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). 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.

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 to target. As well as being aware of these things throughout the duration of our project and any further research we undertook.

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*

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. Plantarumwere 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.

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.

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)
• '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

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.

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.

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.

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 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.

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 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 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

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 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.

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)

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 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 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

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 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


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

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 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.

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.