisoF\_02645 to increase intracellular c-di-GMP concentration and polysaccharide production, reduce bacterial motility and therefore enhance biofilm formation.

Phosphodiesterases (PDEs) degrade c-di-GMP and our goal was to therefore knock down one of the native PDEs in *P. sp.* IsoF.
We chose to knock down PisoF\_02645, a strong PDE according to Hailing Nie et al.[1](#ref1).
Since we want the gene to only be downregulated when there is xylose, we decided to use a CRISPR interference system (CRISPRi), instead of a knockout.

After cloning our sgRNA into the plasmid and transforming it into *P. sp.* IsoF we performed a c-di-GMP assay, a biofilm staining assay and a swimming motility assay.

#### 1.1 c-di-GMP assay

To optimize the protocol for the c-di-GMP assay, we conducted several preliminary measurements.
These pre-measurements aimed to identify the optimal OD and growth conditions to refine our experiment.
Our pre-measurements confirmed what we had previously heard from our experts: a low OD of 0.25 is optimal for this assay, whereas higher OD levels seem to lead to rapid oversaturation.
Additionally, we tested two different media - LB medium and saline solution - and concluded that the saline solution produced more favorable results.
We observed that incubating the strains overnight with rhamnose resulted in poor gene expression, likely due to the depletion of rhamnose over time.
We determined that the optimal induction period for the cultures was up to 5 hours after adjusting them to an OD of 0.25.

Built on these findings, we proceeded to conduct three c-di-GMP assays, focusing on the effects of PDE and Diguanylate cyclase ([see section “Diguanylate cyclases“](#section-6)) activity under the identified optimal conditions.

![ Fluorescence Intensity from c-di-GMP Assay Across Different Strains, Experiment 1 and 2](https://static.igem.wiki/teams/5250/project/results/img-01-1.png)

**Figure 1: Fluorescence Intensity from c-di-GMP Assay across different Strains, Experiment 1 and 2:** The box plot shows the fluorescence intensity measurements for various Pseudomonas sp. IsoF strains in a c-di-GMP assay, representing the c-di-GMP levels in each strain.

Figure 1 shows the first two assays summarized in one graph.
The first strain tested is *P. sp.* IsoF, containing the sgRNA to knockdown the PDE PisoF\_02645.
The second through seventh strains are our DGC constructs, that we created with the intention of enhancing biofilm production.
We included three different negative controls, *P. sp.* IsoF with and without plasmid as well as a strain that contains a plasmid overexpressing PDE.
Since PDE is inhibiting c-di-GMP production, we expected to see only little c-di-GMP production in this strain.
This can’t really be shown from this graph. However, there seems to be a high variance between different values of the PDE, suggesting that more data points would be needed for a conclusive analysis.
Further, we included a positive control, *P. sp.* IsoF YedQ, which is known to increase biofilm production.
However, an increase compared to the control can’t be observed.
After analyzing our controls, we decided to handle our results with caution.
Interestingly, we observed an increase in c-di-GMP levels in the controls between the no rhamnose and rhamnose conditions, suggesting that the addition of rhamnose may naturally elevate c-di-GMP levels or rhamnose could lead to background noise in the fluorescence signal.

Although not confirmed by statistical analysis, our designed PDE knockdown visibly increased c-di-GMP levels when induced with rhamnose compared to the no rhamnose condition.
Furthermore, we did not observe a significant increase compared to the 'no plasmid' control.
This contradicts the result from the biofilm staining assay, so to address this issue, we would need to conduct further experiments with a larger sample size.

#### 1.2 Biofilm staining assay

We performed three biofilm staining assays.
The first assay only contained the strain with the sgRNA, knocking down the PDE.
The other two included the strains expressing the different DGCs and also the PDE knockdown.

After two days of incubation, we examined the plates with a fluorescence microscope.
We took pictures of the fluorescence from different cultures and processed them with ImageJ to quantify the fluorescence levels.

![Biofilm staining assay 1 point plot](https://static.igem.wiki/teams/5250/project/results/img-02-1.png)

**Figure 2: Biofilm staining assay 1 point plot**

Figure 2 represents data from the first biofilm assay where we only measured the PDE activity.
The figure shows the mean fluorescence of each strain.

*P. sp.* IsoF wt, *P. sp.* IsoF dCas9 and *P. sp.* IsoF with an empty pBBR1MCS5 plasmid served as negative controls.
*P. sp.* IsoF YedQ contained a plasmid with the constitutively expressed DGC YedQ and served as a positive control.

*P. sp.* IsoF PDE knockdown refers to *P. sp.* IsoF that contains a plasmid with our designed sgRNA.

The dCas9 on the bacteria’s genome is controlled by a rhamnose promoter and thus activates our CRISPRi system upon induction.
We investigated the effect of activating the CRISPRi system on polysaccharide production by using *P. sp.* IsoF dCas9 sgRNA in both the presence and absence of rhamnose.
To determine whether rhamnose itself affects the production of biofilm components, we also tested the control strain, *P. sp.* IsoF dCas9 once induced with rhamnose and once not induced.
The controls showed lowest fluorescence, indicating the least amount of polysaccharide production.
In contrast, *P. sp.* IsoF YedQ displayed the highest fluorescence, reflecting the most polysaccharide production.
*P. sp.* IsoF dCas9 with the sgRNA showed higher fluorescence than the controls, but lower than *P. sp.* IsoF YedQ.
This suggests that the sgRNA inhibits the PDE PisoF\_02645, leading to increased c-di-GMP levels and higher polysaccharide production.
This is also shown in the raw data, as can be seen in Table 1. The table shows the mean fluorescence of the different strains.
The strain that contains the sgRNA and therefore inhibits the PDE on its genome, has increased fluorescence signal, and thus produces more polysaccharides, compared to the negative controls but shows less signal than the positive control.
The presence of the sgRNA alone leads to an increase in polysaccharide staining but as shown in the graph, inducing the strain containing the sgRNA with rhamnose did not result in a big difference compared to the non-induced strains.
Polysaccharide production did not increase substantially when dCas9 was induced compared to when it wasn’t, suggesting that the rhamnose promoter in front of dCas9 may be leaky.
Discussions with our advisor confirmed that similar results were observed in other gene knockdown experiments.
No statistical tests could be performed as we only had one replicate per strain for this assay.

![Table with mean fluorescence](https://static.igem.wiki/teams/5250/project/results/img-03-1.png)

**Table 1: Table with mean fluorescence**

In the second biofilm staining assay we measured the strains containing the different DGCs and the strain containing the PDE knockdown.
All strains, including the controls, were once inoculated with rhamnose and once without rhamnose, to determine whether rhamnose itself affects biofilm component production.
Each strain and condition was tested in duplicates.
The data, shown in Figure 3, shows similar results to the first assay in Table 1.
Fluorescence was higher in the *P. sp.* IsoF strain containing the sgRNA to knock down PisoF\_02645 (labeled as “PDE knockdown” in this assay) than in the control strains.
While rhamnose induction caused a change in fluorescence, the increase was not as strong as in other induced strains.
Two different rhamnose promoters were used, one for the knockdown and one for the different DGCs.
This result again supports the assumption that the first rhamnose promoter is indeed leaky, whereas the second promoter seems to be less leaky.

Using a linear model with the bacterial strain as the explanatory variable (10 categories) and mean fluorescence as the response variable, we observed a significant increase in fluorescence in the PDE knockdown (next to other DGC strains described below).
The p-value of the strain containing the PDE knockdown is 0.002.
The adjusted R-squared for the model is 0.9733, with an F-statistic of 78.35.
The explained variance of the bacterial strain variable is 18’046.4, while the unexplained variance is 259.1, with an overall p-value of 2.062e-07.

![Biofilm staining assay 2 point plot](https://static.igem.wiki/teams/5250/project/results/img-04-1.png)

**Figure 3: Biofilm staining assay 2 point plot**

#### 1.3 Swimming Motility assay

To assess the swimming motility of the different bacterial strains we measured the growth diameter after incubating them overnight on 0.3% semisolid agar plates.
We performed two swimming motility assays, although we were not able to record two datasets for all strains as some of the plates melted.
As PDEs degrade c-di-GMP, we expect to see less motility in strains with an overexpressed PDE and vice versa more in strains where the PDE is knocked down.

Figure 4 shows that the knocked down PDE shows a decreased motility when rhamnose is added, as expected. With the PDE knocked down, c-di-GMP is not degraded, increasing the potential for biofilm formation, which aligns with our expectations.

![Figure 4: Swimming Motility Assay point plot](https://static.igem.wiki/teams/5250/project/results/img-05-1.png)

**Figure 4: Swimming Motility Assay point plot**

**Conclusion:** All three experiments confirm that PisoF\_02645 was successfully knocked down, leading to an increased c-di-GMP concentration, enhanced polysaccharide production and restricted motility.
We achieved our goal of knocking down PisoF\_02645 which in consequence enhances biofilm formation.

### 2. Diguanylate cyclases

The second enzyme family we want to target are the diguanylate cyclases (DGCs).
DGCs produce cyclic di-GMP.
We tested 5 strains with different DGCs: two wildtype sequences, one native to *P. sp.* IsoF (PisoF\_00565) and one native to *P. aeruginosa* (WspR).
Additionally a mutant version of WspR (WspR R242A) and two mutated sequences of PisoF\_00565, that we designed and mutated by ourselves (DGC PisoF R196A and DGC PisoF R240A) were tested.
The aim of these mutations is to enhance the enzyme’s activity by inhibiting their I-site.

#### 2.1 c-di-GMP assay

**Aim 1:** We want to prove that the introduction of a DGC can indeed increase biofilm production in *P. sp.* IsoF.

**Aim 2:** We want to determine whether we can enhance the enzyme’s activity by modifying the allosteric binding site of c-di-GMP by mutating the sequence of PisoF\_00565 by ourselves or by using the already established mutated sequence of WspR.

To test our DGC constructs, we applied the same c-di-GMP assay as described for the PDE, with the findings illustrated in Figure 1 (shown again for clarity).

![ Fluorescence Intensity from c-di-GMP Assay Across Different Strains, Experiment 1 and 2](https://static.igem.wiki/teams/5250/project/results/img-01-1.png)

**Figure 1: Fluorescence Intensity from c-di-GMP Assay across different Strains, Experiment 1 and 2:** The box plot shows the fluorescence intensity measurements for various *P. sp.* IsoF strains in a c-di-GMP assay, representing the c-di-GMP levels in each strain.

First, we wanted to assess whether the c-di-GMP level increases when the strains are induced with rhamnose, as our constructs are only activated when induced with rhamnose.
If a change in c-di-GMP levels is observed, it would suggest that this change results from the introduced DGC.

Our mutations, DGC PisoF\_00565 R196A and DGC PisoF\_00565 R240A, labeled as DGC PisoF R196A and DGC PisoF R240A in the graph, showed a significant increase in activity when induced with rhamnose compared to the condition without rhamnose.
This suggests that our constructs were functioning as expected and that the introduction of a DGC increases c-di-GMP levels. (R196A: t = 6.4794, df = 7.7563, p-value = 0.0002199, R240A t = 6.4794, df = 7.7563, p-value = 0.0002199).

When comparing the difference in average c-di-GMP levels between the rhamnose and no rhamnose conditions, we observed a larger difference between our DGC mutations and our control, confirming that this variation is not merely due to background noise:

* No Plasmid (Rhamnose) - No Plasmid (No Rhamnose): 96.00 RFU
* DGC R196A (Rhamnose) - DGC R196A (No Rhamnose): 240.33 RFU
* DGC R240A (Rhamnose) - DGC R240A (Rhamnose): 144.533 RFU

The other *P. sp.* IsoF strains containing a DGC (DGC PisoF, DGC WspR and DGC WspR R242A) also showed a visible increase in c-di-GMP levels, however the difference was not significant.
This indicates that different DGCs lead to different increases in intracellular c-di-GMP levels but that in general the intracellular c-di-GMP concentration is elevated by introducing a DGC.

Further we aimed to assess whether the mutations of the DGC PisoF we created indeed leads to increased activity compared to the wild-type DGC.
The mutation of the DGC PisoF R196A, in which we exchanged an arginine at position 196 with alanine, showed a significantly higher c-di-GMP level than the wildtype DGC PisoF sequence (t = 2.5872, df = 8.9978, p-value = 0.02936).

**Our mutated enzyme therefore shows higher activity than the wildtype enzyme. This indicates that we successfully mutated DGC PisoF\_00565, specifically the enzyme’s I-site, by removing its negative regulation through c-di-GMP. So by altering the sequence of the DGC we prevented c-di-GMP from binding to the negative allosteric site, which enhanced the enzyme’s activity.**

This has never been done or shown before for any DGC native to *P. sp.* IsoF.
Our second mutation, DGC PisoF R240A on the other hand did not show a significant increase compared to the wildtype enzyme, suggesting that the I-site was not successfully modified.

Next, we compared our constructs to a control *P. sp.* IsoF dCas9 without plasmid to quantify the change in c-di-GMP production.
Several of our tested strains show a notable increase in c-di-GMP production compared to the control *P. sp.* IsoF dCas9 no plasmid, however only the mutated DGC WspR (DGC WspR R242A) showed statistical significance for it (t = 2.4238, df = 7.3234, p-value = 0.04433).
The high variability in the DGC WspR might suggest that while the construct has a promising potential to achieve high c-di-GMP levels, further optimization is required to achieve more consistent results.
Although our mutations of the DGC native to *P. sp.* IsoF (DGC PisoF R196A and DGC PisoF R240) did not show a significant increase in c-di-GMP levels compared to the empty plasmid, we believe that obtaining more data points could provide a clearer picture, as there appears to be a noticeable difference in c-di-GMP levels visually.
Further, when compared to *P. sp.* IsoF dCas9 empty plasmid, DGC PisoF R196A shows a significant increase (t = -2.9931, df = 5.3119, p-value = 0.02814) indicating inconsistencies in the data, since no difference between the two controls is expected.

A third experiment was excluded from the previous analysis because the values observed in the no-rhamnose condition were significantly different from those in the first two experiments.
This discrepancy suggested that the experiments might not be directly comparable, possibly due to unintended variations in the experimental setup.
The results of the third experiment can be seen in Figure 5.

![Figure 5: Fluorescence Intensity from c-di-GMP Assay Across Different Strains, Experiment 3.](https://static.igem.wiki/teams/5250/project/results/img-06-1.png)

**Figure 5: Fluorescence Intensity from c-di-GMP Assay Across Different Strains, Experiment 3.**

Since there was only one data point per strain and condition, no statistical test was conducted.
Differences to the empty plasmid are only visible for the two mutated DGCs, DGC PisoF R196A and DGC PisoF R240A.
The difference in fluorescence signal of PisoF R196A compared to the empty plasmid is 239.00 and 329.00 for DGC PisoF R24 compared to the empty plasmid respectively.
This supports the findings from the data shown in Figure 1.
Introducing a DGC elevates c-di-GMP concentrations and again, it seems like the mutation of PisoF\_00565 at position 196 worked and the modified enzyme seems to have increased activity.

Based on the data of the different assays, we conclude that introducing a DGC increases the intracellular c-di-GMP concentration.
Additionally, the mutated enzyme DGC PisoF\_00565 R196A seems to have an increased activity compared to the wildtype DGC PisoF\_00565, indicating that its I-site was successfully mutated and c-di-GMP stopped from binding to it.

However, our findings also underscore the need for additional experiments to validate and better characterize these effects.

#### 2.2 Biofilm staining

Figure 3 (shown again below) represents the fluorescence data of the strains containing the five different DGCs (alongside the strain containing the sgRNA to knockdown PDE).
The figure illustrates the mean fluorescence for each strain.
*P. sp.* IsoF dCas9 and *P. sp.* IsoF with an empty pBBR1MCS5 plasmid served as negative controls, while *P. sp.* IsoF YedQ once again served as a positive control.
Each strain and condition (with and without rhamnose) was measured in duplicate.

![Figure 3: Biofilm staining assay 2 point plot](https://static.igem.wiki/teams/5250/project/results/img-04-1.png)

**Figure 3: Biofilm staining assay 2 point plot**

As observed in the first biofilm staining assay, the control strains showed the lowest fluorescence, suggesting minimal polysaccharide production.
In contrast, the bacterial strain expressing the constitutively active DGC YedQ showed the highest fluorescence, as expected.
Among the other tested strains, three showed a significant increase in fluorescence, indicating enhanced polysaccharide production: *P. sp.* IsoF DGC PisoF\_00565, *P. sp.* IsoF DGC WspR and *P. sp.* IsoF DGC WspR R242A.

The strain expressing the mutated WspR DGC (*P. sp.* IsoF WspR R242A) showed the highest fluorescence level, even exceeding the positive control (*P. sp.* IsoF YedQ).
This suggests that the mutated DGC WspR leads to the greatest increase in polysaccharide production and, consequently, the most significant enhancement in biofilm formation.

In contrast, the mutated variants of DGC PisoF\_00565 (DGC PisoF R196A and DGC PisoF R240A) did not show the same increase in fluorescent signal as the other DGCs.
While DGC R240A displayed a modest increase in fluorescence compared to the controls, it was not as distinct as in the other tested DGCs.

Interestingly, DGC PisoF R196A showed no measurable increase in fluorescence. This result contradicts the findings from the c-di-GMP assay, where the DGC PisoF R196A led to an increase in intracellular c-di-GMP levels.

Throughout different experiments, we observed that the strains carrying different DGCs had reduced growth efficiency when induced with rhamnose.
We have two hypotheses of why this could be the case. First, it could be that rhamnose is toxic to bacteria in high concentrations.
Alternatively, the overexpression of a DGC, leading to the overproduction of c-di-GMP, might be costly for the bacteria and thus inhibits their growth.
This could also explain why the strains containing different DGCs did not show a significant increase in polysaccharide production.
During the biofilm staining assay, we were unable to measure the OD of each strain, meaning that we don’t know how well each strain grew on the plate.
As a result, we could not normalize the polysaccharide production based on an OD measurement.

To further analyze the results, we applied the same linear model used in the previous experiments, with bacterial strain as the explanatory variable (10 categories) and mean fluorescence as the response variable.
The model showed a statistically significant increase in fluorescence for the following strains:

* *P. sp.* IsoF DGC PisoF\_00565 (p-value: 5.21e-07)
* *P. sp.* IsoF DGC WspR (p-value: 1.17e-06)
* *P. sp.* IsoF DGC WspR R242A (p-value: 2.16e-07)
* *P. sp.* IsoF DGC YedQ (p-value: 7.29e-07)

As noted, the strains containing the mutated forms of DGC PisoF\_00565 (R196A and R240A) did not lead to a significant increase in fluorescent signal.

The adjusted R-squared for the model is 0.9733, with an F-statistic of 78.35.
The explained variance from the bacterial strain variable is 18’046.4, while an unexplained variance of 259.1 remains, with an overall p-value of 2.062e-07.

In conclusion, the data from these biofilm staining assays confirm that certain DGCs, particularly WspR and its mutated form WspR R242, significantly enhance polysaccharide production.
WspR R242 demonstrates the greatest increase in biofilm formation overall.
Surprisingly, the mutated forms of DGC PisoF\_00565, R196A and R240A did not lead to a notable increase in polysaccharide production.

These results confirm that introducing certain DGCs can significantly increase and therefore promote polysaccharide production, which represents an enhanced and stronger biofilm formation.
Elevating polysaccharide production through the introduction of a DGC provides an efficient and straightforward approach to boost biofilm formation, without the need to focus only on specific genes in the genetic pathway to produce one or two polysaccharides.

#### 2.3 Swimming Motility DGCs

As DGCs drive biofilm formation by producing c-di-GMP, we expect to see a shift in our DGC strains from a motile to a more sessile state in which biofilm-formation can take place.

Figure 4 (shown again below) shows that the DGC WspR and the mutated form WspR R242A show the highest motility (indicated by the swimming motility diameter) when the DGC is not induced by rhamnose.

![Figure 4: Swimming Motility Assay point plot](https://static.igem.wiki/teams/5250/project/results/img-05-1.png)

**Figure 4: Swimming Motility Assay point plot**

The high motility levels suggest that these strains may be in a state with less biofilm formation.
However, in the presence of rhamnose, their motility drastically decreases in the first assay (the effect is not as drastic in the second one).
This indicates that when c-di-GMP is further increased due to DGC activity, motility decreases as expected, which aligns with an increased potential for biofilm formation.
The sharp drop in motility suggests that WspR likely contributes heavily to biofilm formation when expressed.

Our mutations DGC PIsoF R240A and R196A show lower motility in both conditions, yet the rhamnose condition shows higher motility than no rhamnose condition.
This suggests that this DGC may not be as important as the WspR in decreasing the motility for example.
Our controls *P. sp.* IsoF no plasmid, empty plasmid and PDE overexpression show higher levels than our rhamnose induced DGCs.

These results confirm that for some DGCs, such as DGC WspR and DGC WspR R242A, the relationship between DGC activity, motility, and the biofilm formation process is strongly correlated.
While it is not clear why our DGC mutations R196A and R240A show higher motility with rhamnose, this is also seen when rhamnose is added to the DGC YedQ.
This may suggest that rhamnose could promote bacterial motility in other metabolic contexts, separate from DGCs.

**Conclusion:**
We successfully achieved our goal of promoting biofilm formation through the implementation of DGCs in *P. sp.* IsoF.
Introducing a DGC into *P. sp.* IsoF increased intracellular c-di-GMP levels, enhanced the production of polysaccharides and reduced bacterial motility.
All key factors for a robust biofilm formation.
These findings suggest that the introduction of a DGC effectively enhances biofilm formation.
Elevating intracellular c-di-GMP levels through the introduction of a DGC therefore provides an efficient and straightforward approach to boost biofilm formation by regulating different genes, without the need to focus only on specific genes in the pathway.

## Plant experiments

---

###

**Aim:** We inoculated plant roots with three different bacterial strains of *P. sp.* IsoF and with LB medium to assess their effects on the root and shoot weight in normal and drought conditions.

Raw data from the plant experiment was obtained on the date of inoculation - time point 0, and two weeks later.

The plants were divided in 4 groups that underwent different treatments: PisoF with an empty pBBR1MCS5 plasmid, PisoF YedQ, PisoF PDE overexpression and LB medium.
Half of the plants were regularly watered, in the other half we induced drought.
For more information about the set up of the plant experiment and the processing of the plants go to the [Experiments Page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).

#### Time point 0

One plant out of each treatment for the drought and normal/watered conditions was taken out and processed on the first day after 30 minutes of inoculation.
The initial measurements are summarized in the following graph:

![Figure 6: Initial measurements of the plants at time point 0 (30 minutes after inoculation)](https://static.igem.wiki/teams/5250/project/results/img-07.png)

**Figure 6: Initial measurements of the plants at time point 0 (30 minutes after inoculation):**
The net weights of the wet and dry aerial part, as well as the net weight of the rhizosphere is included in the measurements.

Figure 6 represents the time point 0 measurements that serve as a baseline for our analysis. Based on these values we can make observations about the growth that the rest of the plants in the same treatment group exhibit.

#### Wet weight Measurement after 14 days

We conducted the second measurement after 14 days. The remaining plants from each treatment in the drought or wet conditions respectively were processed.
The data points are depicted in Figure 7:

![Figure 7: Net dry and wet weight of the treatment groups under normal and drought conditions.](https://static.igem.wiki/teams/5250/project/results/img-08.png)

**Figure 7: Net dry and wet weight of the treatment groups under normal and drought conditions.**
In green is the net wet weight of all plants processed at Time point 0.

As expected, the drought conditions result in an overall lower mass of the aerial part of the plant than the normal conditions.
However, under drought conditions there is barely a difference in the mean values between the treatments.
This contradicts our hypothesis that the bacterial treatments would result in a higher biomass than the LB medium treatment.
We think this might be due to the drought treatment being too harsh, possibly resulting in conditions too dry for the bacteria to have any beneficial effect.
In addition, the variance in the data points we obtained from the LB treatment was too high for us to effectively draw a conclusion from it.
The same applies to all the data points obtained from the wet measurements.

One notable observation is that the PDE overexpression treatment exhibits lower biomass values than the PisoF empty pBBR1MCS5 plasmid and the PisoF DGC overexpression plasmid in both drought and normal conditions.
This is consistent with our expectations, since the PDE overexpression plasmid leads to more c-di-GMP degradation, less biofilm and therefore less plant growth.

In order to be able to draw conclusions about the plant growth under different treatments and conditions, we took the average biomass of all plants measured at time point 0 and compared it to the mean values from each box plot in the graph above.
In the drought condition there is no growth observed. In fact, the wet weight of the biomass decreases after 14 days of drought. This is likely due to the fact that many plants were severely dehydrated, which suggests that water loss is the cause of the decreased measured weight.
In normal (non-drought) conditions, on the contrary, one can observe growth in all treatments. However, the variation between the individual data points we obtained within each treatment is high.
Therefore, it is difficult to draw precise conclusions about the effect of the treatments on the weight of the biomass, especially since sample size was rather low. If this experiment is repeated in the future, we would need to make sure to include more plants in the experiment for a more reliable statistical analysis.
Overall though we can observe the highest increase in growth after the DGC overexpression treatment, which is consistent with our hypothesis.

#### Dry weight Measurement after 14 days

After we processed the wet weight, we kept the plants in the oven for a few days until they were completely dry.
Then we measured them again to obtain the dry weight of all plants.
These measurements are illustrated in Figure 8 below:

![Figure 8: Net dry weight of the aerial part of the treatment groups under normal and drought conditions.](https://static.igem.wiki/teams/5250/project/results/img-09.png)

**Figure 8: Net dry weight of the aerial part of the treatment groups under normal and drought conditions.**
In green is the net dry weight of all plants processed at Time point 0.

There is a substantial difference between the dried weight of the plants from the drought condition and those grown in normal conditions.
This most likely arises due to the severe dehydration of the plants grown in drought.
The mean values of the dry plants are almost identical, which suggests that the treatments with our bacterial strains did not necessarily prevent the drought stress imposed on the plants.

Additionally, the results in the group of plants treated with bacteria under normal watered conditions are also inconclusive.
Visually, one can note that the highest mean value is at the PisoF DGC YedQ treatment, which is consistent with our expectations.
PisoF DGC YedQ is known to form more polysaccharides and an induced biofilm.
The PDE overexpression plasmid treatment which we use as a negative control also exhibits a mean value, lower than the two other bacterial treatments.
Against our expectations, the strain with an empty pBBR1MCS5 plasmid shows the same mean value as all plants processed at Time point 0.

However, there is high variability between the individual data points,therefore no meaningful observation can be made about the effect of the different treatment groups.
For future experiments and better statistical analysis, we would need to include more plants in our treatment groups.

Since the dry weight, but not the wet weight, increases with YedQ, we can infer that YedQ—and consequently, enhanced biofilm formation—improves water retention potential.
With a more optimized experimental setup, such as an extended growth period and younger, more actively growing plants, this effect might more effectively translate into improved plant growth.

#### CFU count

We conducted a colony forming unit count from the rhizosphere of each of the treated plants.
With the aim of establishing the number of *P. sp.* IsoF colonies 14 days after the treatment implementation we took a sample of the rhizosphere and performed a dilution series.
The diluted samples were plated and counted.
Different amounts of soil were stuck to the roots in each sample and we adjusted the count for 1g of rhizosphere.

![Figure 9: CFU/g rhizosphere from plants extracted at Timepoint 0](https://static.igem.wiki/teams/5250/project/results/img-10.png)

**Figure 9: CFU/g rhizosphere from plants extracted at Timpoint 0**

Figure 9 shows that the DGC YedQ and PDE overexpression strains have the highest number of surviving colonies, followed by the PisoF pBBR1MCS5 plasmid and LB.
The plates we used had a gentamicin resistance to help us select for our strains that carry the plasmids we introduced with a gentamicin resistance.
This aligns with our expectations and proves that *P. sp.* IsoF can survive in conditions mimicking the environment and retain the plasmids that have been introduced to it.
The difference between the wet and dry conditions can be explained by the fact that some of the bacteria might have been washed away when the wet plants were watered.

In addition, the data we obtained 14 days after inoculation represented in Figure 10 shows more relevant results.

![Figure 10: CFU/g rhizosphere from plants extracted after 14 days of growth](https://static.igem.wiki/teams/5250/project/results/img-11.png)

**Figure 10: CFU/g rhizosphere from plants extracted after 14 days of growth**

When it comes to a difference between the drought and normal conditions, we can observe the same trend as in the plants processed at Time point 0.
There are less CFUs in the wet rhizosphere potentially due to the bacteria being washed away in the process of watering.
The plants in the drought condition show that the bacterial treatments also result in a higher CFU count in the rhizosphere compared to the mean of the LB treatment.
On the other hand, the variance within the LB-treated samples is too high for us to make a notable observation.

For both the drought and the regularly watered condition the difference in the mean CFUs of the different bacterial strain treatments is barely recognizable.
This suggests that the genes being expressed on the pBBR1MCS5 backbone barely cause more strain to the bacteria than the presence of the empty backbone and the colonies are able to survive despite being genetically engineered.

**Conclusion:**
It can be said that while we did not observe a substantial increase in the biomass of the treated plants that underwent drought conditions, the results we observed from the regularly watered plants supported our main hypothesis - the enhanced biofilm of *P. sp.* IsoF is beneficial for plant growth.
Moreover, our CFU count experiment confirmed that our biofilm overexpression modules do not cause more of a metabolic strain on the bacteria in conditions mimicking the environment than the empty plasmid.
However, more samples are needed overall to conduct proper statistical tests and come to final conclusions.

## Killswitch

---

**Aim:** We want to design and finetune an established killswitch, ensuring the control and environmental safety of our product.

In order to finetune every part of the killswitch, first, the toxin expression has to be optimized.
We needed to find the sweet spot of toxin production: high enough to kill the cell, but low enough for the antitoxin production to override the toxin’s effect.
Expression levels are adjusted using three different strengths of ribosome binding sites (RBSs).

### Toxin Assay 1: OD Measurement

#### Toxin assay in *E. coli* SY327 (1)

![Figure 11](https://static.igem.wiki/teams/5250/project/results/ks1.png)

**Figure 11:**
As a negative control we used *E. coli* SY327 with an empty backbone of the same plasmids used for our toxins (pJUMP29-1D(sfGFP)).
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03).
We inoculated the strains with either 1% rhamnose or no rhamnose (+0%).

The toxin test did not work, as there was no significant difference in cell growth between the cells induced with rhamnose and the ones that grew without rhamnose.

Before testing our toxin again, we needed to rule out that the issue lies with the rhamnose promoter.

#### Rhamnose promoter characterisation in *E. coli* SY327

To test whether the promoter was the issue and at which rhamnose concentration its expression would be the strongest, we conducted a promoter characterisation.
We did so using a plasmid that had our rhamnose promoter with RFP as a reporter gene.

We performed a preliminary test which, after an overnight incubation with 1% rhamnose, exhibited a red color, confirming that the rhamnose promoter was active.

To quantify the promoter’s activity at different rhamnose concentrations and the potentially toxic or nutritious effect of rhamnose on the cell, we performed an overnight growth assay, measuring OD600 and fluorescence at excitation 530±25 nm and emission 590±35 nm.

![Figure 12](https://static.igem.wiki/teams/5250/project/results/ks2.png)

**Figure 12:**
Depicted is the fluorescence, and thus promoter activity at different rhamnose concentrations (1%, 1.5%, 2%, 2.5% and 3%). The values are adjusted to an OD600 of 1.00 to account for the fluorescence being stronger at higher cell numbers.
We additionally measured the autofluorescence of *E. coli* SY327 wildtype when inoculated with the same rhamnose concentrations.
We subtracted said autofluorescence from the promoter activity data to isolate the fluorescence obtained from the RFP production alone.

The promoter was detectably activated after 2hrs of inoculation with rhamnose, regardless of the concentration.
The highest fluorescence per OD and thus the strongest promoter activity was at 1% rhamnose, followed by 1.5%.
This means the promoter’s maximum activity with the minimum negative effect of rhamnose was at 1%.

From this test we conclude that the issue does not lie with the promoter and that the toxin test needs to be repeated.

#### Toxin Assay in *E. coli* SY327 (2)

![Figure 13](https://static.igem.wiki/teams/5250/project/results/ks3.png)

**Figure 13:**
Displayed are the OD600 of *E. coli* SY327 wildtype (wt) and said strain with an empty backbone (empty plas) of the same plasmids used for our toxins (pJUMP29-1D(sfGFP)) at 0%, 1% and 1.5% rhamnose.
As a positive control (pos cont), we added Kanamycin to the wildtype strain which is sensitive to this antibiotic.
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03).

In this second toxin test on *E. coli* SY327, we again observed no significant difference in the strains’ growth between the ones with an induced toxin production and the ones without.
We subtracted the growth of the empty plasmid control (pJUMP29-1D(sfGFP)) from the strains tested at the respective rhamnose concentrations.
This way, we would see the difference in growth caused by our toxin plasmid alone - expecting at least some slowed growth.

![Figure 14](https://static.igem.wiki/teams/5250/project/results/ks4.png)

**Figure 14:**
We subtracted the growth of the empty plasmid control (pJUMP29-1D(sfGFP)) from the strains tested at the respective rhamnose concentrations (0%, 1% and 1.5%).
This way, we see the difference in growth caused by our toxin plasmid alone.
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03).

At first, the medium toxin strength seemed to present the slowest growth in the first three hours.
On second examination this is purely due to the fact that the medium strength strains already started off with an OD600 0.06 lower than the control at the respective rhamnose concentrations. Secondly, there is no significant difference between 0% and 1% or 1.5% rhamnose, indicating that the variability is not due to a difference in toxin production.
Regardless, ccdB is expected to fully inhibit the cell’s growth, as it is a potent gyrase toxin, and would show a more drastic difference than seen above.[2](#ref2)

We concluded from this that the most likely cause for our toxin’s lack of effect is a gyrase mutation in *E. coli* SY327.
Upon further research and consultation with Dr. Zaira Heredia Ponce we found that *E. coli* SY327 is based on *E. coli* S17-1, which carries a gyrA462 allele - a gyrase mutation making cells immune to ccdB toxins[3](#ref3).
This allele has been deliberately transferred to several strains derived from *E. coli* S17-1, though we could not find evidence to support it was also transferred to *E. coli* SY327.
Still, we found this to be reason enough to assume our current *E. coli* strain had a gyrA mutation, wielding it immune to the ccdB toxin and thus explaining our toxin’s lack of effect. We therefore had to transform our plasmids into a new strain and repeat the toxin test.
After some brief research and consulting which strains were readily available by our host lab, we chose to go with *E. coli* DH5α.

#### Toxin assay in *E. coli* DH5α

![Figure 15](https://static.igem.wiki/teams/5250/project/results/ks5.png)

**Figure 15:**
Shown is the OD600 of *E. coli* DH5α with the toxin strengths and a control at respective rhamnose concentrations (0%, 1% and 1.5%).
As a control (GFP plas) we used a plasmid consisting of the same backbone as our toxins but with GFP, to have a comparable metabolic strain of the toxin, but without its toxic effect (p03Rhyz11).
As a positive control (pos cont), we added Kanamycin to the GFP plas strain which is sensitive to this antibiotic.
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03).

Once more, the cell’s growth seems to be unaffected by the ccdB toxin, as no significant difference was found between the control, the different toxin strengths, or their induction with rhamnose.
We revisited our plasmid construct to eliminate all possibilities of a design error.
While in our original research we found no gyrA mutations in *E. coli* DH5α, an AddGene blog entry by Mathew Ferenc shed light on a gyrase mutation (gyrA96) mutation carried by *E. coli* DH5α which, though not proven to provide resistance to ccdB, may affect the toxin’s effectivity.[4](#ref4)
We used Ferenc’s blog to screen for other potentially sensitive *E. coli* strains and noted the strains which our lab had available.
We decided to continue the testing of our toxin in HB101, MC1061 and CSH50.

#### Toxin assay in *E. coli* HB101, *E. coli* MC1061 and *E. coli* CSH50

![Figure 16](https://static.igem.wiki/teams/5250/project/results/ks6.png)

**Figure 16:**
Shown is the OD600 of *E. coli* HB101 with the toxin strengths and a control at respective rhamnose concentrations (0%, 1% and 1.5%). As a control (GFP plas) we used a plasmid consisting of the same backbone as our toxins but with GFP, to have a comparable metabolic strain of the toxin, but without its toxic effect (p03Rhyz11).
We also tested the *E. coli* HB101 wildtype strain (wt) to observe the effects of rhamnose on it.
As a positive control (pos cont), we added Kanamycin to the wildtype strain which is sensitive to this antibiotic.
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03).

![Figure 17](https://static.igem.wiki/teams/5250/project/results/ks7.png)

**Figure 17:**
We measured the OD600 of *E. coli* MC1061 with the toxin strengths and a control at respective rhamnose concentrations (0%, 1% and 1.5%).
As a control (wt) we used the *E. coli*MC1061 wildtype strain.
As a positive control (pos cont), we added Kanamycin to the wildtype strain which is sensitive to this antibiotic.
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03).

![Figure 18](https://static.igem.wiki/teams/5250/project/results/ks8.png)

**Figure 18:**
Shown is the OD600 of *E. coli* CSH50 with the toxin strengths and a control at respective rhamnose concentrations (0%, 1% and 1.5%).
As a control (wt) we used the *E. coli* CSH50 wildtype strain.
As a positive control (pos cont), we added Kanamycin to the wildtype strain which is sensitive to this antibiotic.
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03).

In all three *E. coli* strains tested above (HB101, MC1061 and CSH50), the cell’s growth seems to be unaffected by the ccdB toxin, as no significant difference was found between the controls, the different toxin strengths, or their induction with rhamnose.
There were still some differences between the tests such as *E. coli* MC1061 strains having more variability, *E. coli* HB101 overall growing to higher concentrations and the positive control in *E. coli* CSH50 seemingly growing slightly despite the presence of Kanamycin.
However, none of these differences in results affect the conclusion that our ccdB toxin system does not kill or inhibit any of our cell’s growth.
As at this stage of our project we were starting to run out of time, we decided to perform a last toxin test in *P. sp.* IsoF for the sake of thoroughness.

#### Toxin Testing in *P. sp.* IsoF

![Figure 19](https://static.igem.wiki/teams/5250/project/results/ks9.png)

**Figure 19:**
Shown is the OD600 of P. sp. IsoF with the different toxin strengths and a control at respective rhamnose concentrations (0%, 1% and 1.5%).
As a control (empty plas) we used the P. sp. IsoF with the same backbone from the toxin-carrying strains (pBBR1MCS5).
As a positive control (pos cont), we added Kanamycin to the wildtype strain which is sensitive to this antibiotic.
The three strengths (weak, medium and strong) refer to the strengths of the RBSs associated with the toxins in the respective strain (pBBR pT41Rhyz01, pBBR pT41Rhyz02, pBBR pT41Rhyz03).

As expected, the *P. sp.* IsoF cells responded to the ccdB toxin very similarly to all previously tested *E. coli* strains.
The cells, regardless of toxin strength or rhamnose concentration, grew to OD600 ~1 and stagnated thereafter.
There was no significant difference between any of the strains.
By now, it was evident that the issue with our toxin was neither in the plasmid design nor the strains, but rather had to lie in our way of testing.
Alternatively, the amount of transcription induced by our rhamnose promoter could simply be insufficient to produce enough toxin to have an effect.
Upon revisiting the iGEM parts registry, we discovered that, for testing similar constructs, the plates themselves can also be induced with Rhamnose - something we hadn’t previously implemented.
We’ve decided to incorporate this method into our next experiment.

### Toxin Assay 2: rhamnose induced plates

As mentioned earlier, we adjusted our toxin testing protocol by incorporating rhamnose into the plates, with the goal of performing a CFU (colony-forming unit) assay.

![Figure 20](https://static.igem.wiki/teams/5250/project/results/ks-10-min.jpg)

![Figure 20](https://static.igem.wiki/teams/5250/project/results/ks-11-min.jpg)

**Figure 20:** Comparison of E. coli CSH50 PT41Rhyz03 (left) and P. sp. IsoF (right) grown on agar plates containing varying rhamnose concentrations (0%, 1%, and 1.5%).

Figure 20 shows the plates after letting the bacteria grow over the weekend.
Based on visual inspection of the plates we determined that there is an inhibition of growth in *E. coli* CSH50 strain but not in the *P. sp.* IsoF strain.
However, the CFU count in some of the plates was too high for us to effectively count.
Therefore, we decided to conduct 2 more repeats of the toxin assay with a starting OD600 reduced tenfold to obtain quantitative data.
The results are summarized in the following histogram:

![Figure 21](https://static.igem.wiki/teams/5250/project/results/copy-of-graph-toxin-assay-2.webp)

**Figure 21:**
CFU count of the E. coli. CSH50 strain and P. sp. IsoF strain containing the toxin plasmid against a control strain of both bacterial species containing an empty plasmid.

Figure 21 demonstrates a lower CFU count in the *E. coli* CSH50 plates expressing the CcdB toxin.
Compared to the control strain containing the empty plasmid, we can conclude that the toxin effectively inhibits growth in *E. coli*.
There is a tenfold decrease in the CFU count in the *E. coli* 1.5% rhamnose plate compared to the 0% rhamnose plate.
Another observation we made is that the growth inhibition is dependent on the concentration of rhamnose we used in the plates - the higher the rhamnose concentration, the lower the CFU count.
However, there is no visible effect from the toxin on the *P. sp.* IsoF strain.
Both the control strain and the toxin expressing strain give identical results.

We undertook various troubleshooting measures, including testing different strains that lacked resistance, evaluating the functionality of our rhamnose promoter, examining our construct, and experimenting with different conditions.
Eventually we were able to show that the toxin we used inhibits growth in *E. coli*.
Nevertheless, due to time constraints we were not able to test whether the CcdA antitoxin neutralizes the toxin activity.

**Conclusion:** Despite our extensive efforts, we were unable to establish a functional kill switch.

## Sensing

---

**Aim:** We want to test whether the promoter in our construct can successfully sense Xylose.

To assess the promoter's sensing capability, we conducted a fluorescence measurement experiment over a 24-hour period, during which we recorded fluorescence and optical density (OD) at various time points. Unfortunately, we were unable to detect any significant increase in fluorescence compared to the control samples at any time.
After troubleshooting the issue, we identified a design error in our construct and are currently awaiting new results.

## Future Steps

---

##### Sensing:

We will perform an inverse PCR to include the operator that was missing an the reason our sensing assay failed. As a backup plan we will order the operator as oligonucleotides and perform a digestion ligation after oligo annealing to implement the operator into the plasmid.
In case one of those cloning methods is successful, we will perform the sensing assay described in experiments again.

##### Biofilm components:

Since our controls didn't show the expected results, further experiments should be performed to confirm our findings.

##### Kill Switch:

Instead of redoing another toxin experiment with the CcdB/CcdA system, that has initially been established for *E. coli* strains, we could switch to another toxin-antitoxin system, that is established in *Pseudomonas species*.

##### Final construct:

If we had more time we would have recloned the level 2 plasmids and introduced them into *P. sp.* IsoF to test our genetic circuit.

We would first research the concentration of xylose exceeded by plant roots.
We could then induce the strain with different xylose concentration, one of them being the exceeded concentration and again measure the c-di-GMP concentration, polysaccharides production and bacterial motility to see if an increase in xylose would lead to an enhanced biofilm formation.
If this is the case we would know our construct works as intended and if the xylose concentration in the rhizosphere would be enough to trigger our system.

Further, we could remove the xylose source again by washing the culture with NaCl and adding new LB medium to let them recover.
We could then plate the strains and see how many colonies survived. In case our construct works as intended we expect to not have any colonies on our plate, since the bacteria should be killed when it is not in proximity of plant roots.
Removing the xylose by washing the cells would mimic the distancing of the bacteria from the rhizosphere of the plant root.
Lastly, we could test the full construct again on plants under drought and normal conditions to see whether our system would also function in vivo.

## References

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