iGEM 2025 — Team unimuenster

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block
image %}
![gruppen-photo](https://static.igem.wiki/teams/5946/images/header/header-labbook-min.webp)
{% endblock %} {% block page\_content %}

#### January

##### Calendar-Week 2: 06.01.-12.01.25

**General Matters**: We received the applications for
the Team 2025. We interviewed the people that were
interested in being part of iGEM 2025. After the
interviews, we send the invitations to the new team
members for the first team-meeting.

##### Calendar-Week 3: 13.01.-19.01.25

**General Matters**: The first teammeeting took place
on the 14.01. In the beginning, we played a game to get
to know each other. Afterwards, we decided on a date and
time for the weekly teammeeting and also for a kick-off
weekend. We talked about the structure of the iGEM
season and planned the research phase. Since
communication and organization are essential for team
work, we introduced the tools we will use for
communication and organization (Discord, Notion,
Sciebo). To get some insights in iGEM projects, a short
presentation about a former iGEM project should be
prepared in groups and presented in 2 weeks.

##### Calendar-Week 4: 20.01.-26.01.25

**General Matters**: In the second teammeeting, we
introduced the iGEM-Notion page and initiated the start
of the focus groups. Furthermore, we presented the
planning of the kick-off weekend.

##### Calendar-Week 5: 27.01.-02.02.25

**General Matters**: We heard and held the
presentations about a former iGEM Project in the
team-meeting

###### Focus Groups:

Sponsoring: During our first team meeting, we were informed
about an estimated budget required for registration and
participation in iGEM and the Grand Jamboree, distinguishing
between essential and ideal funding needs. We then received
valuable feedback and inspiration from SynBio members (past
IGEM Münster participants), which helped us refine our next
steps. Our key action points moving forward:

* Conducting a lab inventory
* Organizing a group photo
* Expanding our list of potential sponsors beyond previous
  years' contacts
* Exploring opportunities to attend fairs for networking
* Designing a sponsorship flyer

**Marketing & social media**: Our group was formed and on
Saturday 1st we made a small brain dump, which we regarded
thoroughly the next day. We decided on a timeline for future
posts, group leaders and the responsible person for specific
social medias. Guidelines for posting were set.

**Human Practices**: The Human Practices Team was formed.

**Meetup**: We decided that Florian Becker should be the
team leader. We decided to look up which events could
interfere with potential meetup dates and made a list.

#### February

##### Calendar-Week 6: 03.02.-09.02.25

**General Matters**: In our team-meeting we updated
each other about the progress we did in our research
projects to come up with a project idea.

###### Focus Groups:

**Sponsoring**: The team agreed on a regular schedule and
assigned first contacts of potential sponsors to individual
team members. Furthermore, goals were set for the next group
meeting, including the revision of an existing Excel file
from previous iGEM Münster teams, containing a list of past
and potential sponsors, as well as the creation of an
official sponsorship email account.

**Marketing & social media**: The team members did the
organization for the group photo and continued to work on
the social media concept.

**Human Practices**: The team focused on researching data
regulations relevant to potential interviews, reviewing
guidelines from other iGEM teams. Additionally, the team
explored creative approaches to integrating inclusion
throughout the project.

**Meetup**: The team proposed a potential date for the
Meetup. We plan to survey other iGEM teams to gauge their
interest and estimate the expected size of the event.

**Teambuilding**: We appointed a group leader, discussed
the team fund, the trip to Carolinensiel and ordering/buying
drinks and snacks.

##### Calendar-Week 7: 10.02.-16.02.25

**General Matters**: We decided on a weekend
(01.05.-04.05.) to go to Carolinensiel. This weekend is
planned to spend some together and grow as a team. We
also talked about the medal criteria and what they mean
for our iGEM roadmap.

###### Focus Groups:

**Sponsoring**: This week, the team made further progress
in organizing sponsorship efforts. We created a shared Gmail
account for sponsorship communication and divided the list
of potential sponsors among team members for outreach. The
deadline for flyer creation has been set for February 16,
2025. Additionally, two important tasks remain: confirming
the delivery address for sponsorship parcels and verifying
the bank account details for sponsor payments.

**Marketing & social media**: The team agreed on a layout
for the posts on social media. The need for a new
introduction of iGEM was highlighted and regarded, as posts
from the previous years are too hard to reach.

**Human Practices**: The team continued studying Human
Practices rules, with a focus on data security for interview
transcriptions. Special attention was given to
confidentiality and privacy compliance to ensure ethical
handling of data.

**Meetup**: The group decided on organizing a smaller
Meetup with potential international attendees. The
preliminary schedule includes arrival on Friday, a poster
session and lectures on Saturday, and departure on Sunday.
Requests for initial venue options, including Münster Castle
and the MPI, as well as accommodations, have been made.

##### Calendar-Week 8: 17.02.-23.02.25

**General Matters**: We planned another research
weekend. Furthermore, we presented each other the
research topics we are working on.

###### Focus Groups:

**Sponsoring**: The sponsoring flyer’s design is
finalized, but text updates, an abstract about past iGEM
Münster projects, and final checks are still pending. It was
decided that sponsorship efforts will emphasize general
benefits for sponsors rather than predefined packages.
Initial outreach for donations and sponsorships has already
begun. In a first meeting with the IMMB secretary (our
financial advisor), we clarified financial processes and the
university's support for the iGEM team. Additionally, bank
account details from the previous iGEM Münster team were
transferred, and a review of the remaining financial
resources was conducted.

**Marketing & social media**: We have continued to
improve the introduction for iGEM and the layout for posts
on social media.

**Human Practices**: We continued our research about data
security in interviews.

**Meetup**: Ideas for possible speakers and topics were
collected. In addition, initial teams, accommodation and
rooms are being requested.

##### Calendar-Week 9: 24.02.-02.03.25

**General Matters**: We had another research weekend
to dive even deeper into the different project ideas. To
get more profound feedback on which project ideas are
feasible, we planned an evening to present the ideas to
professors, postdocs, and former iGEMers to ask for
their opinion and tips.

###### Focus Groups:

**Sponsoring**: We finalized the sponsoring flyer and
discussed several key topics, including new team photos, the
possibility of crowdfunding, and the savings from previous
iGEM teams.

**Marketing & social media**: The preparations and ideas
for posts, possible formats and publications in social media
have been prepared individually.

**Human Practices**: The meeting has been postponed by
one week. Further research was carried out.

**Meetup**: The kick-off meeting with Ms. Pott for the
project management module has taken place.

#### March

##### Calendar-Week 10: 03.03.-09.03.25

**General Matters**: We have written a “Quality
Improvement Funds” application in which we have applied
for the application fee to be covered by this pot of
money.

###### Focus Groups:

**Sponsoring**: Outreach to potential sponsors started.
Additionally, we set goals for the number of sponsorship
requests each team member should send per week.

**Marketing & social media**: We optimized the subgroup’s
organization by choosing assignees for the launching posts
about ongoing series and the leader for each social media
platform. To keep a recognizable view in the feed, color
palettes were chosen.

**Human Practices**: We discussed preparations for the
next Human Practices meeting scheduled in two weeks. The
familiarization with Human Practices principles and
guidelines will continue.

**Meetup**: This week, the team planned task timelines
for project organization and discussed the possibility of
rescheduling the Meetup, as the preferred venue at Münster
Castle is unavailable for July 4-6, 2025.

##### Calendar-Week 11: 10.03.-16.03.25

**General Matters**: Discussed further organizational
issues.

###### Focus Groups:

**Sponsoring**: All sponsoring inquiries up to this point
have been discussed among the focus group members, and a
standard protocol for weekly updates has been established.
Additionally, it was suggested that all team members reach
out to a select number of potential sponsors to enhance
engagement.

**Marketing & social media**: The group launched its
introductory post, followed by the completion of the
individual member pictures. Additionally, its first
continuous series, Fun Fact Friday, on Instagram was
published to awaken the general public’s interest in
scientific facts.

**Human Practices**: Further research was carried out.

**Meetup**: The team agreed to hold the Meetup at Münster
Castle from July 25 to 27, 2025.

##### Calendar-Week 12: 17.03.-23.03.25

**General Matters**: Expert round took place this
week. Three topics were presented and discussed in a
round with professors, PhDs and other students
interested in synthetic biology. Meeting with our PI to
organize further workflow and lab-start → S1
instruction?

###### Focus Groups:

**Sponsoring**: The focus group met with Petra Bölling,
Head of the University of Münster's Funding Department, to
explore additional financial support opportunities and seek
further assistance with sponsorship matters. During the
regular focus group meeting, the newly established protocol
for weekly updates was implemented, and the first
sponsorship commitments were recorded.

**Marketing & social media**: The group launched its
second continuous series, Wisdom Wednesday, on Instagram to
help the general public better understand diverse laboratory
techniques. To ensure accuracy and improve team organisation
specific members were assigned to handle future posts in the
ongoing series, while supervisors took charge to oversee
such posts.

**Human Practices**: This week, the team reviewed
successful Human Practices strategies used by past iGEM
teams. We discussed how to adapt these approaches to address
the three current topics under consideration.

**Meetup**: Possible programme items for the MeetUp
weekend were discussed.

##### Calendar-Week 13: 24.03.-30.03.25

**General Matters**: We decided on a project.
Luckily, we were very happy with all project ideas,
making the decision easy. We are excited to work on the
Phage project for the next months.

###### Focus Groups:

**Sponsoring**: The weekly update on sponsoring enquiry
was completed. Due to the resignation of a team and focus
group member, the assigned potential sponsors had to be
redistributed among the remaining members. Additionally, the
final cost estimate for this year’s project was created.

**Marketing & social media**: The team published its
second FunFactFriday post on Instagram and prepared further
posts.

**Human Practices**: The team reviewed how previous iGEM
teams implemented their projects and integrated Human
Practices.

**Meetup**: This week, tasks for the upcoming weeks were
assigned among team members. Additionally, the team
discussed design elements and catering options for the
Meetup.

#### April

##### Calendar-Week 14: 31.03.-06.04.25

**General Matters**: The focus this week was on
refining our project ideas and aligning them with the
feedback we had gathered from experts. Each subgroup
prepared initial outlines of their concepts for
discussion in the team meeting.

###### Focus Groups:

**Human Practices**: We reviewed the latest expert
interviews and documented insights into our structured
format (summary, aim of contact, insights, implementation).
These were shared in the team meeting and integrated into
our ongoing decision-making process.

**Wet Lab**: The lab group started compiling a list of
protocols needed for the upcoming experimental phase.
Members researched appropriate strains, vectors, and growth
conditions that would align with our project.

##### Calendar-Week 15: 07.04.-13.04.25

**General Matters**: During the weekly team meeting,
we presented our first consolidated project ideas and
discussed their feasibility in terms of time and
resources. Feedback was collected and documented to help
guide the selection process.

**Sponsoring**: The sponsoring group started contacting
local companies using the newly designed sponsorship flyer.
Additionally, a database of potential sponsors was expanded.

##### Calendar-Week 16: 14.04.-20.04.25

**General Matters**: With project selection
approaching, we evaluated the pros and cons of each
idea. Emphasis was placed on innovation, feasibility,
and alignment with iGEM values.

**Marketing & Social Media**: The group prepared
Easter-themed social media content to maintain engagement
and visibility of the team online.

##### Calendar-Week 17: 21.04.-27.04.25

**General Matters**: We finalized our internal voting
process for project selection. The procedure was agreed
upon during the meeting to ensure transparency and
fairness.

**Meetup**: Preparations for our JuniorJam meetup
advanced, with initial logistics planning and venue
inquiries.

#### May

##### Calendar-Week 18: 28.04.-04.05.25

**General Matters**: We had an introduction in lab
methods held by Prof. Prüfer and performed a laboratory
workshop to gain insight into common methods in
synthetic biology and cloning.

###### Focus Groups:

**Sponsoring**: We met with a sales representative from
INTEGRA to discuss sponsorship opportunities, scheduled and
planned further meetings with industry representatives, and
had our weekly sponsorship update.

**Marketing & Social Media**: We continued producing
posts.

**Human Practices**: We discussed special projects that
we want to tackle, e.g., an art exhibition.

**Meetup**: We contacted the Greek team after building
the first schedule ideas.

**Teambuilding**: We went through with the planned trip
to Carolinensiel, which had a positive impact on the team.

###### Lab Groups:

**Endolysin Platform**: We discussed the things we
researched in the meantime.

**Biofilm destruction**: We did further research in depth
on how to induce biofilms and which bacteria and phages to
use best.

**Drylab**: We continued learning about the methods that
we will use.

##### Calendar-Week 19: 05.05.-11.05.25

**General Matters**: We completed the registration of
our team roster.

###### Focus Groups:

**Sponsoring**: General updates and progress of the
individual members were discussed, as well as the next
steps. We decided not to continue writing a specific
application for funding, as the chances of success were
slim.

**Marketing & Social Media**: Ideas for a logo and a name
were presented to the team and a meeting was arranged.

**Human Practices**: We continued finding and contacting
interview partners.

**Meetup**: We continued to improve the schedule and
discussed potential speaker candidates.

###### Lab Groups:

**Endolysin Platform**: We continued the search for
feasible endolysin candidates and inducible plasmids and
planned the wetlab work.

**Biofilm destruction**: We met with a scientist
experienced in Biofilm destruction to exchange ideas and
questions. The team also discussed further assistance
offered by another German scientist. Individual findings and
questions were shared for better understanding.

**Drylab**: We continued researching machine learning and
modeling.

##### Calendar-Week 20: 12.05.-18.05.25

**General Matters**: We held a PowerPoint and
presentation workshop to prepare for our appearance at
the BFH Meetup next week. Brainstorming for possible
project titles was also carried out.

###### Focus Groups:

**Sponsoring**: General updates and progress of
individual members were discussed, along with next steps for
foundations. We had an online meeting with a representative
of Greiner Bio One regarding possible lab material donations
and sponsoring packages for our meetup.

**Marketing & Social Media**: Final stages of logo design
were carried out, a provisional project name was elected,
and business cards were designed and printed.

**Human Practices**: Potential interview partners
continued to be sought and contacted.

**Meetup**: The team continued to work out the timetable
and discussed the evening program.

**Teambuilding**: Finances of Carolinensiel were
discussed and further activities planned.

###### Lab Groups:

**Endolysin Platform**: Continued searching for feasible
endolysin candidates and inducible plasmids and planning the
wetlab work.

**Biofilm destruction**: More research on planned lab
work; emailed Prof. Hust and Prof. Horz with questions
regarding phages.

**Drylab**: Continued learning about the methods that
will be used.

##### Calendar-Week 21: 19.05.-25.05.25

**General Matters**: We attended the BFH Meetup and
presented our project for the first time. Valuable
feedback was received for integration in upcoming weeks.

###### Focus Groups:

**Sponsoring**: Each group member outlined their
individual work process for updates. New exercises were
introduced and assigned. A visit with a representative from
Neofroxx resulted in seven boxes of pipette tips, with
photos taken.

**Marketing & Social Media**: Continued creating posts
and working on social media presence.

**Human Practices**: Prepared questions for the next
interview.

**Meetup**: Discussed speakers, possible judges, and
evening location.

###### Lab Groups:

**Endolysin Platform**: Continued searching for feasible
endolysin candidates and inducible plasmids and planning
wetlab work.

**Biofilm destruction**: Continued research for planned
lab work and discussed open questions regarding peptide
usage.

**Drylab**: Progressed on coding of the Top-model and
divided tasks for an upcoming sponsoring opportunity.

##### Calendar-Week 22: 26.05.-01.06.25

**General Matters**: We had a big discussion with all
wetlab groups to restructure and rethink lab work. We
decided to focus on the in vitro expression approach and
fuse Wetlab 1 and 2 into a new group: Endolysin
Platform. Drylab approach was also reconsidered.

###### Focus Groups:

**Sponsoring**: Talk with Louis Schanzmann (former
iGEMer) regarding sponsoring contacts and worked on funding
applications.

**Marketing & Social Media**: Continued building social
media presence.

**Human Practices**: Prepared and conducted next
interview; initial ideas for the ethics lecture collected.

**Meetup**: Attended BFH meetup and discussed adaptations
for our own meetup.

###### Lab Groups:

**Endolysin Platform**: Fused Wetlab Teams (Cell-free
expression and Recombinant expression) into Endolysin
Platform. New aims: establish an endolysin screening
platform, test efficacy of combinations of endolysins and
endolysins with antibiotics, create fusion proteins with
peptides to improve bacterial lysis. Focus shifted to
cell-free expression due to time constraints and platform
limits.

**Biofilm destruction**: Further research and discussion
for planned lab work.

**Drylab**: Further conceptualized workflow by building a
pipeline.

#### June

##### Calendar-Week 23: 02.06.-08.06.25

**General Matters**: In our team meeting, we
discussed possible gold medal criteria, which ones to
focus on, and brainstormed how to answer all questions
from the judging handbook to ensure our project will be
valuable for iGEM in the future.

###### Focus Groups:

**Sponsoring**: An application is being submitted to
Xynabio retroactively, and several new funding requests
(including Rituals and the Mobility Fund) have been sent
out. Diapharm is currently unresponsive, and the status of
the module funds is to be clarified with Andrea.

**Marketing & Social Media**: Deliberated the optimal
arrangement for posts on the laboratory group's presentation
and considered changing post frequency, aiming for quality
over quantity.

**Human Practices**: Discussed current project status and
potential interview candidates.

**Meetup**: Prepared for an upcoming workshop and
discussed progress.

###### Lab Groups:

**Endolysin Platform**: Discussed research progress and
ideas; received first plasmid (pALICE01) for the cell-free
expression system and designed constructs including a tag
and POI.

**Biofilm destruction**: Created a potential vector for
ordering and conducted further research for planned lab
work.

**Drylab**: Distributed tasks and connected with the
Biofilm destruction lab group for cooperation.

##### Calendar-Week 24: 09.06.-15.06.25

**General Matters**: Discussed village selection and
identified three possible options to decide next week
(Therapeutics, Infectious Diseases, Conservation).

###### Focus Groups:

**Sponsoring**: Received the thermocycler from Analytik
Jena and continued acquiring sponsoring contacts.

**Marketing & Social Media**: Further work on posts.

**Human Practices**: Prepared an interview questionnaire
for the next interview.

**Meetup**: Attended the Project Management Module
workshop and made further plans for the meetup.

###### Lab Groups:

**Endolysin Platform**: Planned lab work for the upcoming
week and worked on plasmid design in SnapGene. Prepared
media for casting plates.

**Biofilm destruction**: Discussed genes needed on
vectors and planned primer preparation.

**Drylab**: Discussed progress on tasks and took the
group picture.

##### Calendar-Week 25: 16.06.-22.06.25

**General Matters**: Decided on the village
“Infectious Diseases” as first preference, with
“Therapeutics” or “Conservation” as alternatives.

###### Focus Groups:

**Sponsoring**: Received a new scale and discussed meetup
requirements for possible sponsors/donors.

**Marketing & Social Media**: Continued work on posts.

**Human Practices**: Conducted an interview with a lawyer
and biologist regarding authorization of phage therapy and
considered other potential interview partners.

**Meetup**: Reached consensus on financial remuneration
and registration period for the meetup and considered other
possible speakers.

###### Lab Groups:

**Endolysin Platform**: Shifted focus from
high-throughput screening to high-yield expression platform
to leverage advantages of cell-free in vitro expression.
Transformed competent *E. coli* Turbo with modified
pALICE01 plasmid and used cPCR for verification.

**Biofilm destruction**: Discussed peptide functions and
selected the best-performing and least hemolytic options.

**Drylab**: Continued working on designated tasks.

##### Calendar-Week 26: 23.06.-29.06.25

###### Focus Groups:

**Sponsoring**: Funding efforts ongoing. Secured €800
from AStA and €750 from Sparkasse for the meetup. Applied
for additional funding from the city utility. Jamboree fee
pending; Prof. Schmid following up.

**Marketing & Social Media**: Media design workshop
conducted.

**Human Practices**: Conducted further interviews and
discussed next steps.

**Meetup**: Updated on progress and made key planning
decisions.

**Teambuilding**: Various activities planned.

###### Lab Groups:

**Endolysin Platform**: Performed first transformations
with pALICE01 plasmid and began cloning pALICE\_lysin
plasmids via restriction-ligation.

**Biofilm destruction**: Ordered vector and started
planning lab work.

**Drylab**: Continued working on designated tasks.

#### July

##### Calendar-Week 27: 30.06.-06.07.25

###### Focus Groups:

**Sponsoring**: Raised €43,000 in donations and funds so
far. Requested further contacts recommended by Invitris and
Louis. Companies like Eppendorf and Zymmo Research want to
meet at the institute.

**Marketing & Social Media**: Designed team T-shirts and
discussed team logo color. Prepared a post highlighting a
speaker for the meetup.

**Human Practices**: Prepared the ethics lecture.

**Meetup**: Made important planning decisions and decided
to invite all European teams.

###### Lab Groups:

**Endolysin Platform**: Continued experimental work to
obtain sufficient plasmid quantities for the ALICE reaction.
Started initial design for a Golden Gate-based modular
expression platform.

**Biofilm destruction**: Began lab work and made
bacterial stocks.

**Drylab**: Updated on progress and connected with the
Endolysin Platform group.

##### Calendar-Week 28: 07.07.-13.07.25

###### Focus Groups:

**Sponsoring**: Discussed individual progress and
outstanding foundation inquiries. Created a sponsor list for
the meetup as a weekly goal.

**Marketing & Social Media**: Agreed on T-shirt design
and ordered them. Prepared further posts for the meetup
weekend.

**Human Practices**: Discussed further processing of
interviews.

**Meetup**: Continued planning.

**Wiki**: Planned general structure and searched for
inspiration from previous years.

###### Lab Groups:

**Endolysin Platform**: Progressed in cloning vectors and
endolysins; obtained first verified and purified constructs.

**Biofilm destruction**: Made electrocompetent cells and
performed gDNA extraction.

**Drylab**: Ran unbiased simulations (1–3 μs) and
molecular dynamics (MD) simulations with energy groups to
analyze Coulomb and Lennard-Jones interactions. Observed
CM15 inserting into the OC region after 4000 ns, indicating
higher effectiveness when separated from the endolysin;
SMAP29 unaffected by attachment.

##### Calendar-Week 29: 14.07.-20.07.25

###### Focus Groups:

**Sponsoring**: Several sponsors confirmed support,
including product donations, software access, and initial
funding. Some contacts are pending. Additional outreach
ongoing.

**Marketing & Social Media**: T-shirts ordered and
further posts for the meetup weekend uploaded.

**Human Practices**: Continued planning the art
exhibition and interview preparation.

**Meetup**: Planned and prepared the meetup weekend;
created a timetable with task distribution.

**Wiki**: Continued collecting ideas for Wiki design.

###### Lab Groups:

**Endolysin Platform**: Delivered and prepared primers
for Golden Gate. Amplified and purified endolysins and Strep
vector for Golden Gate.

**Biofilm destruction**: Performed first transformation
with M13 KE vector and gDNA extraction.

**Drylab**: Continued unbiased simulations for rough
membranes and investigated AMP interactions with
rough/smooth LPS. Observed SMAP29 insertion with N-terminal
end.

##### Calendar-Week 30: 21.07.-27.07.25

###### Focus Groups:

**Sponsoring**: Crowdfunding platform almost ready to
launch.

**Marketing & Social Media**: Prepared and posted several
updates for the meetup.

**Human Practices**: Planned further interviews.

**Meetup**: Successfully organized the meetup weekend.

**Wiki**: Continued working on the wiki.

###### Lab Groups:

**Endolysin Platform**: Conducted an interview with Prof.
Christine Heilmann on autolysins and performed further
research.

**Biofilm destruction**: Continued lab work and improved
transformations.

**Drylab**: Researched Lipid A layer insertion and
methods to detect membrane disruption.

#### August

##### Calendar-Week 31: 28.07.-03.08.25

###### Focus Groups:

**Sponsoring**: Continued working on current exercises.

**Marketing & Social Media**: Organised meetup pictures;
polished team introductions (some still missing).

**Human Practices**: Collected questions for further
interviews.

**Meetup**: Meetup was carried out successfully.

**Wiki**: Continued work on the Wiki.

###### Lab Groups:

**Endolysin Platform**: Busy with the meetup.

**Biofilm destruction**: Busy with the meetup.

**Drylab**: Worked on visualisation, plotting, and
presentation.

##### Calendar-Week 32: 04.08.-10.08.25

###### Focus Groups:

**Sponsoring**: Continued current exercises.

**Human Practices**: Conducted further interviews.

**Wiki**: Started preparing the promotion video.

**Teambuilding**: -

###### Lab Groups:

**Endolysin Platform**: Performed Golden Gate Assembly of
HY-133 endolysin and Alice01 plasmid. Started Alice reaction
with PlyEc2, E84, PlyYouna2, and T4.

**Biofilm destruction**: Tried the next electroporation.

**Drylab**: Performed additional calculations.

##### Calendar-Week 33: 11.08.-17.08.25

###### Focus Groups:

**Sponsoring**: Continued current exercises.

**Marketing & Social Media**: Finalised and uploaded
promotion video.

**Human Practices**: Conducted further interviews.

**Wiki**: Focused on promotion video.

###### Lab Groups:

**Endolysin Platform**: Conducted initial endolysin
activity assays on PlyEc2 and *E. coli* K12 without
success; optimisation and troubleshooting planned for next
week.

**Biofilm destruction**: Multiplied and cleaned
phages/DNA.

**Drylab**: Used PackMemb tool to analyze lipid defects;
modified for LPS instead of phospholipid.

##### Calendar-Week 34: 18.08.-24.08.25

###### Focus Groups:

**Human Practices**: Conducted further interviews.

**Wiki**: Made design choices and began implementation in
code.

###### Lab Groups:

**Biofilm destruction**: Attempted first Biofilm
destruction experiment.

**Drylab**: Wrote Python program to convert parameters
from GROMACS ITP file for PackMem; studied lipid packing
defects.

#### September

##### Calendar-Week 35: 25.08.-31.08.25

###### Focus Groups:

**Sponsoring**: Continued current exercises.

**Human Practices**: Followed up on interviews and
discussed Wiki concept.

**Wiki**: Continued coding, focusing on menu bar and
header.

###### Lab Groups:

**Endolysin Platform**: Continued troubleshooting protein
expression in ALICE-Kit (including work at Prüfer Lab);
finalised design and primer order for fusion constructs.

**Biofilm destruction**: Purified phages, isolated and
amplified DNA.

**Drylab**: Finalised calculations; used PackMem to study
peptide insertion into LPA phase.

##### Calendar-Week 36: 01.09.-07.09.25

###### Focus Groups:

**Human Practices**: Prepared art exhibition; discussed
Wiki design.

**Wiki**: Discussed content for team page.

###### Lab Groups:

**Endolysin Platform**: Finalised troubleshooting;
performed western blot to confirm protein expression. No
expression detected; lab work discontinued.

**Biofilm destruction**: Prepared samples for screening.

##### Calendar-Week 37: 08.09.-14.09.25

###### Focus Groups:

**Human Practices**: Discussed Wiki development for human
practices.

**Wiki**: Discussed areas for improvement (e.g., home
page); updated progress and current issues.

###### Lab Groups:

**Biofilm destruction**: Prepared samples for screening
in S2 laboratories on enteroaggregative and K12
*E. coli*
strains.

##### Calendar-Week 38: 15.09.-21.09.25

###### Focus Groups:

**Human Practices**: Continued writing Wiki texts.

**Wiki**: Continued working on design and implementation.

###### Lab Groups:

**Biofilm destruction**: Conducted first screening
experiments on enteroaggregative *E. coli*.

##### Calendar-Week 39: 22.09.-28.09.25

###### Focus Groups:

**Marketing & Social Media**: Continued creating posts.

**Human Practices**: Continued writing Wiki texts.

**Wiki**: Discussed landing page content and assigned
responsibilities for subpages.

###### Lab Groups:

**Biofilm destruction**: Repeated experiment with longer
biofilm formation; obtained final results from biofilm
destruction screening.

[Members](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27members%27%29%20%7D%7D)

[IHP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)

[Education](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D)

{% endblock %}

{% extends "layout.html" %} {% block title %}Engineering{% endblock %} {% block
image %}
![gruppen-photo](https://static.igem.wiki/teams/5946/images/header/spitzenkasten.webp)
{% endblock %} {% block page\_content %}

## ![abstract-icon](https://static.igem.wiki/teams/5946/images/icons/abstract-icon.webp) Abstract

With our project, we aim to establish a cell-free endolysin
expression platform that enables the reliable and rapid
identification of lead variants, particularly against
Gram-negative bacteria. In addition, we seek to develop a
toolkit based on this system to facilitate endolysin
modification and the generation of target-optimized
variants.

Considering the potential application of our endolysins in
human infections, we identified biofilms as a major obstacle
and therefore aimed to disrupt its integrity via a
synergistic approach employing phage-based endolysin
transport in combination with antimicrobial and
signal-modulating peptides.

AMPs (antimicrobial peptides) can also enable endolysins to
diffuse through the OM (outer membrane). This process is
still not well understood, thus, we developed
[**AMP-MD**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D), a molecular dynamics (MD) platform to study how AMPs
interact with the OM.

To successfully implement our research idea, we used the
engineering cycle based on the design-build-test-learn
(DBTL) model.

#### Endolysin Expression Platform

##### Cytosolic endolysin constructs

As every project ours too started with a design phase. Here we
created our experimental plasmid design and chose our endolysin
candidates. With a clear outline of that, a project blueprint
was established comprising all characteristics of the biological
system and parts. This was then implemented in the build phase
where we assembled and verified our plasmids and started the
protein expression. Subsequently, the expression samples of our
endolysins were tested with regard to antimicrobial activity
against diverse organisms. With the testing results in at hand
we could learn from the generated data and use the gained
insights for further improvement and shaping of our project in
the next iterations of this engineering cycle.

![eng-cycle](https://static.igem.wiki/teams/5946/images/pages/engineering/eng-cycle.svg)

### Design: Experimental & Plasmid design

During the preliminary design phase, we
selected the endolysin PlyEc2 ([bba-25h8ekrx](https://registry.igem.org/parts/bba-25h8ekrx)). This phage lysin demonstrates promising
cell lysis for a broad spectrum of
Gram-negative organisms, including
*Pseudomonas aeruginosa, Acinetobacter
baumannii*, and *Escherichia coli*
[(Xu et al., 2021)](#ref-xu2021). Consequently, it served as an ideal
starting point for developing our production
system using a cell-free expression kit.

In the selection of a cell-free expression
kit, the ALiCE® kit demonstrated notable
potential. This kit is based on the crude
lysate of *Nicotiana tabacum* Bright
Yellow 2 (BY-2) cells and exhibits a high
amount of producible protein
[(Buntru et al., 2014;](#ref-buntru2014)[LenioBio®, 2024)](#ref-leniobio2024). Since protein expression in bacteria is
often difficult for endolysins due to their
toxic properties
[(Cremelie et al., 2024)](#ref-cremelie2024)
cell-free expression with high yield, like
ALiCE®, enables a promising alternative.
Furthermore, the kit was kindly provided to
us by the AG Prüfer, rendering a promising
and accessible expression system, also for
establishing our planned toolbox, the
details of which will be explained in the
[AMP-Endolysin fusion constructs](#wl1-cycle3)
section.

During the first cycle, our primary
objective was to assess the potential of
producing functional protein using ALiCE®.
To accomplish this, we designed the
necessary plasmids and decided on growth
inhibition assays as our primary screening
method.

Additionally, the necessary preparations
were made to work with the biosafety level 2
(BSL-2) strain of
*Pseudomonas aeruginosa*
PAO1 since growth inhibition by PlyEc2 had
already been described for this model strain
[(Xu et al., 2021)](#ref-xu2021). *P. aeruginosa* PAO1 was
deliberately selected because
strain-specific differences of the endolysin
would limit the significance of results
obtained with other S1 Pseudomonas strains.
Furthermore, our project contributes to the
fight against antibiotic resistance, with
ESKAPE pathogens being particularly
relevant. In front of this background, the
inclusion of a clinically relevant strain
such as *P. aeruginosa* was of utmost
importance.

### Build: Cloning of expression plasmids and subsequent protein expression

The pALiCE01 standard vector was utilized as
a backbone in cloning PlyEc2 (Figure 1), as
this endolysin is neither membrane-bound nor
subject to post-translational modifications,
theoretically enabling the cytosolic
production of our protein of interest (POI),
using the ALiCE® system
[(LenioBio®, 2024)](#ref-leniobio2024).

For the assembly, we used
restriction-ligation cloning, as the
supplied plasmid is optimized for this. We
utilized *E. coli* Turbo for
transformation and plasmid amplification.
The nucleotide sequence of the endolysin was
ordered as a G-block from IDT. View the
[results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
page to discover more about our exact
cloning procedures.

![fig8](https://static.igem.wiki/teams/5946/images/pages/engineering/wl1/ec-figure-1-plasmid-map-plyec2-without-strepii.webp)

**Figure 1:** Plasmid map
of the pALiCE01\_PlyEc2 plasmid.

After successful plasmid sequencing, we
purified the plasmids using
phenol-chloroform purification to ensure a
high degree of purity, essential for the
ALiCE® system. We then started with
expressing our desired proteins.

### Test: Growth inhibiton assay with *Pseudomonas aeruginosa* PAO1

A growth inhibition assay was conducted with
*Pseudomonas aeruginosa* PAO1 to assess
the functionality of the produced endolysin
(Figure 2). No growth inhibition was
observed for the PlyEc2 reaction supernatant
or for the non-template reaction control
(negative control). All the assay variants
exhibited comparable growth to that of the
positive control without any added
component.

![fig8](https://static.igem.wiki/teams/5946/images/pages/engineering/wl1/ec-figure-2-growth-inhibition-pao1.webp)

**Figure 2:** Initial
growth inhibition assay of PlyEc2 on
*Pseudomonas aeruginosa* PAO1.
PlyEc2 supernatant (SN) with an
estimated concentration of 20 µg/ mL and
60 µg/ mL were tested on 106
*P. aeruginosa* PAO1 cells.
Additionally, the non-template controls
(NTC) and the LB control were used as
negative controls. LB +
*P. aeruginosa* PAO1 itself and in
combination with gentamecin were
utilized as positive control. Cells were
grown in LB medium, at 37 °C and 250
rpm.

### Learn: Findings for the next cycle iteration

Contrary to our expectations, our initial
tests revealed no signs of growth inhibition
and the lack of a reliable and feasible
detection method for our non-tagged PlyEc2
prevented the determination of the exact
protein concentration. Initially, we were
thus unable to rule out an insufficient
protein concentration, its total absence, or
the lack of endolysin functionality despite
it being present.

Based on these findings, we decided to
integrate a StrepII-tag into our vector
constructs in order to be able to confirm
the absence or presence of our protein and
to clarify the concentration of our POIs.

In addition to the possibility that our
lysin was not produced at all, or at least
not produced functionally, we also
investigated whether the strain specificity
of the tested endolysin could be a possible
cause for the lack of growth inhibition.

‹
›
‹
›
‹
›
‹
›

##### StrepII-tag endolysin constructs

![eng-cycle](https://static.igem.wiki/teams/5946/images/pages/engineering/eng-cycle.svg)

### Design: Experimental & Plasmid design

The modified pALiCE01 vector now contained a
StrepII-tag combined with a (GGGGS)x2
linker. This vector was additionally adapted
for both restriction-ligation and Golden
Gate cloning, provided that specific primers
are utilized.

Furthermore, we decided to employ an
additional endolysin, designated as
PlyYouna2 ([bba-25uoafah](https://registry.igem.org/parts/bba-25uoafah)). This endolysin originates from the Youna
phage and is specialized for the lysis of
Gram-positive bacteria, including
*Bacillus megaterium* and
*B. subtilis*. However, studies
demonstrate its additional efficacy in the
lysis of specific Gram-negative organisms,
including e.g., *E. coli* or
*Pseudomonas putida*
[(Son et al., 2023)](#ref-son2023).

While the lysis of Gram-positive bacteria by
externally applied endolysins is specific,
it is facilitated by the lack of an outer
membrane, which simplifies access and
disruption of the peptidoglycan layer from
the exterior
[(Zheng and Zhang, 2024)](#ref-zheng2024). The bacterial strains utilized in our
experiments are BSL-1 strains, which can be
easily integrated into our standard
laboratory protocols due to their RG1
classification and their well-established
utilization in neighbouring working groups.

Additionally, we decided to switch to plate
spot assays instead of growth inhibition
assays as the initial screening method, to
enable a faster but still reliable workflow.

### Build: Cloning of the plasmids and protein expression

Using the modified pALiCE01 vector including
a StrepII-tag and the (GGGGS)2 linker we
cloned the following plasmids for each
endolysin via Golden Gate Cloning (Figure
3):

* pALiCE01\_StrepII\_PlyEc2
* pALiCE01\_StrepII\_PlyYouna2

![fig8](https://static.igem.wiki/teams/5946/images/pages/engineering/wl1/ec-figure-3-plasmid-maps-with-strepii.webp)

**Figure 3:** Plasmid map
of A) pALiCE01\_StrepII\_PlyEc2 and B)
pALiCE01\_StrepII\_PlyYouna2.

The correct assembly was confirmed by
plasmid purification, followed by
sequencing. Afterwards protein expression
via the ALiCE® system was performed.

### Test: Plate spot assays and validation of purification and expression methods

Initially, our SDS-PAGE results did not
allow a clear determination of the presence
or absence of our endolysins due to
contamination of the non-template control
(NTC), which was of the same expected size
as that of the endolysins.

Additionally, the plate spot assay for
PlyYouna2 on B. megaterium revealed lysis in
both the supernatant and the NTC. However,
lysis was no longer detectable after native
protein purification with the MagStrep
Strep-Tactin™ XT Beads. The same was
observed for PlyEc2 on
*P. aeruginosa* (Figure 4).

![fig8](https://static.igem.wiki/teams/5946/images/pages/engineering/wl1/ec-figure-4-plate-spot-assay.webp)

**Figure 4:** Plate spot
assay of PlyYouna2 on
*Bacillus megaterium* (A) and of
PlyEc2 on
*Pseudomonas aeruginosa* PAO1(B).
For A) the supernatant (SN), pellet and
natively purified PlyYouna2 as well as
50 µg/µL ampicillin, the non-template
control (NTC) supernatant and the
elution buffer of the purification (1x
BXT) were tested for lysis. For B) the
PlyEc2 supernatant, 50 µg/µL ampicillin
and the non-template control (NTC)
supernatant were tested regarding lysis.

Repeating the SDS-PAGE followed by Western
blot revealed that our endolysin had not
been produced (Figure 5).

To ensure the functionality of the ALiCE®
kit, another Western blot was performed to
verify the successful production of the
positive control protein (eYFP) using our
StrepII-tag (Figure 5). This experiment
herein also confirmed the successful
purification of proteins, such as the
labelled eYFP, using the MagStrep
Strep-Tactin™ XT Beads. Furthermore,
phenol-chloroform plasmid purification was
excluded as a potential causative agent for
our failed endolysin expression. Plasmids
purified using this method could be used
effectively to produce and purify eYFP
successfully.

![fig8](https://static.igem.wiki/teams/5946/images/pages/engineering/wl1/ec-figure-5-final-westernblot-new.webp)

**Figure 5:** Ponceau
staining on the left and Western blot on
the right. A) shows the results of the
StrepII-tagged eYFP derived from the
standard pALiCE01 plasmid already
delivered purified. Further it exhibits
the PlyEc2 samples of the whole lysate
(WL), supernatant (SN), resuspended
pellet (P), and the natively purified
protein (A). B) shows the results of the
StrepII-tagged eYFP derived from the
standard pALiCE01 that was previously
purified with the phenol-chloroform
purification method. It contains the WL,
SN, P and A as well as the input (IP)
and the wash of the native protein
purification. Additionally, IP and wash
of PlyEc2 and the non-template control
(NTC) are visible. The NTC is an ALiCE®
reaction sample where no plasmid was
used as template. It exhibits the
negative control of the ALiCE® reaction.
Further, the SDS-PAGE gel of
**B)** contained a leaky pocket. As a
result, a sample flowed across the gel,
resulting in the slight continuous
staining visible in the WB.

### Learn: Findings from the troubleshooting process

During the troubleshooting process, the
functionality of the ALiCE® kit was
confirmed, as well as the efficacy of the
phenol-chloroform plasmid purification and
the MagStrep Strep-Tactin™ XT Beads
purification. Furthermore, the accuracy of
the plasmid assembly was again validated
through sequencing.

Through advanced research and consultation,
we discovered that the lysis, which was
observed in the NTC, could be attributed to
antibacterial substances present in the
lysate of the tobacco root cells utilized in
the kit
[(Al-Lahham et al., 2020)](#ref-allahham2020).

Finally, this indicated that the kit cannot
be used to establish an adequate negative
control for the expression of antimicrobial
proteins. Consequently, we deemed the kit to
be unsuitable for our intended application.
However, the implementation of an
alternative cell-free kit based on a
prokaryotic lysate, which was actually
planned since May, was hindered by the
protracted regulatory process involved,
rendering further efforts in this direction
infeasible due to time constraints.

‹
›
‹
›
‹
›
‹
›

##### AMP-Endolysin fusion constructs

Based on our Integrated Human Practices work, we identified
antimicrobial peptides (AMPs) as a possibility for improving
endolysins to enhance their effectiveness against Gram-negative
bacteria. Because of the broad range of AMPs that could
potentially be fused to endolysins, we developed a
molecular-dynamics-based (MD) model to understand their
insertion mechanism into the outer membrane of Gram-negative
bacteria and evaluate which properties may enhance their
effectiveness against these bacteria when fused to an endolysin.
Based on these studies, we selected the AMPs K14 ([BBa-25xu6jmw](https://registry.igem.org/parts/bba-25xu6jmw)) and Sushi S1 ([BBa-k5057004](https://registry.igem.org/parts/bba-k5057004)) to design our fusion constructs. Both the cationic CM15
derivate K14 as well as the Sushi S1 AMP exhibit low or no
haemolytic activity
[(Yau et al., 2001;](#ref-yau2001)[Ding, Li and Ho, 2008;](#ref-ding2008)
[Sato and Feix, 2008)](#ref-sato2008).

For an efficient and rapid assembly, we initially aimed to
create a toolbox approach based on the cytosolic ALiCE® vector
using Golden Gate Assembly. The individual building blocks,
consisting of AMPs, linkers, and endolysins with respective
position tags, could then be easily exchanged to effectively
create enhanced endolysin variants. In silico designs for both
the ALiCE® and pEt28b(+) vectors with these building blocks had
already been designed at that point (see
[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)
for more details).

However, due to non-reliable protein expression results with the
ALiCE® system, the absence of the second cell-free expression
kit, and time constraints, we had to stop at this point.

For more information about the model, please visit our
[model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page
and view the [model cycles](#dl).

#### Biofilm destruction

##### Identifying the needs for a holistic alternative to antibiotics

Our initial approach aimed to research and develop a
comprehensive alternative to conventional antibiotics in light
of rising antimicrobial resistance (AMR). During literature
review, phage therapy appeared especially promising, as well as
phage-encoded endolysins, identified as potent active agents due
to their ability to degrade bacterial cell walls and disrupt
biofilms. However, besides our work on utilizing endolysins
potential, our human practices endeavors also led us to focus on
potential for clinical applications in humans. In this regard,
biofilms were repeatedly pointed out as key complicating factors
during infections caused by resistant bacteria. It became
evident that biofilms pose a significant barrier to bacterial
control across multiple settings. Thus, in line with our
objective of finding a holistic alternative potentially
surpassing conventional antibiotics, we expanded our focus to
include the targeted eradication and disruption of biofilms in
respect to future application.

##### Theory of biofilm infiltration and destruction by phages and auxiliary substances

![eng-cycle](https://static.igem.wiki/teams/5946/images/pages/engineering/eng-cycle.svg)

### Design:

During our literature research on the
targeted destruction of bacterial biofilms,
we developed the idea of introducing
modified endolysin variants to
biofilm-forming bacteria using infiltrating
phages. To increase this infiltration
capacity of the phages into the biofilm, we
planned to fuse biofilm-damaging peptides to
their capsid proteins. At the same time, we
discovered that pathogenic and
non-pathogenic biofilms differ structurally
and functionally. To test our concepts on
clinically relevant strains like
enteroaggregative *Escherichia coli*,
we initiated a collaboration with the
Dobrindt research group. We chose M13 phages
as the vector, because they are
well-established for phage display and
enable clear analysis of endolysin effects
due to their lysogenic cycle.

### Build:

For the implementation, we selected peptide
sequences on the condition that they would
be suitable for possible later use in
humans. Accordingly, we did not allow the
selected peptides to be hemolytic or
otherwise toxic. We decided to test
different modes of action, including
antimicrobial peptides (AMPs),
anti-biofilm-active peptides (ABPs), and
peptides that disrupt bacterial growth
signals within the biofilm specifically. We
derived some sequences from human proteins
in the hope of minimizing immunological
reactions in clinical applications. We then
designed suitable primers for these peptides
and produced the first bacteriophages.

### Test:

The production of the phages was successful,
as was the DNA extraction using
phenol-chloroform purification. However,
difficulties arose during DNA amplification
of the phage vector using Q5 polymerase for
overlap extension PCR, a step that was also
intended to enable the cloning of the
peptides.

### Learn:

After several attempts, we evaluated
alternative polymerases for PCR. We finally
achieved the desired amplification with the
Phusion polymerase. At the same time, we
conducted several human practices interviews
on the regulatory situation of phages in
Germany and decided to continue working with
wild-type phages rather than genetically
modified (GMO) phages.

‹
›
‹
›
‹
›
‹
›

##### Planning of BSL-2 work on pathogenic bacteria and initial testing

![eng-cycle](https://static.igem.wiki/teams/5946/images/pages/engineering/eng-cycle.svg)

### Design:

Therefore, we decided to focus on the
combined effect of the peptides on biofilm
destruction, both individually and in
combination with wild-type M13 phages.
Although these phages are exclusively
lysogenic, they can also inhibit horizontal
gene transfer, preventing the exchange of
resistance genes within the biofilm. In
preparation for the upcoming experiments, we
examined the legal and safety requirements
for working in BSL-2 laboratories. We
prepared the necessary documentation,
consulted the company doctor, and clarified
all necessary aspects of the safety
briefings to ensure our personal safety.

### Build:

For the first series of tests, we selected
peptide concentrations based on the minimum
inhibitory concentrations (MIC values)
specified in the literature and designed
combinations of phages and peptides for use
in a 96-well plate.

### Test:

The first experiment was performed on a PVC
96-well plate. The biofilm was first
cultured for 24 hours, followed by further
24 hours of incubation with the
peptide/phage combinations. After staining
with crystal violet, the amount of biofilm
was quantitatively determined by absorption
measurements. All peptides were used at
double the MIC concentration.

### Learn:

In the first run, only slight biofilm
formation was observed. The
enteroaggregative *E. coli* strain
55989 exhibited limited growth, but no
statistically reliable conclusions could be
drawn from the results.

‹
›
‹
›
‹
›
‹
›

##### Testing biofilm destruction and reflecting on the results

![eng-cycle](https://static.igem.wiki/teams/5946/images/pages/engineering/eng-cycle.svg)

### Design:

As the results of the previous test cycle
were only of limited significance, we
analyzed potential sources of error and
possible revisions to the test design.

### Build:

During this analysis, we determined that,
under certain conditions, biofilms require
more than 24 hours to achieve sufficient
density and stability for reproducible
measurements. We also decided to use an
alternative plate material since biofilm
adhesion can be influenced by the plastic
surface. We also planned to use higher
peptide concentrations to quantify a more
pronounced effect.

### Test:

The optimized experiment was performed on
polystyrene 96-well plates. The biofilms
were allowed to form for 48 hours before
being treated with the peptide/phage
combinations. In addition to twice the MIC
concentration, we also tested the 10-fold
concentrations of the respective peptides
from the first testing.

### Learn:

The modified test conditions led to
significantly more usable
[results](https://2025.igem.wiki/unimuenster/results#biofilm-reduction-tests), which we then analyzed in detail. This
enabled us to identify
[plausible explanations for the observed
effects](https://2025.igem.wiki/unimuenster/results#michelle-disschussion). However, the measured standard deviations
were still relatively high in some cases.
For subsequent cycles, it would be essential
to repeat the experiments in order to be
able to make more statistically reliable
statements. In addition, possible
synergistic effects and the adaptability of
the biofilm to different environmental
conditions should be investigated.

‹
›
‹
›
‹
›
‹
›

#### Modeling of AMP-membrane interactions

Our initial goal was to quantify the effectiveness of different
antimicrobial AMPs based on their insertion free energy into the
membrane through Umbrella Sampling. This approach failed because
of the sensitivity of Umbrella Sampling in large systems.
Therefore, we decided to focus more on understanding the
mechanism of the insertion of peptides into the membrane. This
new approach of our
[modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%7D%7D) led
to exciting results, and our model could even be used to explain
phenomena in our wetlab.

##### Umbrella Sampling

![eng-cycle](https://static.igem.wiki/teams/5946/images/pages/engineering/eng-cycle.svg)

### Design:

Our first idea was to quantify the
effectiveness of different antimicrobial
AMPs based on their insertion free energy
into the membrane through Umbrella Sampling.

### Build:

We built the membrane system (*E. coli*
R3 O111/CM15) with CHARMM-GUI
[(Jo et al., 2008)](#ref-jo2008)
and inserted the CM15 peptide on top of the
membrane in the extracellular space.

### Test:

Then we performed a pull simulation and
extracted the Umbrella Sampling starting
windows. For each starting window, a biased
simulation was performed with a harmonic
potential restricting the peptide in the z
direction. From the biased distributions of
each umbrella window, the insertion free
energy was calculated. We also tested
different starting configurations, yielding
different free energies.

### Learn:

A quantitative description of the insertion
free energies from different peptides in the
same membrane is not possible with umbrella
sampling due to the high sensitivity of
umbrella sampling to small changes in the
starting configurations.

‹
›
‹
›
‹
›
‹
›

##### Unbiased simulations

![eng-cycle](https://static.igem.wiki/teams/5946/images/pages/engineering/eng-cycle.svg)

### Design:

After our failed goal to quantify the
effectiveness of different AMPs with
umbrella sampling, we decided to focus on
the mechanism of AMP insertion into the
membrane and how it increases the membrane
permeation.

### Build:

We built a new membrane/peptide (*E. coli*
R3 O111/6 x CM15) system and equilibrated
the system.

### Test:

We performed unbiased simulations of the
system and observed a free insertion into
the outer membrane. Further, we studied the
interactions between the peptide and outer
membrane and quantified the increase of
membrane permeation with the “PackMem”
package
[(Gautier et al., 2018)](#ref-gautier2018).

### Learn:

The unbiased simulations helped us
understand the mechanism of membrane
insertion of peptides. We observed an
increase in lipid packing defects after the
peptide was inserted into the
Core-saccharide region of the membrane.

‹
›
‹
›
‹
›
‹
›

#### References

Al-Lahham, S. et al. (2020) “Antioxidant, antimicrobial and
cytotoxic properties of four different extracts derived from the
roots of Nicotiana tabacum L.,”
*European Journal of Integrative Medicine*, 33, p.
101039. Available at:

<https://doi.org/10.1016/j.eujim.2019.101039>.

Buntru, M. et al. (2014) “Tobacco BY-2 cell-free lysate: an
alternative and highly-productive plant-based in vitro
translation system,” *BMC Biotechnology*, 14(1), p. 37.
Available at:

<https://doi.org/10.1186/1472-6750-14-37>.

Cremelie, E., Vázquez, R. and Briers, Y. (2024) “A comparative
guide to expression systems for phage lysin production,”
*Essays in Biochemistry*, 68(5), pp. 645–659. Available
at:

<https://doi.org/10.1042/EBC20240019>.

Ding, J.L., Li, P. and Ho, B. (2008) “The Sushi peptides:
structural characterization and mode of action against
Gram-negative bacteria,”
*Cellular and Molecular Life Sciences*, 65(7), pp.
1202–1219. Available at:

<https://doi.org/10.1007/s00018-008-7456-0>.

Gautier, R. et al. (2018) “PackMem: A Versatile Tool to Compute
and Visualize Interfacial Packing Defects in Lipid Bilayers,”
*Biophysical Journal*, 115(3), pp. 436–444. Available at:

<https://doi.org/10.1016/j.bpj.2018.06.025>.

Jo, S. et al. (2008) “CHARMM-GUI: A web-based graphical user
interface for CHARMM,”
*Journal of Computational Chemistry*, 29(11), pp.
1859–1865. Available at:

<https://doi.org/10.1002/jcc.20945>.

LenioBio® (2024) “Instruction Manual for Cell-Free Protein
Expression with ALiCE® for Research.”

Sato, H. and Feix, J.B. (2008) “Lysine-Enriched
Cecropin-Mellitin Antimicrobial Peptides with Enhanced
Selectivity,” *Antimicrobial Agents and Chemotherapy*,
52(12), pp. 4463–4465. Available at:

<https://doi.org/10.1128/AAC.00810-08>.

Son, B. et al. (2023) “Isolation and Characterization of a
Weizmannia coagulans Bacteriophage Youna2 and Its Endolysin
PlyYouna2,” *Journal of Microbiology and Biotechnology*,
33(8), pp. 1050–1056. Available at:

<https://doi.org/10.4014/jmb.2303.03021>.

Xu, S. et al. (2021) “Decontamination of
*Escherichia coli* O157:H7 on fresh Romaine lettuce using a
novel bacteriophage lysin,”
*International Journal of Food Microbiology*, 341, p.
109068. Available at:

<https://doi.org/10.1016/j.ijfoodmicro.2021.109068>.

Yau, Y.H. et al. (2001) “High Therapeutic Index of Factor C
Sushi Peptides: Potent Antimicrobials against
*Pseudomonas aeruginosa*,”
*Antimicrobial Agents and Chemotherapy*, 45(10), pp.
2820–2825. Available at:

<https://doi.org/10.1128/AAC.45.10.2820-2825.2001>.

Zheng, T. and Zhang, C. (2024) “Engineering strategies and
challenges of endolysin as an antibacterial agent against
Gram-negative bacteria,” *Microbial Biotechnology*,
17(4), p. e14465. Available at:

<https://doi.org/10.1111/1751-7915.14465>.

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)

[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

{% endblock %}

{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block
image %}
![gruppen-photo](https://static.igem.wiki/teams/5946/images/header/img-0131-min.webp)
{% endblock %} {% block page\_content %}

To ensure that all our experiments and results are reproducible, we
provide all protocols that were used.

#### Endolysin platform protocols

Creation of chemically competent cells

Transformation of the chemically competent cells

Plasmid DNA purification

Restriction enzyme digestion

QIAGEN PCR purification

Gel extraction of plasmid backbone

Golden Gate Assembly

Ligation

Bead protein purification

Bactericidal Effect

#### Biofilm destruction protocols

IPTG/X-Gal agar plates

LB, Top Agar, and SOC media

Electrocompetent *E. coli* K12 ER2738

*E. coli* K12 ER2738 glycerol stock

Propagating plaques and isolating phages

Phage lysis and DNA purification

Biofilm assay

#### Other protocols

Buffers, LB Medium, Agarose Gel and Antibiotics

IDT Gene Preparation

cPCR and PCR

SDS Page

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)

[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)

{% endblock %}

{% extends "layout.html" %} {% block title %}Results{% endblock %} {% block
image %}
![gruppen-photo](https://static.igem.wiki/teams/5946/images/header/header-results.webp)
{% endblock %} {% block page\_content %}

#### Endolysin expression platform

![fig3](https://static.igem.wiki/teams/5946/images/pages/results/wl1/res-graphische-zusammenfassung.webp)

##### Aim of this project

With our project, we aimed to establish a cell-free endolysin
expression platform that enables the reliable and rapid
identification of lead endolysin variants, particularly against
Gram-negative bacteria. In addition, we sought to develop a
toolkit based on this system to facilitate fast and easy
endolysin modification and the generation of optimized variants.

##### Plasmid Endolysin Constructs

The standard plasmid pALiCE01 was used as the backbone for
assembling our expression vector, with the endolysin PlyEc2 ([bba-25h8ekrx](https://registry.igem.org/parts/bba-25h8ekrx)) serving as the protein of interest (POI) ([Xu et al., 2021](#ref-xu2021);
[LenioBio®, 2024](#ref-leniobio2024)). The construct was assembled via restriction enzyme digestion
using KpnI and NotI. The correct assembly was confirmed by
Sanger sequencing (Figure 1).

![fig3](https://static.igem.wiki/teams/5946/images/pages/results/wl1/result-figure-1-palice01-plyec2-sequencing-result.webp)

**Figure 1:** Positive sanger sequencing result
of the pALiCE01\_PlyEc2 plasmid (Created with SnapGene).

For efficient endolysin production using the ALiCE® cell-free
expression system, highly purified plasmid DNA was required. To
achieve this, six individual plasmid purification samples were
pooled and further purified using phenol-chloroform
purification. The resulting purified plasmid was subsequently
used for cell-free protein synthesis of our POI using the ALiCE®
system, and the reaction was terminated after 48 hours. The
centrifuged supernatant (SN) was collected and employed in
growth inhibition assays using *P. aeruginosa* PAO1.

For the initial growth inhibition experiments, we estimated that
the ALiCE® system produced approximately 2 mg/mL of endolysin
per reaction, as described in ([LenioBio®, 2024](#ref-leniobio2024)). Based on this assumption, final concentrations of 20 µg/mL
and 60 µg/mL were tested, considering that the minimal
inhibitory concentration (MIC) of PlyEc2 had been reported to be
around 12.5 µg/mL. However, no growth inhibition was observed
under any condition, neither for the PlyEc2 supernatants nor for
the non-template control, without plasmid DNA added to the
reaction mix. The treated *P. aeruginosa* PAO1 cultures
exhibited growth comparable to that of the positive control
(Figure 2).

![fig3](https://static.igem.wiki/teams/5946/images/pages/results/wl1/results-figure-2-growth-inhibition-pao1-kopie.webp)

**Figure 2:** Initial growth inhibition assay
of *P. aeruginosa* PAO1 together with PlyEc2. The assay
was conducted in triplicates at 37 °C, 250 rpm in LB medium
with approximately 106 cells per well of exponentially grown
*P. aeruginosa PAO1*. NTC: non-template control; SN:
supernatant

Contrary to our expectations, the initial growth inhibition
assays revealed no detectable inhibitory activity. Due to the
lack of a reliable and feasible protein detection method, the
actual concentration of the expressed endolysin could not be
determined. Consequently, an insufficient protein yield, absence
of expression, or loss of functionality could not be excluded at
this stage. To overcome this limitation, we introduced a StrepII
tag into our constructs, enabling both verification of protein
expression and quantification of our POI.

#### StrepII-tagged Endolysin Plasmids

For our second cloning approach we utilized a modified version
of the pALiCE01 plasmid as the backbone, containing a
StrepII-tag and a (GGGGS)2 linker. In addition to our initial
endolysin PlyEc2, we selected PlyYouna2 ([BBa-25uoafah](https://registry.igem.org/parts/bba-25uoafah)), an
endolysin primarily active against Gram-positive bacteria, but
reported to exhibit activity against certain Gram-negative
strains as well ([Son et al., 2023](#ref-son2023)). In order to allow for standardized cloning, we utilized a
Golden Gate Assembly approach for our vector construction. The
plasmid maps of our constructs can be viewed in Figure 3.

![fig3](https://static.igem.wiki/teams/5946/images/pages/results/wl1/results-figure-3-plasmid-maps-with-strepii-kopie.webp)

**Figure 3:** Plasmid maps of
pALiCE01\_StrepII\_PlyEc2 and pALiCE01\_StrepII\_PlyYouna2 (Created with SnapGene).

The resulting plasmids were successfully assembled, verified by
sanger sequencing (Figure 4), and purified as described
previously. These constructs were subsequently used for
cell-free expression of the respective endolysin via the ALiCE®
system. Additionally, two controls were included: a non-template
control, without plasmid DNA, and a positive control expressing
StrepII-tagged eYFP from the standard pALiCE01 plasmid.

![fig3](https://static.igem.wiki/teams/5946/images/pages/results/wl1/results-figure-4-seq-results.webp)

**Figure 4:** Positive sanger sequencing
results of A) pALiCE01\_StrepII\_PlyEc2 and
B) pALiCE01\_StrepII\_PlyYouna2 (Created with SnapGene).

To verify protein production, the supernatants (SN) of all
ALiCE® reactions and the pellets of the endolysin samples
(resuspended in 1× PBS) were analyzed by SDS-PAGE, accompanied
by plate spot assays (Figure 5). The plate spot assay for PlyEc2
was performed with P. aeruginosa PAO1, whereas
*Bacillus megaterium* ATCC 14581 was used for PlyYouna2, as
both species are known to be susceptible to the corresponding
endolysins ([Xu et al., 2021](#ref-xu2021);
[Son et al., 2023](#ref-son2023)).

For the assays, bacterial lawns from exponentially grown liquid
cultures were plated on LB agar plates. After a short drying
period, 10 µL of the SN samples, resuspended pellets, NTC
extracts, and appropriate antibiotics were applied. For
PlyYouna2, additional samples of natively purified protein
(isolated via MagStrep Strep-Tactin™ XT Beads and eluted in the
appropriate elution buffer) were also tested.

A comparative analysis of the two approaches yielded analogous
results. The endolysin-containing supernatants (SN) exhibited
growth-inhibitory effects, similar to the negative control with
ampicillin on *B. megaterium* ATCC 14581. As expected,
ampicillin did not inhibit *P. aeruginosa* PAO1 growth due
to naturalist intrinsic β-Lactam resistance ([Alam et al., 2019](#ref-alam2019)). Repeating the assay with gentamicin confirmed the expected
growth inhibition (data not shown). Unexpectedly, the NTC SN
samples also caused growth inhibition, whereas the natively
purified PlyYouna2 and the 1x BXT elution buffer showed no
inhibitory effect.

![fig3](https://static.igem.wiki/teams/5946/images/pages/results/wl1/results-figure-5-plate-spot-assay.webp)

**Figure 5:** Plate spot assay of PlyYouna2 on
*Bacillus megaterium* ATCC 14581 (A) and of PlyEc2 on
*Pseudomonas aeruginosa* PAO1(B). For A) the
supernatant (SN), pellet and natively purified PlyYouna2 as
well as 50 µg/µL Ampicillin, the non-template control (NTC)
supernatant and the elution buffer of the purification (1x
BXT) were tested for lysis. For B) the PlyEc2 supernatant,
50 µg/µL Ampicillin and the non-template control (NTC)
supernatant were tested regarding lysis.

Protein expression of both endolysins was further analyzed via
SDS-PAGE. For PlyEc2 (SN) and PlyYouna2 (SN and pellet), bands
were visible at the expected molecular weights (PlyEc2: ~25 kDa;
PlyYouna2: ~31.8 kDa). However, the NTC also displayed a
discernible band at a comparable position, an unexpected
observation under standard conditions (Figure 6).

![fig3](https://static.igem.wiki/teams/5946/images/pages/results/wl1/results-figure-6.webp)

**Figure 6:** SDS-PAGE analysis of different
ALiCE® reactions, including supernatants (SN) and
resuspended pellets of PlyEc2 (left), PlyYouna2 and control
(right) samples. NTC = non-template control & eYFP =
positive control with standard pALiCE01 vector. Protein
sizes were quantified using PageRuler Prestained protein
ladder (10 - 180 kDa).

These findings initially suggested a potential contamination of
the NTC with one of the endolysin plasmids. However, since the
purified PlyYouna2 no longer exhibited growth inhibition. This
raised alternative hypotheses: defective purification,
non-functional protein production, or inefficient expression.

To clarify the cause of the observed lack of activity, another
SDS-PAGE was conducted for PlyYouna2, including both native and
denaturing MagStrep Strep-Tactin™ XT Bead purification (Figure
7). The anticipated protein band at 31.8 kDa was not detected in
any fraction (whole lysate (WL), SN, pellet, or native purified
samples). A malfunction of the ALiCE® system was disproven, as
the positive control (eYFP) showed strong fluorescence and a
clear ~29 kDa band compared to the NTC.

![fig3](https://static.igem.wiki/teams/5946/images/pages/results/wl1/results-figure-7.webp)

**Figure 7:** SDS-PAGE analysis of different
ALiCE® reactions, including supernatants (SN) and
resuspended pellets of PlyEc2 (left), PlyYouna2 and control
(right) samples. NTC = non-template control & eYFP =
positive control with standard pALiCE01 vector. Protein
sizes were quantified using PageRuler Prestained protein
ladder (10 - 180 kDa).

For PlyEc2, an additional SDS-PAGE and Western blot (WB) were
performed to verify the functionality of the ALiCE® system and
to address the reliability of phenol-chloroform plasmid
purification and MagStrep Strep-Tactin™ XT Beads purification.
Alongside the standard eYFP plasmid, an ALiCE® reaction using
phenol-chloroform-purified eYFP plasmid was included. As shown
in Figure 8, clear protein bands at ~29 kDa were observed in the
WL, SN, and natively purified protein for both eYFP reactions.
These findings were confirmed by WB analysis. In contrast, no
band at the expected size of 25 kDa was detected for PlyEc2 in
either Ponceau staining or WB, indicating that no endolysin was
produced. Nevertheless, these results confirm the overall
functionality of the ALiCE® system and rule out
phenol-chloroform plasmid purification or MagStrep Strep-Tactin™
XT Beads purification as sources of error.

![fig3](https://static.igem.wiki/teams/5946/images/pages/results/wl1/results-figure-8-final-westernblot-new.webp)

**Figure 8:** Ponceau staining on the left and
Western blot on the right. A) shows the results of the
StrepII-tagged eYFP derived from the standard pALiCE01
plasmid already delivered purified. Further it exhibits the
PlyEc2 samples of the whole lysate (WL), supernatant (SN),
resuspended pellet (P), and the natively purified protein
(A). B) shows the results of the StrepII-tagged eYFP derived
from the standard pALiCE01 that was previously purified with
the phenol-chloroform purification method. It contains the
WL, SN, P and A as well as the input (IP) and the wash of
the native protein purification. Additionally, IP and wash
of PlyEc2 and the non-template control (NTC) are visible.
The NTC is an ALiCE® reaction sample where no plasmid was
used as template. It exhibits the negative control of the
ALiCE® reaction. Further, the SDS-PAGE gel of B) contained a
leaky pocket. As a result, a sample flowed across the gel,
resulting in the slight continuous staining visible in the
WB. Protein sizes were quantified using Precision Plus
ProteinTM Standard protein ladder (10 - 250 kDa).

##### Establishment of a modular toolbox

In parallel to the expression of individual endolysins, we began
developing a modular toolbox approach based on the ALiCE®
cell-free expression system. This toolbox is designed to enable
the rapid generation of endolysin fusion constructs by allowing
the exchange of individual genetic modules, thereby facilitating
the efficient identification of new lead variants. The fusion of
antimicrobial peptides (AMPs) with endolysins is of particular
interest, as this combination promises to enhance lysis
efficiency against Gram-negative bacteria ([Carratalá et al.,
2023](#ref-carratala2023)). In this regard, our molecular dynamics (MD) simulations
also contributed to this hypothesis (see [model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)).

To implement this approach, we introduced position markers
within the construct, allowing for the straightforward exchange
of sequence elements without the need for new primer design.
This modular design enables flexible combinations of AMP linkers
and endolysins, allowing rapid testing of numerous variants in a
standardized format (Figure 9). For more information see
[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D).

However, since no endolysin expression was achieved in the
ALiCE® system, production of new fusion constructs could not be
initiated. An alternative expression kit had been planned since
May, but its implementation was ultimately not possible due to a
lengthy approval process and time constraints.

![fig3](https://static.igem.wiki/teams/5946/images/pages/results/wl1/results-figure-9-palice01-k14-plyec2-strepii-map-with-primer-new.webp)

**Figure 9:** Plasmid map of
pALICE01\_K14\_PlyEc2\_StrepII with required primers for the
GGA. Further information about the primers is available
under Contribution (Created with SnapGene).

#### Biofilm destruction

##### Aim & Approach of this project

During our literature review and consideration of potential applications for our endolysin variants, we identified biofilms as a major challenge in the fight against bacterial infections. Not only do they increase the resistance and persistence of bacteria in infectious diseases, but they are also a serious problem on implants, in food production, and in water and piping systems.

Biofilms form when numerous bacterial cells aggregate to produce a polymeric slime layer (extracellular matrix, ECM) around themselves. This matrix enables adhesion to surfaces and protects the cells from external influences such as antibiotics, disinfectants, and the immune system
([Biofilms Research Group “Biofilms” Jena University Hospital (2025)](#ref-biofilm);
[Ban-Cucerzan et al., 2025](#ref-ban-cucerzan2025)).
Therefore, a truly sustainable alternative to antibiotics must also overcome the protective barrier formed by biofilms, which is why we sought to expand our research to include this critical aspect.

Conveniently, bacteriophages have the ability to infiltrate biofilms. Their advantage lies in their autonomous replication within the biofilm, the efficient lysis of bacterial cells, and the secretion of hydrolases and endolysins, which weaken the biofilm matrix. This enables biofilms to be completely destroyed, or at least made significantly more sensitive to antimicrobial agents
([Mayorga-Ramos et al., 2024](#ref-mayorga-ramos2024)).
However, the composition of the biofilm matrix often limits the infiltration ability of phages, among other things due to its physical barrier effect and the heterogeneous metabolic states within the bacterial population
([Meneses et al., 2023](#ref-meneses2023)).

Therefore, we wanted to investigate ways of increasing the infiltration capacity of phages by using phage display, by generating various fusion proteins via cloning that were then to be expressed on the phage surface. In order to cover a broad spectrum of activity, we combined different approaches aimed at weakening the integrity of the biofilm.

As a transport vehicle for our endolysins, we chose the filamentous M13 phage, which, due to its high density of over 2,500 pVIII major coat proteins, offers an ideal platform for phage display and has already been extensively studied in this context
([Chang et al., 2023](#ref-chang2023)).
In addition, M13 binds to the F pilus of *Escherichia coli* strains, thereby inhibiting horizontal gene transfer, which in turn can reduce the rapid adaptation of bacteria to stress factors and the development of resistance. Furthermore, the M13 phage is not lytic but lysogenic, making the effect of endolysins easier to detect. At the same time, infection by M13 can slow down the bacterial cell cycle
([Kok et al., 2024](#ref-kok2024)).

In parallel, we investigated various peptides. First, we used two antimicrobial peptides (AMPs): K14, a short, cationic peptide with amphiphilic properties
([Sato & Feix, 2008](#ref-sato2008)), which, due to its positive charge, effectively binds to the negatively charged bacterial cell membrane, destabilizing and permeabilizing it.

In addition, we used Lf11-324, a derivative of lactoferricin, whose biofilm-inhibiting effect is based on interaction with the cell membrane, destabilization of the extracellular matrix (ECM), and an antibacterial effect mediated by iron deprivation
([Zweytick et al., 2011](#ref-zweytick2011);
[Gifford et al., 2005](#ref-gifford2005)).
Since lactoferricin is a component of the human immune system, this peptide seemed particularly suitable to us, as phage coating with this molecule could also attenuate possible undesirable immune reactions in future medical applications.

To specifically weaken the biofilm matrix, we also tested the lipid-bound peptide VFR12, a derivative of human thrombin, to investigate whether binding to lipophilic components of the biofilm could impair its stability
([Singh et al., 2013](#ref-singh2013)).

Finally, we also wanted to specifically disrupt signal transduction processes that influence bacterial growth. For this purpose, we chose the immunomodulator peptide IDR-108
([de la Fuente-Núñez et al., 2014](#ref-de-la-fuente-nunez2014)), which modulates the bacterial stress response, disrupts intercellular cohesion in the biofilm, and has been shown to activate the immune response in the host
([Steinstraesser et al., 2012](#ref-steinstraesser2012);
[Mansour et al., 2015](#ref-mansour2015)).

In the end, we decided not to use genetically modified phages, but only wild type (WT) phages in combination with the raw peptides, since our HP feedback considered them more appropriate for application in humans from a regulatory point of view in Germany.

##### Biofilm reduction tests

After overcoming a few minor, unexpected difficulties at the beginning (see [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D#wl1-cycle4)), and after all legal requirements and the necessary administrative work for working in the S2 laboratory had been completed and personal safety was ensured through safety training and a medical clearance by the responsible physician.

The selected peptides were to be tested on biofilms of the enteroaggregative and pathogenic *E. coli* 55989 strain (S2) as well as, for the control group, on biofilms of *E. coli* K-12.

The measured absorption at 580 nm correlates with the amount of biofilm formed and therefore served as an indirect indicator of the effectiveness of our phage and peptide treatments. All measurements were performed in at least biological triplicates (n = 3). As expected, the enteroaggregative *E. coli* strain 55989 formed more biofilm on average than the non-pathogenic control strain K-12.

Interestingly, the samples treated with M13 phage alone showed a slight reduction in biofilm mass. However, when the phage was used in combination with one of the peptides IDR-108, Lf11-324, or VFR12, the biofilm-reducing effect of the peptides appeared to be partially reversed—both in the enteroaggregative and K-12 strains. However, these values showed a high standard deviation, indicating biological variability or minor technical fluctuations. The peptides alone, applied at a concentration ten times the minimum inhibitory concentration (MIC), thus led to a significantly greater reduction in biofilm quantities than in combination with M13 ([Figure 10](#michelle-fig10)).

Contrary to our expectations, Lf11-324 caused only a slight reduction in biofilm compared to the pure M13 samples, while VFR12 achieved a biofilm reduction of over 20% and IDR-108 reduced the amount of biofilm by more than a third compared to the buffer control in the enteroaggregative strain([Figure 10](#michelle-fig10)).

![figure10](https://static.igem.wiki/teams/5946/images/pages/results/wl2/test-55989.webp)

**Figure 10:** Biofilm decimation via peptides and phages for the enteroaggregative *E. coli* strain 55989. Biofilms were grown for 48 h and treated with the respective test substance for 24 h. Then washed, dyed with crystal violet, washed again, and the absorption was measured at 580 nm. Values compared to buffer control treated with TBS.

The K-12 strain showed a differentiated picture: VFR12 had little effect on the amount of biofilm, Lf11-324 had a similar effect to that on the pathogenic strain, while IDR-108 led to a reduction of almost 50% ([Figure 11](#michelle-fig11)).

![figure11](https://static.igem.wiki/teams/5946/images/pages/results/wl2/test-k12.webp)

**Figure 11:** Biofilm decimation via peptides and phages for the control *E. coli* strain K-12. Biofilms were grown for 48 h and treated for 24 h with respective substances, then washed, dyed, and absorption measured at 580 nm. Values compared to TBS buffer control.

The combinations of peptides yielded consistent results: in the enteroaggregative strain, the combination of Lf11-324 and VFR12 led to only a slight reduction, while Lf11-324 + IDR-108 reduced the biofilm by almost 20% and IDR-108 + VFR12 by almost 25% – in each case compared to the buffer control. In the K-12 strain, the combinations Lf11-324 + VFR12 and VFR12 + IDR-108 showed only minor effects, while the combination of Lf11-324 + IDR-108 led to a reduction in biofilm quantity of over 30% ([Figure 12](#michelle-fig12)).

![figure12](https://static.igem.wiki/teams/5946/images/pages/results/wl2/peptide-combination.webp)

**Figure 12:** Biofilm decimation via peptide combinations for the enteroaggregative *E. coli* strain 55989 and control K-12. Biofilms grown for 48 h, treated for 24 h, then washed, dyed, and absorption measured at 580 nm. Values compared to buffer control treated with TBS.

We observed the most unexpected result with peptide K14. When applying twice the MIC concentration – based on the MIC previously described for *E. coli* BL21 – only a slight reduction in biofilm quantity was observed in both strains (55989 and K-12). However, at ten times the concentration, this effect was reversed: there was a significant increase in biofilm formation – by over 150% in the 55989 strain and even by more than 270% in K-12 compared to the buffer control. When M13 was added, the value for 55989 remained almost unchanged, while the biofilm value for K-12 continued to increase, reaching almost 350% of the control value ([Figure 13](#michelle-fig13)).

![figure13](https://static.igem.wiki/teams/5946/images/pages/results/wl2/k14.webp)

**Figure 13:** Effects of the AMP K14 on biofilm quantity of enteroaggregative *E. coli* strain 55989 and control K-12. Biofilms grown for 48 h, treated for 24 h, then washed, dyed, and absorption measured at 580 nm. Values compared to buffer control treated with TBS.

##### Discussion and outlook

The slight reduction in biofilm quantity caused by M13 WT phages, which actually replicate lysogenically and do not lyse bacteria, can be explained by their binding to the F pilus, which, in addition to interrupting horizontal gene transfer, can slow down cell division and bacterial growth. Although infection and insertion of the genetic phage material do not kill the bacteria, it does lead to altered gene expression, which negatively affects the bacterium's metabolism and thus the synthesis of extracellular polymeric substance (EPS)
([Amankwah et al., 2021](#ref-amankwah2021)).

However, the fact that the peptides IDR-108, VFR12, and Lf11-324 have greater biofilm destruction properties on their own than in combination with phages may be due to the phages either occupying their binding sites on the bacterial surface or sterically hindering them, or simultaneously interacting with the peptides themselves, which could reduce their availability and effectiveness. The fact that Lf11-324 actually has little effect on biofilm, even though its effectiveness against bacteria was rated as high in previous studies, may be due to the fact that it is a cationic molecule, which means it can be bound by eDNA and other anionic molecules in the biofilm and at the same time may diffuse less easily to the bacterial membrane
([Vieira-da-Silva, 2023](#ref-vieira-da-silva2023)).

VFR12, on the other hand, does not bind as easily to anionic molecules due to its chemical properties, allowing it to diffuse better to the bacteria and accumulate in the membrane, destabilizing it and making it permeable, which explains the lower biofilm values
([Lei et al., 2019](#ref-lei2019);
[Zhang et al., 2017](#ref-zhang2017)).
IDR-108 not only has an antimicrobial effect by penetrating and destabilizing the bacterial cell wall, which also has a negative impact on EPS synthesis, but also blocks the (p)ppGpp signal, thereby disrupting its protective mechanism and reducing the expression of biofilm-synthesizing genes
([de la Fuente-Núñez et al., 2014](#ref-de-la-fuente-nunez2014)).
These two simultaneous attack pathways also explain why IDR-108 is so much more effective.

The different effects on K-12 biofilms could be explained as follows: Lf11-324 acts mainly directly on the bacterial membranes, which is why this effect does not have to differ significantly between different biofilms. VFR12, however, acts predominantly via lipid interactions, and K-12 biofilms are known to be less complex in structure than the biofilms of enteroaggregative bacteria
([Beloin et al., 2008](#ref-beloin2008)).
If fewer lipids are available for interaction, this may explain the low effect. The fact that IDR-108 is more effective in K-12 may be because the stress response and intracellular regulatory mechanisms are highly conserved in K-12 strains and are also essential for biofilm formation
([Hobley et al., 2015](#ref-hobley2015)). Since K-12 generally forms less biofilm than enteroaggregative strains, the effect may be even stronger here
([Beloin et al., 2008](#ref-beloin2008)).
This background also explains the different effects of the peptide combinations, and especially the good results of IDR-108 and VFR12 for 55989, since they disrupt the biofilm with multiple different attacking methods due to their different modes of action.

The slight biofilm-inhibiting function of K-14 at low concentrations was expected, as K-14 can insert itself in bacterial membranes due to its chemical properties and likely cause pore formation, as a result of which the cell can no longer maintain its chemical equilibrium
([Lesniewski, 2022](#ref-lesniewski2022)).
However, the high absorption at high concentrations has not been described in the literature before and could have various reasons: Either complex formation, which distorts the result when stained with crystal violet, or aggregation, which binds the individual bacteria more strongly together
([Domnin et al., 2025](#ref-domnin2025)).
The more likely explanation, especially given the differences in the strains, would be an effect on cellular stress signals. As a laboratory strain, K-12 is significantly more sensitive to stress and altered gene regulation than pathogenic strains
([Bhatia et al., 2022](#ref-bhatia2022)).
This also explains the sharp increase in biofilm formation in K-12, which is also a protective mechanism when high concentrations of K-14 are combined with M13 phages, as these also influence the cellular processes of bacteria and can trigger stress and altered gene regulation. However, another approach to explain the relatively little negative effect on biofilms in low concentrations compared to the other tested peptides is the high charge density of K14, as explained in our model part. There, the insertion in the membrane was shown in our model for the CM15 that has only one different amino acid to be much slower than for other AMPs. Many K14 molecules together could also hinder each other during the insertion process.

However, much more research is needed before clear statements can be made on this subject. In general, our method is very sensitive in its application, and the trends derived from it must be interpreted with caution. In order to obtain more meaningful results, the experiments must be repeated several times. In addition, further different concentrations should be tested, and kinetics should be applied. It would also be interesting to investigate the synergistic effects of different phages, AMPs, ABPs, and antibiotics, as well as changes in environmental factors that can significantly influence biofilm formation. However, this would require more time and money to continue the experiments than we had available.

#### References

Alam, S.T. et al. (2019) “Antimicrobial Biophotonic Treatment of
Ampicillin-Resistant *Pseudomonas aeruginosa* with
Hypericin and Ampicillin Cotreatment Followed by Orange Light,”
*Pharmaceutics*, 11(12), p. 641. Available at:

<https://doi.org/10.3390/pharmaceutics11120641>.

Carratalá, J.V. et al. (2023) “Design strategies for positively
charged endolysins: Insights into Artilysin development,”
*Biotechnology Advances*, 69, p. 108250. Available at:

<https://doi.org/10.1016/j.biotechadv.2023.108250>.

LenioBio® (2024) “Instruction Manual for Cell-Free Protein
Expression with ALiCE® for Research.”

Son, B. et al. (2023) “Isolation and Characterization of a
Weizmannia coagulans Bacteriophage Youna2 and Its Endolysin
PlyYouna2,”
*Journal of Microbiology and Biotechnology*, 33(8), pp.
1050–1056. Available at:

<https://doi.org/10.4014/jmb.2303.03021>.

Xu, S. et al. (2021) “Decontamination of *Escherichia coli*
O157:H7 on fresh Romaine lettuce using a novel bacteriophage
lysin,”
*International Journal of Food Microbiology*, 341, p.
109068. Available at:

<https://doi.org/10.1016/j.ijfoodmicro.2021.109068>.

Biofilms Research Group (2025) “Biofilms,” *Jena University Hospital*. Available at:
[https://www.uniklinikum-jena.de/infektionsmedizin/Forschung/Laborforschung/Forschergruppe+Biofilms.html](https://www.uniklinikum-jena.de/infektionsmedizin/Forschung/Laborforschung/Forschergruppe%2BBiofilms.html) (Accessed on: October 4, 2025).

Ban-Cucerzan, A. et al. (2025) “Persistent Threats: A Comprehensive Review of Biofilm Formation, Control, and Economic Implications in Food Processing Environments,” *Microorganisms*, 13(8), p. 1805. Available at:
<https://pmc.ncbi.nlm.nih.gov/articles/PMC12388178/> (Accessed: 4 October 2025).

Mayorga-Ramos, A., Carrera, R., Pacheco, L. & Cruz, W. (2024) “Bacteriophage-mediated approaches for biofilm control,” *Frontiers in Cellular and Infection Microbiology*, 14, Art. 1428637. Available at:
<https://doi.org/10.3389/fcimb.2024.1428637>.

Meneses, L., Maktabi, M., Khursigara, C.M. & Fong, J. (2023) “A systematic review of the use of bacteriophages for in vitro biofilm control,” *Microorganisms*, 11(9), p. 2345. Available at:
<https://doi.org/10.1007/s10096-023-04638-1>.

Chang, C. et al. (2023) “Engineered M13 phage as a novel therapeutic and bionanomaterial platform,” *Nanomedicine*, 18(5), pp. 534–550. Available at:
<https://pmc.ncbi.nlm.nih.gov/articles/PMC10102448/> (Accessed: October 4, 2025).

Kok, D.J. et al. (2024) “Biogenesis and virus assembly of the filamentous coliphage M13 and its effect on the F-pili of *Escherichia coli*,” University of Hohenheim, Dissertation. Available at:
<https://hohpublica.uni-hohenheim.de/items/6e2f112f-d11f-40ca-8447-a465e297165a> (Accessed: 04.10.2025).

Sato, H. & Feix, J.B. (2008) “Lysine-enriched cecropin-mellitin antimicrobial peptides with enhanced selectivity,” *Antimicrobial Agents and Chemotherapy*, 52(12), pp. 4463–4465. Available at:
<https://doi.org/10.1128/AAC.00810-08>.

Gifford, J.L., Hunter, H.N. & Vogel, H.J. (2005) “Lactoferricin: A lactoferrin-derived peptide with antimicrobial, antiviral, antitumor and immunological properties,” *Cellular and Molecular Life Sciences*, 62(22), pp. 2588–2598. Available at:
<https://doi.org/10.1007/s00018-005-5373-z>.

Zweytick, D. et al. (2011) “Studies on lactoferricin-derived *Escherichia coli* membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides,” *Journal of Biological Chemistry*, 286(24), pp. 21266–21276. Available at:
<https://doi.org/10.1074/jbc.M110.195412>.

Singh, S. et al. (2013) “Lipopolysaccharide interactions of C-terminal peptides from human thrombin,” *Biomacromolecules*, 14(5), pp. 1482–1492. Available at:
<https://doi.org/10.1021/bm400150c>.

de la Fuente-Núñez, C. et al. (2014) “Broad-spectrum anti-biofilm peptide that targets a cellular stress response,” *PLoS Pathogens*, 10(5), e1004152. Available at:
<https://doi.org/10.1371/journal.ppat.1004152>.

Steinstraesser, L. et al. (2012) “Innate defense regulator peptide 1018 in wound healing and wound infection,” *PLoS ONE*, 7(8), e39373. Available at:
<https://doi.org/10.1371/journal.pone.0039373>.

Mansour, S.C., de la Fuente-Núñez, C. & Hancock, R.E.W. (2015) “Peptide IDR-1018: Modulating the immune system and targeting bacterial biofilms,” *Frontiers in Immunology*, 6, Article 353. Available at:
<https://doi.org/10.3389/fimmu.2015.00353>.

Assmann, J. (2020) “Antibiotic resistance and biofilm formation in pathogens of ...” [online]. Available at:
<https://refubium.fu-berlin.de/bitstream/handle/fub188/27670/Dissertation_Assmann.pdf> [Accessed October 5, 2025].

Amankwah, R. et al. (2021) “Bacterial biofilm destruction: Phage-based strategies,” *NSA*, September 13, pp. 2–4. Available at:
<https://www.dovepress.com/bacterial-biofilm-destruction-a-focused-review-on-the-recent-use-of-ph-peer-reviewed-fulltext-article-NSA> (Accessed: 05.10.2025).

Marx, L. et al. (2021) “Bridging the antimicrobial activity of two lactoferricin derivatives in *E. coli* and lipid-only membranes,” *Frontiers in Medical Technology*, 3, p. 625975. Available at:
<https://www.frontiersin.org/journals/medical-technology/articles/10.3389/fmedt.2021.625975> (Accessed: 5 October 2025).

Vieira-da-Silva, B. & Castanho, M.A.R.B. (2023) “The structure and matrix dynamics of bacterial biofilms as revealed by antimicrobial peptides’ diffusion,” *Journal of Peptide Science*, 29(6), e3470. Available at:
<https://doi.org/10.1002/psc.3470>.

Lei, J. et al. (2019) “The antimicrobial peptides and their potential clinical applications,” *Frontiers in Microbiology*, 10, 1430. Available at:
<https://doi.org/10.3389/fmicb.2019.01430>.

Zhang, T.-W. et al. (2017) “Hydrophobic residues are crucial for the helix formation, hemolytic, and bactericidal activities of the amphipathic antimicrobial peptide TP4,” *PLOS ONE*, 12(10), e0186442. Available at:
<https://doi.org/10.1371/journal.pone.0186442>.

Beloin, C., Roux, A. & Ghigo, J.M. (2008) “*Escherichia coli* biofilms,” *Current Topics in Microbiology and Immunology*, 322, pp. 249–289. Available at:
<https://doi.org/10.1007/978-3-540-75418-3_12>.

Hobley, L., Harkins, C., MacPhee, C.E. & Stanley-Wall, N.R. (2015) “Structuring the biofilm matrix: An overview of individual and collective contributions to common structures,” *FEMS Microbiology Reviews*, 39(5), 649–669. Available at:
<https://doi.org/10.1093/femsre/fuv015>.

Lesniewski, M. (2022) “Thioether-basierte Immobilisierung und Charakterisierung von NKLysin-basierten antimikrobiellen Peptiden,” Masterarbeit, Department Biotechnologie, Hochschule für Angewandte Wissenschaften Hamburg.

Domnin, P.A. et al. (2025) “In Vitro Evaluation of Antimicrobial Amyloidogenic Peptides for the Treatment of Early and Mature Bacterial Biofilms,” *International Journal of Molecular Sciences*, 26(18), p. 8767. Available at:
<https://doi.org/10.3390/ijms26188767>.

Bhatia, R.P. et al. (2022) “Transcriptomic profiling of *Escherichia coli* K-12 in response to a compendium of stressors,” *Scientific Reports*, 12(1), p. 8788. Available at:
<https://doi.org/10.1038/s41598-022-12463-3>.

[Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)

[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)

[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)

{% endblock %}