iGEM 2023 — Team exeter


# ENGINEERING SUCCESS

Engineering involves using a set of principles to build and design products efficiently. These can also be applied to synthetic biology systems. The engineering cycle implements these principles, and outlines a structure that scientific research can follow.
Throughout our project we went through many iterations of this cycle. It consists of four stages: design, build, test, learn.

Click the arrows below to learn more about how we integrated the cycle into our research through the questions we considered at each stage.

Why did/didn't it work?
What can we take away from the test and how can this be
implemented in the redesign?
Is there any other useful data that can be obtained
from this cycle?

How will it be built?
Make a model
Transform DNA
Build a protoype
What components can we currently build?
How does this component fit with the rest of the
design?

Did what was built work?
Were the results obtained what
was expected?
Did the model follow what is expressed
in literature?
What component is the part where it stops working?
What form is the data in and how exactly is this going to
be analysed?

What do we want to make?
Can what we want to make be modularised?
Can we split it into smaller milestones?
What parameters need to be defined?
How can we test it?
Do we have the equipment/facilities
to test it?
Can this be modelled?
What do we need to make it?

**Iterating through this [cycle](#path2) lead us to [successfully](https://2023.igem.wiki/exeter/results) construct a part which expressed a protein in *E. coli* called AHL- lactonase. We used LCMS to demonstrate the degradation of biofilm inducer C4-HSL by this protein.**

Although more information can be found in [2. Building our DNA](#path2)...

Here are the graphs that demonstrate our success:

![](https://static.igem.wiki/teams/4694/wiki/results-docs/graphe.png)
![](https://static.igem.wiki/teams/4694/wiki/results-docs/graphf.png)

Details of areas of our project where we encountered and completed various engineering cycles can be found below. Use the menu to move between topics or just scroll through.

**[1. RIP](#route)
[2. Building our DNA](#path2)
 [3. Adhesion of *L. plantarum*](#path3)
 [4. Biofilm Assays](#path4)
 [5. Human Practices](#path5)**

### 1. RIP

At the beginning of our project we found literature about the RIP RAP TRAP mechanism. Building off this we planned to use RIP to inhibit the biofilm formation in *S. epidermidis*. However through the building of various models, we soon noticed some issues with the mechanism. This Prompted us to undertake more literature research and start reformulating ideas of what mechanisms we wanted to target to inhibit the biofilm formation. Click on each section of the flowchart to read more about what happened.

After examining the literature we wanted to form a [model](https://2023.igem.wiki/exeter/model) to inform the wetlab aspects of our project at a later date

##### Design:

* To build a model that attempts to quantify the expression of RNAIII and its subsequent effect on biofilm formation.
* To build this model the odeint function in the Scipy package will be used

##### Build:

* The model was built in two sections. the first being a set of differential equations for the RIP-RAP-TRAP mechanism based off of Henri-Michaelis-Menten kinetics (see [modelling](https://2023.igem.wiki/exeter/model)).
  And a second set of differential equations to describe the activity of the Agr operon: its autoactivation and expression of RNAIII

##### Test:

* Alphafold2 and ChimeraX were used to test the parameters of the model. Specifically, the binding of RIP, RAP and TRAP – relating to constants K1 and K2. The predicted aligned error between RAP and TRAP was high, flagging the likelihood that RAP and TRAP do not bind to each other.
* Whilst building the model we knew the expected results of each section of the model and how one would affect each other. In this process we experienced some unexpected results that showed that the model was not working as expected such as negative concentrations and massive negative spikes.

##### Learn:

* Before even putting our model into its full throw and attempting to find model parameters. Our model showed that an increase of RIP would lead to a decreased expression of RNAIII and thus a decreased expression of delta toxin (a toxin known to decrease the formation of biofilms). The results of these two models led to some serious questions over our project and resulted in us having to head back to the drawing board and to scour more literature.
* From the test stage (testing the model parameters) we found that the systems do not function as expected based on the literature – with RAP and TRAP not binding meaning TRAP was not phosphorylated through the expected mechanism.
* This fed into our subsequent research stage – using these findings to inform our subsequent project design as we were aware to be hyper-critical when examining the literature.

Returning to the literature to re-evaluate our ideas

##### Design:

* As the modelling had highlighted some serious flaws with the RIP-RAP-TRAP mechanisms we were sceptical about the basis of our project
* Our PI also went through some of the literature we had found and also had some concerns as to the integrity of the mechanisms.

##### Learn:

* This process taught us all to be more thorough and investigative when reading journal articles. We took this mindset with us when looking into [alternative mechanisms](https://2023.igem.wiki/exeter/description) to target. As well as being aware of these things throughout the duration of our project and any further research we undertook.

### 2. Building our DNA

Click on each section of the flowchart to read more about what happened.

Transformation of our 10 DNA parts, with P32, P48, and PldhL1 promoters.

#### Design:

* Having learnt from our mistakes when we cloned and transformed our last batch of DNA, RIP, We took more care and properly planned a step by step plan of exactly what we were going to do and when.
* We had ordered 10 DNA parts that we wanted to eventually test against biofilms. These parts were to be cloned each with 3 different promoters (P48, P32 and PldhL1) with the pX\_1845 plasmid backbone and then transformed into DH5a.

#### Build:

* The TypeIIS method was used to assemble our DNA into genes.
* Then each of these newly assembled genes were transformed into *E. coli* (DH5a). This is due to DH5a being a typical cloning strain, as DH5a preserves and amplifies the genes.
* We then mini prepped the DNA out of the DH5a and sent off each gene for sequencing to see if we had successfully built our desired genes. Comparison of the sequenced data and the genes we built would be done using SnapGene.

#### Test:

* Having received the data for the sequencing, we found that some of our genes were not built properly, for example, there may have been a few nucleotides missing, or a substitution, or an inversion in the base sequence.
* This was a small setback for us as we were unsure on what to do with the genes that hadn’t assembled properly.

#### Learn:

* From this we learnt that science doesn’t always produce the results that we want.
* There were positives and negatives to this. The majority of genes were built properly with 2 or 3 being the exception of this.
* We spoke to our PI and had a few ideas of what to do next. We finalised on the idea to carry on with the genes that had positive sequencing, and with the genes that had negative sequencing we would try and build differently with a new plasmid backbone. [\*this can be found later in the cycle\*](#new-backbone)

We continued with the transformations of the DNA that's sequencing came back positive. Transforming into *E. coli* C2925 then *L. plantarum*. The expression of the genes would then be tested using a Western Blot

#### Design:

* Following our sequencing that came back positive, this DNA was transformed into *E. coli* C2925. This is due to the fact that *L. plantarum* will be our final host bacteria. For DNA to be transformed into this, the DNA needs to be un-methylated which is what the *E. coli* strain C2925 does.
* If this was successful, the DNA could then be transformed into *L. plantarum*. If this transformation was successful then we would need to see if *L. Plantarum* was expressing our desired genes, which would be done via a western blot.

#### Build:

* From the correct sequencing, we transformed this into C2925 and left the plates growing overnight. This DNA had successfully transformed.
* After mini prepping the DNA out of C2925, and making glycerol stocks, we then transformed into *L. plantarum*
* The *L. plantarum* growth plates were left over the weekend to grow as they need 2-3 to form colonies. The Plates had shown that that the majority had grown with the exception of 2 or 3 again. With these plates that had grown, the expression of these genes by *L. Plantarum*were tested using a western blot.

#### Test:

* For each of the transformations that worked into *L. plantarum*, we did a western blot of both the Lysate and supernatent.
* Neither of the western blots gave us the results that we wanted. The western blot showed bands, but they were the same bands for each protein, therefore the results were deemed as inconclusive.

#### Learn:

* We didn’t get the result that we wanted, but we still had a few ideas on how to improve.
* We spoke to our PI and she suggested to us 2 ideas: 1. Carrying out a western blot on a culture with a higher OD600. 2. Carrying out a western blot on the whole cell, as well as the lysate and supernatant.
* We also decided to possibly carry out a western blot on our glycerol stocks of c2925 to see if *E. coli* was expressing our proteins.

As some of the DNA didnt clone correctly we recloned it using a different backbone (with a lower copy number). We also wanted to reclone it due to the Western Blots being inconclusive. Our PI gave some suggestions on how to improve the chances of a sucessful western blot which we also implemented in the next stages of the cycle.

#### Design:

* With some of DNA that didn’t work with our pX\_1845 plasmid backbone we planned to reclone it. Instead, we would use the TypeIIS method to clone our DNA with the pX\_1645 plasmid backbone, which is a lower copy number that pX\_1845.
* If this worked this DNA could be used to transform into *L. plantarum*, to then use to test against biofilm formation.

#### Build:

* The TypeIIS method was used to form our desired genes in the thermocycler.
* After, we transformed the genes into *E. coli* DH5a
* We then mini-prepped the DNA out of *E. coli*, used the Qubit machine to calculate the concentration, and sent out samples for sequencing.

#### Test:

* The results came back from sequencing however the genes could not be processed. This was most likely due to a contamination, or that the genes just didn’t form the way they were supposed to.

#### Learn:

* From this we decided to explore other options by using different promoters.
* We decided to clone all our DNA again with 2 different promoters that are STRONG *E. coli* promoters called the ‘T7’ promoter and the ‘PJ23100’ promoter.

Due to the incorrect assembly we decided to focus on using strong *E. coli* promoters. This allowed us to speed up the process of being able to test for expression of our desired genes as we only had to transform into *E. coli*. Using LCMS we saw **significant degradation** of the C4-HSL biofilm inducer.

#### Design

* After the pX\_1645 plasmids didn’t assemble properly with our DNA, we looked at other options.
* Instead, we decided to clone all of our DNA with two strong *E. coli* promoters, the IPTG inducible ‘T7’ promotor and the strong constitutive promoter ‘PJ23100’.

#### Build

* Our supervisor used the TypeIIS method to clone our DNA and then transformed into *E. coli* DH5a
* Then the DNA was mini prepped out of *E. coli* and a glycerol stock of each one was made.

#### Test

* The results of the western blot showed that the 'T7' and 'PJ23100' promotor both expressed 'his-LuxS' and 'his-AHL lactonase'
* This was good news. We weren't sure as to why the other proteins weren’t being expressed, but it could be due to *E. Coli* just not expressing them.
* From this, LCMS was used to see if his-LuxS’ and ‘his-AHL lactonase’ were in fact degrading the biofilm inducing molecules. [(See LCMS)](https://2023.igem.wiki/exeter/experiments)
* **'his-AHL lactonase' degraded our biofilm inducers**. The results showed **significant degradation**  of our biofilm inducer C4-HSL over time, with a P value of 0.027 between the lysate of the ‘his-AHL Lactonase’ and the lysate of the control (mCHERRY). [Click here to see the full results of the LCMS](https://2023.igem.wiki/exeter/results)

#### Learn

* From doing further research into AHL degradation, we concluded that AHL lactonase may have a species-specific degradation effect.
* AHL lactonase showed significant degradation of C4-HSL, which is part of the *P. auerginosa* QS system (a bacterium which is responsible for catheter associated biofilm formation)
* AHL lactonase were not degrading the N-(beta-ketocaproyl)-L-Homoserine lactone. However, this inducer was isolated from *V. fischeri*.
* This may conclude that AHL Lactonase has a species-specific effect.

\*Our positive [results](https://2023.igem.wiki/exeter/results)\*:

![](https://static.igem.wiki/teams/4694/wiki/results-docs/graphe.png)
![](https://static.igem.wiki/teams/4694/wiki/results-docs/graphf.png)

These graphs show that the AHL lactonase produced by our transformed *E. coli* are degrading C4-HSL at a significantly higher rate than both mCherry (p=0.027) and LuxS (p=0.002). Therefore, we have shown that we can successfully degrade a key signalling molecule in the quorum sensing systems of certain Gram-negative bacterial species such as *P. aeruginosa*.

### 3. Adhesion of *L. plantarum*

Click on each section of the flowchart to read more about what happened.

 Our plan was to form biofilms using just *L. plantarum* as a potential method of adhering our coating to the catheter.

#### Design:

* To make our coating sucessful we needed to find a way to adhere that *L. plantarum* to the catheter surface.
* As stated in literature, biofilms formed by the bacteria would stick to a silicon surface. So the initial plan was to form biofilms in *L. plantarum* as a mechanism for adhesion.

#### Build:

* We formed biofilms on glass slides, petri dishes and in 96 well plates using MRS media.
* Crystal violet assays were used at 24, 48 and 72 hours.
* BHI media was used to determine biofilm strength after several weeks of biofilm formation.

#### Test:

* Biofilms were weak on polystyrene petri dishes and well plates originally. No biofilms formed on glass slides when assayed using crystal violet.
* BHI media did not increase biofilms and in fact hindered their growth.

#### Learn:

* We chose not to attempt to form biofilms on glass slides.
* 72-hour biofilms were especially weak, so we removed these from the analysis design.
* We chose to stop using BHI.
* We did further research and developed a new protocol

#### Test:

* This protocol formed better biofilms under crystal violet scrutiny.

There was literature suggesting the CapA gene would lead to increased adhesion. Using what we learnt from adhereing the *L. plantarum* by forming biofilms, we wanted to test if the presence of CapA had any effect on these biofilms. Unfortunately it did not, however adhesion was still the same so we showed that our transformation method of *L. plantarum* did not inhibit its adhesive biofilm properties

#### Design:

* We found literature that suggested an increase in exopolysaccharide expression would lead to sticky capsules and more reliable adhesion. This us lead to the assumption that better biofilms would form if we inserted the CapA gene.

#### Build:

* The CapA gene was transformed into *L. plantarum* and we compared the growth of biofilms with the Wildtype *L. plantarum* versus the *L. plantarum* containing CapA

#### Test:

* Through the comparison of the optical density of biofilms made of *L. plantarum* containing the CapA gene and biofilms made of a wild type *L. plantarum*, both numerically and visually there wasn't a significant difference in the adhesion of either biofilms.

#### Learn:

* As seen in our [results](https://2023.igem.wiki/exeter/results) section. CapA did not make a significant difference in adhesion of *L. plantarum*
* However, we have shown that our transformed *L. plantarum* still readily forms biofilms on polystyrene plates and therefore we have a method of adhesion.

### 4. Biofilm assays of *S. epidermidis* and *P. fluorescens*

Click on each section of the flowchart to read more about what happened.

Finding a protocol to form biofilms to test on

#### Design:

* In order to test the efficacy of the biofilm inhibiting molecules our tranformed *L. plantarum* will express, we need to be able to form biofilms to test them on
* We researched different protocols to form biofilms in *S. epidermidis* and *P. fluorescens* and finally found a protocol to test and adapt.

#### Build:

* Using the [protocol](https://2023.igem.wiki/exeter/experiments) for each bacteria, two 96 well plates were filled following the template on the protocol
* These were incubated for 24h and 48h

#### Test:

* To test whether we had formed biofilms we used a crystal violet assay. Following what we had learnt from forming biofilms in [Adhesion of *L. plantarum*](#path3) we washed once with PBS before staining with crystal violet, then twice after
* The 24h read of the *S. epidermidis* came back inconclusive as some of the control wells seemed to show growth
* Additionally there was too much variation in the results and the control wells on all the test plates for anything to be able to be tested on the biofilms yet.

#### Learn:

* From these results we learnt that we need to put a much higher focus on consistency in the results
* We also decided that from this point we need to reduce the number of people carrying out these experiments in order to reduce the variation due to human arror across different reads of the same cultures
* We decided to retest the protocols but with a few tweaks in order to try to reduce variance

With a higher focus on consistency and a few changes to the protocol based on what we had learnt from the first round of experiments, we tried again to form better and consistent biofilms.

#### Design:

* After talking to our PI a new protocol was formed
* From the previous results of *S. epidermidis* and *P. fluorescens* as well as the biofilms formed in *L. plantarum* we decided that there was minimal difference between adding 10µl of diluted culture and 2µl of diluted culture to each well. So going forwards all test would be performed using a 1:100 dilution (or adding 2µl of culture to 200µl growth media) in each well.
* Additionally we decided every step should be caried out in exactly the same way so we developed a protocol detailing exactly how each step should be carried out (including where and how the pipette should be placed in the well when adding and removing media (LINK PROTOCOL)
* We also decided to omit the step where a new plate is re-streaked to save time in the protocol, but also because we had seen no difference in the overnights when they had been made from a glycerol stock versus a colony from a streak plate.

#### Build:

* This new protocol was followed and two 96 well plates filled for each bacteria
* Only a 24h read was done this time
* The members of the team carrying out these experiments were made aware of the focus on consistency

#### Test:

* One of the plates for each bacteria was stained using crystal violet.
* The other two plates were handed to Dr Christian Hacker, who had agreed to help us with some Scanning Electron Microscopy (SEM) analysis of the plates
* Through the SEM images we gained confirmation that we had formed healthy biofilms in both *S. epidermidis* and *P. fluorescens*. Additionally the results from the crystal violet assays were a lot more consistent
* See the [results](https://2023.igem.wiki/exeter/results) section for more information

#### Learn:

* We had confirmation that we could sucessfully form consistent biofilms in *S. epidermidis* and *P. fluorescens* a gram positive and a gram negatie bacteria. This meant we could finally start testing the molecules our transformed *L. plantarum* expresses.

Now we knew how to form biofilms in *S. epidermidis* and *P. fluorescens* we could start testing the expressed molecules against them.

#### Design:

* As time was running out, we planned to do many experiments in parallel to make the most of the time we had left to run experiments.
* The first set of experiments planned were to test the lysates of the transformed *L. plantarum* as well as the supernatants of the *L. plantarum* that contained signal-peptides. These lysates and supernatants came from our first set of transformed *L. plantarum* that used P32, P48, and PldhL1 promoters [(see building our DNA)](path2)

#### Build:

* Following the same biofilm formation protocol as before for *S. epidermidis* and *P. fluorescens*, 5 microlitres or 10 microlitres of the supernatant/ lysate was added to the bottom of the well, before adding in the TSB and culture.
* These volumes were chosen as they would be just enough to coat the bottom of the well.

#### Test:

* The plates were Crystal violet assayed and the results analysed.
* The results were not very clear and we couldnt draw conclusions from them. Additionally, some of the wells with added lysate or supernatant showed increased biofilm formation.
* See [results](https://2023.igem.wiki/exeter/results) for more information

#### Learn:

* One theory for why the biofilms increased was that by adding in the lysate/supernatant provided extra nutrients for bacteria.
* We learnt that there will be a tradeoff between how much to add to minimise the nutrients provided and maximise biofilm reduction.
* As the results were not clear, and also using the knowledge gained in [from building our DNA](path2) about these promoters, we were worried that it was because the promoters we were using were not expressing the molecules enough/ at all. And so we decided to repeat the assays using the lysates from the other, stronger, promoters

 As the results were inconclusive and we only had theories as to why, we decided to repeat the tests using lysates containing different promoters. These promotors were also picked due to what we had learnt while [building our DNA.](#path2) The data we collected showed promising results for the lysates containing PJ23100 against *S. epidermidis* biofilms

#### Design:

* Repeat the assays using the cell lysate from the cells containing the PJ23100 and T7 promoters

#### Test:

* The lysates containing the PJ23100 promoters showed promising signs of reducing *S. epidermidis* biofilm formation for the wells where 5µl of lysate solution was added.
* See [results](https://2023.igem.wiki/exeter/results) for a more in depth analysis of this data and the graph.

#### Learn:

* Although we haven't collected as much data as we would have liked on this to draw a strong conclusion, the reduction looks promising and in the future more tests should be done to confirm this further. **However with the data we have acquired we have shown that the molecules expressed with the PJ23100 promoter reduced biofilm formation in *S. epidermidis***

![A graph comparing the optical densities of s. epidermidis biofilms formed in the presence of lysates from bacteria transformed with PJ23100 promoter with different CDS](https://static.igem.wiki/teams/4694/wiki/results-docs/biofilms/s-epidermidispj23100analysis.png)

For the lysate ratio of 0.025, for His-QQ7, SP His-QQ7, His-LuxS and SP-AHL lactonase p < 0.05 when performing a students t-test against the wildtype lysate data
* Further research from this point would be finding the optimum amount of lysate to add to maximise reduction without providing too much nutrients. As well as more tests to conclude which inhibitor out of the ones that worked is the best

### 5. Human Practices

Click on each section of the flowchart to read more about what happened.

 Initially we sought to integrate different stakeholders into our research, using their feedback and ideas to help shape and influence our project. We were aware that due to the nature of our project, this would require an ethics form to be submitted before we could start contacting people. The ethics approval process was long and we encountered a few obstacles in the process.

#### Design:

* Within our attempts to sucessfully acomplish an effective and responsible project, we came across various complications that pushed us to follow what is a reflection of the DBTL cycle.
* Initially we sought to integrate different stakeholders in our research, gaining their insight around our idea. In light of any notable feedback, we would reflect on how we could modify our project in response.
  As our project is theraputically based, and hence would directly impact those within helathcare administering catheters, not to mention the individuals catheterised, we planned open discussion with such people.

#### Build:

* We were aware that submitting our university's bioscience ethics form was required. As well as being conscious that an additional external reiew may be necessary. To address this, we reached out to the Department of Ethics and Governance to seek their guidance.
* Within our email we provided comprehensive explanations of our intentions and methodologies, attaching surveys and social media polls targeted at patient groups we wanted to reach.

#### Test:

* In response to our email, a meeting was arranged to discuss our plans. Unfortunately, the feedback lacked clarity and consistency; this was the primary factor that resulted in the rejection of our bioscience ethics form submission.
* To see further details, please see 'ethics' on our [human practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) page

#### Build:

* With our ethics form rejected, we contacted the department again and arranged to meet with them again along with our PI.

#### Test:

* The feedback from the meeting was inconsistent with what we had previously received. However, we understood the reasons for the form's rejection and sought to rectify them on our re-submission.

#### Learn:

* Due to the extensive ethical application processes and time constraints, our initial plans for integrating healthcare and patient groups within our project were not feasible.
* With the addition of literature research, we learned that there were a vast number of gaps and inconsistencies within ethical review processes in universities.
* It became apparent to us that undergraduates are scarcely prepared in terms of human practice elements within science and research and are taught ethics primarily centered around plagiarism, academic honesty, etc.

 Due to our set-backs with the rejection of the ethics form, we changed our attention to look at the complexities regarding ethical reviews in different institutions. We were also interested in finding out students and iGEM teams awareness of ethical research. We learnt how important responsible research is, and all will be taking these considerations into any future research we may encounter.

#### Design:

* Drawing from our prior knowledge, we opted to centre our attention on the history, complexities, and potential solutions regarding ethical reviews and committees.

#### Build:

* We met with Sarah Hartley to discuss our intentions and came away with a plan for what our next steps would consist of.
* This consisted of deeper literature research, a proposal to carry out a student survey around the awareness of ethical research, meetings with other iGEM teams, and an email to the vice chancellor to discuss the progress made by the university to better teach ethics.
* Gathering all this evidence together, we wanted to write up a report of our findings.

#### Test:

* We gained permission to gather responses for the student survey, and we successfully contacted several international iGEM teams to discuss their experiences with ethical reviews and to gauge the differences between universities.

#### Learn:

* Our experience with attempting to integrate human practice has proivided us with invaluable knowledge of responsible research, that our undergraduate degrees have failed to do. We appreciate the importance of ethical research and seek to employ human practice considerations in any future research we may encounter.

# EXPERIMENTS

Media

[Making LB](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/lb-media-agar.pdf)

[Making MRS media](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/mrs-media-agar.pdf)

[Making BHI](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/bhi-media-agar.pdf)

[Making TSB](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/trptic-soy-broth-media.pdf)

Molecular Biology

[Preparation of competent *E. coli* cells](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/prep-of-competent-e-coli.pdf)

[Transformation of competent *E. coli* cells (heat-shocked based)](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/transformation-of-competent-e-coli.pdf)

[Preparation of competent *L. plantarum cells*](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/prep-of-competent-l-plantarum.pdf)

[Transformation of competent *L. plantarum cells*](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/transformation-of-competent-l-planatarum.pdf)

[Plasmid DNA extraction](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/miniprep.pdf)

[TypeIIS Cloning](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/updated-typeiis-assembly.pdf)

[Western Blot](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/updated-western-blot.pdf)

[Cell Lysis](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/updated-cell-lysis.pdf)

Biofilms

[Forming *L. plantarum biofilms*](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/updated-l-plantarum-biofilm-formation.pdf)

[Forming *S. epidermidis biofilms*](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/updated-s-epidermidis-biofilm-formation.pdf)

[Forming *P. fluorescens biofilms*](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/updated-p-fluorescens-biofilm-formation.pdf)

[Biofilm Washing and staining](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/updated-biofilm-washing.pdf)

Assays

[Lysate and supernatant assays on biofilms](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/updated-lysate-supernatant-assay.pdf)

[LCMS standard prep](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/new-prep-of-lcms-standards.pdf)

[LCMS enzyme assay](https://static.igem.wiki/teams/4694/wiki/experiment-pdfs/new-lcms-enzyme-assay.pdf)

# RESULTS

The primary goal of CathExe is to **overcome the risk factors of catheterisation**, see [here](https://2023.igem.wiki/exeter/description) for more information. We aimed to **prove** that, through the utilisation of a commensal coating, **mature biofilm formation could be inhibited.**

 Throughout our iGEM experience, **many hurdles were met** and had to be overcome. We came to appreciate the ways **science can inevitably go wrong** at times and the importance 'trial and error' can hold within experimental research. An element of this, is **acknowledging the significance of negative, as well as positive results,** as disproving a hypothesis also **holds value.** Negative results indicate areas that do not work, providing **knowledge and prospect** for future development.

 Modern day researchers have a tendancy to provide only positive results achieved, disregarding the negative, this was especially the case when considering the results reported by Balaban *et al.* [[1]](#resultref1) (read [here](https://2023.igem.wiki/exeter/human-practices) and [here](https://2023.igem.wiki/exeter/engineering) for our change of project idea due to controversy in the literature). Ignoring the negative results within research, can **lead still learning, young/beginner scientists to feel a sense of 'false hope' within their own experiments**. This **facilliates discouragement** when an experiment does not take the desired direction. At CathExe, we avoid this and show **all the results we have obtained**, demonstrating how we have **adapted our project** along the way and that **mistakes are okay.**

Please click on any of the buttons below to see what we have achieved:

**Autoinducer Degradation**
**Protein Expression**
**Biofilm Formation**
**Inhibition of Biofilms**

**Autoinducer Degradation**

* [Calibration Curves](#lcms1)
* [LCMS result graphs](#lcms2)
* [Degradation Graphs](#lcms3)

# Liquid Chromatography Mass Spectrometry (LCMS)

Liquid chromatography mass spectroscopy (LCMS) is an analytical technique which combines the separation power of liquid chromatography, along with the detection capability of mass spectroscopy.
In our project, we have utilised LCMS to determine the degradation of each of our standards. 4-Hydroxy-5-methyl-3-furanone was used to replicate the presence of AI-2 in the reaction. C4-HSL and N-(beta-Ketocaproyl)-L-homoserine lactone are examples of different AHLs, so we used these as a test for our AHL lactonase.
First, we created a calibration curve for each of these standards.

#### 4-hydroxy-5-methyl-3-furanone calibration curve

![(calibration curve for 4-Hydroxy-5-methyl-3-furanone showing a linear positive correlation from 1 nM to 100 μM)](https://static.igem.wiki/teams/4694/wiki/wiki-images/hydroxy-methyl-furanone-standard-curve.jpg)

#### C4-HSL calibration curve

![(calibration curve for C4-HSL showing a linear positive correlation from 1 nM to 100 μM)](https://static.igem.wiki/teams/4694/wiki/wiki-images/c4-hsl-standard-curve.jpg)

#### N-(β-Ketocaproyl)-L-homoserine calibration curve

![(calibration curve for N-(β-Ketocaproyl)-L-homoserine lactone showing a linear positive correlation from 1 nM to 100 μM)](https://static.igem.wiki/teams/4694/wiki/wiki-images/b-keto-standard-curve.jpg)

These initial results confirmed that our standards could be detected on the LCMS and provided us with the necessary equations for calculating the concentration of each standard in the assay.
In these equations, y is the area under the curve and x is the calculated concentration in micromolar.

**4-hydroxy-5-methyl-3-furanone calibration:**

$$ y = 68.727454\*x - 70.794631 $$

**C4-HSL calibration:**

$$ y = 636.920486\*x - 168.173302 $$

**N-(β-Ketocaproyl)-L-homoserine calibration:**

$$ y = 7852.257148\*x - 6124.904398 $$

Following detection of LuxS and SP-AHL lactonase on the Western blot following the E. coli transformation
we set up a 0-hour reaction, 6-hour reaction and a 24-hour reaction by adding the mix standard a then lysate of the enzyme.
After the set duration time, we stopped the reaction by adding in a set volume of ice-cold methanol.

The LuxS was not expected to have any degrading action against any of our standards as it produces 4,5,-dihydroxy-2,3-pentanedione (DPD), a precursor of AI-2 [[2]](#resultref2).
As there are no other enzymes in the assay, DPD cannot be converted into AI-2 molecules. Therefore, we compared the activity of SP-AHL Lactonase to both the mCherry and the LuxS as controls.

We tested against a mixed sample, containing all three standards.
As expected, the degradation of 4-hydroxy-5-methyl-3-furanone over time by AHL lactonase was negligible, shown in Graph A below.
This was analysed by assessing the line for being non-zero and the p values, displayed on the Graph A, are greater than 0.05.
The data presented shows the assay with AHL lactonase compared to both mCherry and LuxS. With mCherry and LuxS compared on Graph D.

**The following graphs display the results obtained from measuring 4-hydroxy-5-methyl-3-furanone concentration:**

A

![(scatter plot of 4-Hydroxy-5-methyl-3-furanone concenration over 24 hours for AHL lactonase and mCherry)](https://static.igem.wiki/teams/4694/wiki/results-docs/grapha.png)

B

![(scatter plot of 4-Hydroxy-5-methyl-3-furanone concenration over 24 hours for LuxS and mCherry)](https://static.igem.wiki/teams/4694/wiki/results-docs/graphb.png)

C

![(scatter plot of 4-Hydroxy-5-methyl-3-furanone concenration over 24 hours for AHL lactonase and LuxS normalised)](https://static.igem.wiki/teams/4694/wiki/results-docs/graphc.png)

*(A) Scatter plot of 4-Hydroxy-5-methyl-3-furanone concenration over 24 hours for AHL lactonase and mCherry
(B) Scatter plot of 4-Hydroxy-5-methyl-3-furanone concenration over 24 hours for LuxS and mCherry
(C) Scatter plot of 4-Hydroxy-5-methyl-3-furanone concenration over 24 hours for AHL lactonase and LuxS normalised*

**The following graphs display the degradation of C4-HSL and N-(beta-Ketocaproyl)-L-homoserine lactone by AHL lactonase compared to mCherry and LuxS:**

D

![(scatter plot of C4-HSL concentration over 24 hours for AHL lactonase and mCherry)](https://static.igem.wiki/teams/4694/wiki/results-docs/graphe.png)

E

![(scatter plot of C4-HSL concentration over 24 hours for AHL lactonase and LuxS)](https://static.igem.wiki/teams/4694/wiki/results-docs/graphf.png)

F

![(scatter plot of N-(β-Ketocaproyl)-L-homoserine concentration over 24 hours for AHL lactonase and mCherry)](https://static.igem.wiki/teams/4694/wiki/results-docs/graphg.png)

G

![(scatter plot of N-(β-Ketocaproyl)-L-homoserine concentration over 24 hours for LuxS and mCherry)](https://static.igem.wiki/teams/4694/wiki/results-docs/graphh.png)

H

![(scatter plot of N-(β-Ketocaproyl)-L-homoserine concentration over 24 hours for LuxS and mCherry)](https://static.igem.wiki/teams/4694/wiki/results-docs/graphi.png)

I

![(scatter plot of N-(β-Ketocaproyl)-L-homoserine concentration over 24 hours for LuxS and mCherry)](https://static.igem.wiki/teams/4694/wiki/results-docs/grpahj.png)

*(D) Scatter plot of C4-HSL concentration over 24 hours for AHL lactonase and mCherry
(E) Scatter plot of C4-HSL concentration over 24 hours for AHL lactonase and LuxS
(F) Scatter plot of C4-HSL concentration over 24 hours for LuxS and mCherry
(G) Scatter plot of N-(beta-Ketocaproyl)-L-homoserine concentration over 24 hours for AHL lactonase and mCherry
(H) Scatter plot of N-(beta-Ketocaproyl)-L-homoserine concentration over 24 hours for AHL lactonase and LuxS
(I) Scatter plot of N-(beta-Ketocaproyl)-L-homoserine concentration over 24 hours for LuxS and mCherry.*

Graph D shows that the degradation was increased by approximately 2.5 times what was observed when cell lysate from the *E. coli* transformed with mCherry was used.
Graph E shows that the degrading action of AHL lactonase compared to LuxS was over 4 times greater.
A one-tailed Wilcoxon signed rank test was carried out to validate this and we obtained a p=0.027 for Graph D and p=0.002 for Graph E.
As the p value is less than 0.05, we can reject the null hypothesis (that the samples follow the same distribution) and conclude that there is a significant difference in the distributions of the two samples.

From Graph G it can be concluded that the AHL lactonases were not degrading the N-(beta-Ketocaproyl)-L-homoserine lactone.
This homoserine-lactone was isolated from *V. fischeri* [[3]](#resultref3).
Perhaps indicating that the enzymes we are producing might not be effective against certain bacterial strains.

Graph I shows that the mCherry had greater degradation than LuxS, confirming that the LuxS has no degrading action on N-(beta-Ketocaproyl)-L-homoserine lactone.

From Graph D and Graph E we have shown that there was a significantly increased rate of C4-HSL degradation by AHL lactonase,
which is part of the quorum sensing (QS) system in *P. aeruginosa* [[4]](resultref4) - a bacterium which is responsible for catheter-associated biofilm formation [[5]](resultref5).
Meaning we have successfully shown that we can produce an enzyme which can target the QS system of a biofilm-forming species.

# Western Blot

In the analysis of our DNA expression, we conducted several Western blots, yielding both negative and positive results. When testing whether the  *L. plantarum*  was expressing our DNA molecules, the blots failed to detect any expression. With the limited time scope, we took a step back, testing the transformed  *E. coli* to verify the expression before we had transformed into *L. plantarum*.

![Image of lysates Western Blot result](https://static.igem.wiki/teams/4694/wiki/results-docs/wb1.png)

The western Blot above is the for the lysates of *L. plantarum* after transformations. The blot revealed bands in all samples, but it did not show that the  *L. plantarum* was expressing our desired proteins due to there being a band present in the wild type. This meant that the Western blot was showing a non-specific binding of a protein normally found within  *L. plantarum* with the antibodies.

![Image of supernatant Western Blot results](https://static.igem.wiki/teams/4694/wiki/results-docs/wb2.png)

The above is the western blot for the supernatant of the signal peptide DNA samples to attempt to detect any expression. The membrane did not show any results with no bands on either the wild-type *L. plantarum* or the samples.

![Image of the whole cell Western Blot](https://static.igem.wiki/teams/4694/wiki/results-docs/wb3.png)

The next was a western blot for the *L. plantarum*  whole cells to cover all avenues before preforming western blots on the *E. coli* to verify whether the transformation had been successful in *E. coli* before we had transformed into *L. plantarum*. This again did not yield any results.

**Our next blots showed successful transformation for AHL lactonase and LuxS into *E. coli*.**

A western blot using the plasmids containing the J23100 promoter, a constitutive promoter for E. coli DH5a was carried out. The positive control- His-mCherry - has a band in the correct place and the His-ladder is visible. There were bands in the genes: 5: His-LuxS and 8: Sp-His-AHL lactonase.

Following this, we transformed our DNA into *E. coli* with T7 promoters and carried out western blots to verify the expression again. The image above shows that both J23100 (left) and the T7 (right) are expressing AHL lactonase and LuxS. There is a lack of expression within the wild type, validating the success of the assay.

![](https://static.igem.wiki/teams/4694/wiki/results-docs/western-blot-pj23100.jpg)
Western blot showing protein expression of enzymes downstream of the strong constitutive promoter J23100. Lanes 1 and 2 are ladders (PageRuler Plus and BenchMark Histagged protein ladder), 3 showing a positive control (mCherry) whilst 4 is an E. coli ‘wild type’ of DH5a (negative control). Lanes ‘7-14’ are our enzymes (7 = his-QQ7, 8 = SP-his-QQ7, 9 = his-QQ5, 10 = his-LuxS, 11 = his-AHL lactonase, 12 = SP-his-AHL lactonase, 13 = SP-his-QQ5, 14 = his-CapA)

![](https://static.igem.wiki/teams/4694/wiki/results-docs/western-blot-t7.jpg)
Western blot showing protein expression of our enzymes downstream with the IPTG inducible ‘T7’ promoter. Lanes 1 and 2 are ladders (PageRuler Plus and BenchMark Histagged protein ladder), 3 showing a postive control (mCherry), whilst 4 is E. coli ‘wild type’ of BL21 (DE3) (negative control). Lanes ‘7-14’ are our enzymes (7 = his-QQ7, 8 = SP-his-QQ7, 9 = his-QQ5, 10 = his-LuxS, 11 = his-AHL lactonase, 12 = SP-his-AHL lactonase, 13 = SP-his-QQ5, 14 = his-CapA). It seems like there is a lot of non-specific binding or protein overloading which has led to smearing on this western blot, making it harder to read.

A band at 36KDa for mCherry, with no bands in our negative control validated our western blot.

The his-LuxS SP-his-AHL lactonase proteins expressed under control of the PJ21300 and T7 promoters were visualised at 18.8 KDa and 33.8 KDa respectively. A further band on lane 10 was seen at ~38 KDa suggesting dimerisation.

Reasons why the beginning western blots could have failed:

* His tag could be hidden in the tertiary structure, therefore cannot been detected by the blot.
* We initially used anti-his primary antibody raised in rabbit however, this was an old antibody and we saw much more success when we swapped to anti-his raised in mouse.
* Maybe *L. plantarum* was expressing the proteins but in very small concentrations, therefore the blot could not detect the proteins.

**Biofilm Formation**

* [*L. plantarum*](#bf1)
* [*S. epidermidis*](#bf2)
* [*P. fluorescens*](#bf3)

# Biofilms

Biofilm formation is something that we have been working on in the lab from the very beginning. It started with trying to form biofilms with *L. plantarum* using both MRS and BHI media, to then forming biofilms with *S. epidermidis* and *P. fluorescens* using TSB media.

We used two different methods to verify our results. Firstly, we used the crystal violet (CV) assay to look at the OD595, OD525, and OD475 (when necessary) of each of the wells in the 96-well plate. This allowed us to compare the OD (Optical Density) of our bacteria forming biofilms against a negative control (usually just pure media). As stronger biofilms would be thicker and contain more cells, more cells would be stained, therefore, the higher the optical density, the stronger the biofilm. Secondly, with the help of Christian Hacker, we were able to see our *S. Epidermidis* and *P. Fluorescens* biofilms by using the scanning electron microscope (SEM).

Here are the results:

***L. plantarum***

Bacterial adhesion plays a major role in biofilm formation as previously explained. See our [project description](https://2023.igem.wiki/exeter/description).
Thus, it was assumed that increased bacterial adhesion would lead to increased biofilm formation.
Hence, to test whether overexpressing CapA increased adhesion, a *L.plantarum* biofilm formation assay (LINK to protocol on experiment page) was carried out.

Briefly, 1% and 5% of bacterial culture was innoculated onto 96 well-plates and left for 24h before a crystal violet stain was used to quantify biofilm presence.
Optical density (OD) was measured at 595nm and to compare the data, the OD of the control wells were subtracted from all wells on the respective plate, giving numerical data for each well that could be normalised across different plates and repeats of the experiment.

All this data was then collated to produce this graph:

![](https://static.igem.wiki/teams/4694/wiki/results-docs/biofilms/l-plantarumcapabplot.png)

Two-sample t-tests were used to compare biofilms between WT and CapA bacteria.
There was no significant difference in biofilm optical density between WT and CapA *L.plantarum* at 1% inoculation volume (p = 0.8909) nor 5% inoculation volume (p = 0.07628).

Therefore, our CapA *L.plantarum* did not have a significant effect on biofilm formation, and consequently adhesion.
This is opposite to what has been suggested from literature [(See our Description Page)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D).
This may be due to the fact that we used biofilm formation as a proxy to measure bacterial adhesion, assuming increased adherence directly correlated with increased biofilm formation, although this may not be the case if there is a trade-off between the two.
The possibility that CapA does not affect adhesion at all should not be completely ruled out either, but further work is needed to warrant this.
With more time, we would have wished to examine the catheter surface using SEM to quantify the number of bacteria that adhered, allowing us to more accurately infer whether overexpressing CapA increased adhesion.

Another idea we had while working on the adherence aspect was modifying the silicon surface of catheters as proposed by (1).
This would involve modifying the silicon to contain mannose residues which *L.plantarum* can bind to with high affinity through their surface membrane mannose-specific adhesion (Msa) proteins.
Although we did not have the time and resources to carry this out, this is something we hope will be tested in future work.

It was also noted from these experiments that the ratio of culture: media in each well also made no significant difference to the strength of the biofilms formed (p= 0.55198 and p= 0.26397 for WT and CapA *L.plantarum* respectively), which prompted the use of only a 0.01 ratio in future tests where biofilms needed to be formed.

***S. epidermidis***

![](https://static.igem.wiki/teams/4694/wiki/results-docs/biofilms/s-epidermidisbiofilmproof.png)

After washing away the planktonic cells the optical density of the wells containing *S. epidermidis* was higher than the optical density of the wells containing just media.

Another set of plates containing the same set up underwent SEM imaging. These are just a few of the images obtained:

Control well:

![](https://static.igem.wiki/teams/4694/wiki/SEM-files/s-epidermis-control-first-well-plate-01-large.png)
![](https://static.igem.wiki/teams/4694/wiki/SEM-files/s-epidermis-control-second-well-plate-03-large.png)

Well containing *S. epidermidis*:

![SEM image of S epidermidis biofilms](https://static.igem.wiki/teams/4694/wiki/sem-files/s-epidermis-first-well-plate-01-large.png)
![SEM image of S epidermidis biofilms](https://static.igem.wiki/teams/4694/wiki/sem-files/s-epidermis-first-well-plate-03-large.png)

As seen in the images the control wells look a lot different to the wells containing *S. epidermidis*.
This proved that the controls were uncontaminated and all the data on these plates was highly likely to be reliable.

To quote Christian Hacker - 'If that's not a biofilm I don't know what is', providing confidence in the method used to form them.

Additionally, the presence of the biofilm matrix seen between several of the cells displays the maturity of the biofilms. Here the extracellular matrix has facilitated in maintaining the adherence of the bacterial cells.

**P. fluorescens**

![](https://static.igem.wiki/teams/4694/wiki/results-docs/biofilms/p-fluorescensbiofilmproof.png)

Although the *P. fluorescens* biofilms were slightly weaker and had more variance than those formed by *S. epidermidis*.
The data collected was greater than the controls, validating biofilms had indeed formed, and that the method used worked.

Another set of plates containing the same set up underwent SEM imaging. These are just a few of the images obtained

Control wells:

![SEM image of S epidermidis biofilms](https://static.igem.wiki/teams/4694/wiki/sem-files/p-f-control-first-well-plate-01-large.png)
![SEM image of S epidermidis biofilms](https://static.igem.wiki/teams/4694/wiki/sem-files/p-f-control-second-well-plate-03-large.png)

Wells containing *P. fluorescens*

![SEM image of S epidermidis biofilms](https://static.igem.wiki/teams/4694/wiki/sem-files/p-f-first-well-plate-13-large.png)
![SEM image of S epidermidis biofilms](https://static.igem.wiki/teams/4694/wiki/sem-files/p-f-first-well-plate-14-large.png)

The SEM images confirmed successful biofilm formation in *P. fluorescens* validating the protocol used.

After carrying out these analysis', we had confidence in our biofilm formation protocols.

**Inhibition of Biofilms**

* [Original Promotors Lysate Assays](#ib1)
* [Supernatant Assays](#ib2)
* [Summary](#ib3)

# Inhibition of Biofilms

Biofilm assays allowed us to see if our enzymes were in fact inhibiting the biofilm formation.
The same protocol was carried out for forming biofilms, however before the media and bacterial culture were added to the 96 well plate, a set volume (5µl or 10µl) of the enzyme lysate/supernatant was added onto the bottom of each well.

To quantify this, the crystal violet assay was used again to determine if the enzymes did inhibit the biofilm formation.
We used pure media as a control and a wild type of *S. epidermidis* or *P. fluorescens* to compare against.

To quantify the effectiveness of the expressed molecules, the average of the data collected for the wild type of the *P. fluorescens* or *S. epidermidis* was subtracted from the data collected from the wells containing: media, bacterial culture and cell lysate/ supernatant.
The remaining data sets were averaged and the standard deviations calculated allowing the graphs below to be plotted.
Due to the nature of the data analysis, a relative optical density of below 0 represents a weaker biofilm was formed compared to the biofilm formed not in the presence of lysate/ supernatant.
And a relative optical density above 0 represents a stronger biofilm was formed.

**Original Promotors**
**Lysate Assays**

![](https://static.igem.wiki/teams/4694/wiki/results-docs/biofilms/p-fluorescenslysateassays15th.png)

P32 His-QQ7, P32 His-LuxS, and P32 His-AHL lactonase all had an inhibitory effect of biofilm formation with a smaell variance
However due to the very large variance when lysate from the wildtype was present we cannot demonstrate significant inhibition. However this does make them good candidates for further research.

This graph displays a large variance in the biofilms formed in the presence of lysate containing the Wildtype, PldhL1 His-AHL-lactonase, P32 SP-His-QQ5, P48 SP-His-QQ5 and PldhL1 His-CapA.
Due to the overlapping of error bars between the wild type and subsequent data sets, we cannot draw substantial conclusions relating to biofilm formation.

![](https://static.igem.wiki/teams/4694/wiki/results-docs/biofilms/s-epidermidislysateassays15th.png)

As the error bars for both the 0.05 and 0.025 lysate ratios overlap with that of the wildtype, we cannot draw any conclusions as we cannot prove that the lysate assays result in a reduction of optical density, which is a measure of biofilm formation.

**Supernatant Assays**

The data we collected did not contribute any useful information to our project, so we decided to continue only using lysate assays.

**PJ23100**

![](https://static.igem.wiki/teams/4694/wiki/results-docs/biofilms/p-fluorescenspj23100analysis.png)

As the error bars, with the exception of the 0.025 lysate ratio for His-QQ7 and SP-His-QQ7, for both the 0.05 and 0.025 lysate ratio overlap with that of the wildtype, we cannot draw any conclusions as we cannot prove that the lysate assays result in a reduction of optical density, which is a measure of biofilm formation.

![](https://static.igem.wiki/teams/4694/wiki/results-docs/biofilms/s-epidermidispj23100analysis.png)

For the lysate ratio of 0.025, for His-QQ7, SP His-QQ7, His-LuxS and SP-AHL lactonase p < 0.05 when performing a students t-test against the wildtype lysate data

Looking at 0.025 ratio:

Although the error bars overlap between the different lysates, the results for the 0.025 ratio do look promising as they show a mean decrease in relative optical density of biofilms formed compared to the wildtypes.
For example, His-QQ7, SP-His-QQ7, His-LuxS and SP-His-AHL-Lactonase show a decrease in relative optical density and their error bars do not overlap with the wildtype's. These results are very promising.

Promising results

**T7**

![](https://static.igem.wiki/teams/4694/wiki/results-docs/biofilms/p-fluoresenst7analysis.png)

As the error bars for both 0.05 and 0.025 lysate ratio overlap with that of the wildtype, we cannot draw any conclusions as we cannot prove that the lysate assays result in a reduction of optical density, which is a measure of biofilm formation.

![](https://static.igem.wiki/teams/4694/wiki/results-docs/biofilms/s-epidermidist7analysis-1.png)

As the error bars for both the 0.05 and 0.025 lysate ratios overlap with that of the wildtype, we cannot draw any conclusions as we cannot prove that the lysate assays result in a reduction of optical density, which is a measure of biofilm formation.

**Summary**

In some strains of bacteria, the presence of cell lysis/ cell lysate, can enhance the biofilm formation [[6]](#resultref6) .
However this research seems contended by others where the addition of cell lysate did not affect biofilm formation [[6]](#resultref6).
There is a gap in literature about the effect cell lysate would have on *S. epidermidis* or *P. fluorescens* and thus it is difficult to explain the results we saw without further research into it.
However it can be theorised that as the biofilm formation generally enhanced more when more lysate was added, there is a corelation between the amount of lysate and the effect it will have on the biofilm formation.

Further research would need to be done to find the optimum amount of lysate that would provide enough molecules to inhibit biofilm formation but not enhance biofilm growth too much.

# References ☆

1. Kiran MD, Balaban N. TRAP Plays a Role in Stress Response in Staphylococcus Aureus. *The International Journal of Artificial Organs.* . 2009 Sep;32**(9)**:592–9. DOI:10.1177/039139880903200908
2. Vendeville A, Winzer K, Heurlier K, Tang CM, Hardie KR. Making 'sense' of metabolism: autoinducer-2, LUXS and pathogenic bacteria. Nature Reviews Microbiology. 2005;3(5):383-96.
3. Gupta R, Gupta N. Quorum Sensing, Bioluminescence and Chemotaxis. In: Gupta R, Gupta N, editors. Fundamentals of Bacterial Physiology and Metabolism. Singapore: Springer Singapore; 2021. p. 633-52.
4. Boşgelmez-Tinaz G, Ulusoy S. Characterization of N-butanoyl-L-homoserine lactone (C4-HSL) deficient clinical isolates of Pseudomonas aeruginosa. Microb Pathog. 2008;44(1):13-9.
5. Cole SJ, Records AR, Orr MW, Linden SB, Lee VT. Catheter-associated urinary tract infection by Pseudomonas aeruginosa is mediated by exopolysaccharide-independent biofilms. Infect Immun. 2014;82(5):2048-58.
6. Smakman F, Hall AR. Exposure to lysed bacteria can promote or inhibit growth of neighboring live bacteria depending on local abiotic conditions. FEMS Microbiology Ecology [Internet]. 2022 Feb 1;98(2). Available from: https://doi.org/10.1093/femsec/fiac011

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