iGEM 2022 — Team aalto-helsinki

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_content %}

# Notebook

Welcome to the Notebook of the Aalto-Helsinki team, where we briefly summarise our weekly doings and
accomplishments. For information about the wet lab, please check out our [protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
and [experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).

## February

Week 5

Interviews for the 2022 team as well as selection of new members occurred, with 3.5 team Aalto students and 6.5
University of Helsinki students making the final cut (one team member studies at both!).

Week 6

On 7th February, we all met for the first time over Zoom, a call which was also joined by members of the 2021 Team who
selected us. We started off with a round of introductions and our two new leaders, Melissa and Veera, were revealed to
us. Later that same week, we met up in person for our first social event at the sauna. Using a poll, we chose the most
suitable weekly meeting time going forward, settling on Thursdays at 6-8pm.

The two new team leaders took up contact with last year's academic advisors: Sesilja Aranko, Heli Viskari, Markus Linder
and Ville Paavilainen.

Week 7

Our first 'proper' weekly meeting was held on 17th February. At this point, we were still operating remotely. We went
over the different communication channels that had been created, such as Discord and a Whatsapp group, and introduced
the initial sub-groups that the team would be sub-divided into: fundraising, writing, wet-lab, dry-lab, social media,
human practices, graphic design & videography, and collaborations. The fundraising team had to get to work earlier than
the rest as setting up a pool of sponsors was the first urgent task, so a fundraising kickoff meeting followed the
regular team meeting. A Presemo page was also set up for team members to drop early leads and broad project ideas.

Week 8

This week, we discussed the different track possibilities for our project and checked which initial ideas had been added
to the Presemo page. Here is a selection of the early leads suggested by various team members:

* "Screening method for tumour markers that are linked to most common cancers"
* “Approaches to target biofilms on medical devices”
* “Making use of microbes to generate electricity”
* “Some type of screening method for detecting neurodegenerative diseases before symptoms start to show”

2-3 members were assigned one of the above four ideas (which had received the most votes) to do further literature
review and research by the following week. We went over the overall medal criteria on the iGEM website and set up a PI
meeting photoshoot date, as we would need headshots to be displayed on our sponsorship material.

## March

Week 9

We started our March by meeting our PIs and presenting them the tracks we are interested in and potential topics we have
previously thought about. Additionally, we confirmed with them that we will have lab spaces available and which
prerequisite we would have to meet regarding safety instructions in order to work in the laboratory. After we met our
PIs we limited our topics to six potential topics and also included an additional topic of antibody synthesis in yeast
as a new potential topic. After brainstorming we were also able to take our first group picture and individual pictures
for our web page. At the end of the week, we went ahead with our photoshoot as planned and took headshots for each team
member. The fundraising team finalised the budget plan that we would use in order to apply for funding at HiLIFE.

Week 10

This week we highly focused on the fundraising proposal that we passed to HiLIFE to apply for funding for the entire
year. Furthermore, we continued brainstorming and limited our topics to three topics including “microbes generating
energy”, “antibody production”, “targeting of biofilms” and divided our group according to this topic to perform further
literature research.

Week 11

We decided to just continue with two of our topics, because we saw the opportunity to combine two of our topics
including biofilm inhibition and antibody production, but we excluded the topic of energy generating microbes.
Additionally, we prepared for a course of Prof. Dr. Brendan Battersby that we took part in to find mentors.

Week 12

We met with Prof. Dr. Alexander Frey to discuss our project and possible advances on synthetic production of antibodies.
However, he did not recommend us to continue on this path, because of the anti-immune activity of biofilms. He rather
recommended artificial binding molecules like DARPins or Lectins. We split our group into three literature research
groups for biofilms, DARPins and Lectins. We also had our monthly social event for March, which was karate training with
the team.

Week 13

The first logo sketches were shared and we decided to focus more on DARPins and quorum quenching. In order to do this we
were planning to research quorum sensing and DARPins in more detail for the next week.

## April

Week 14

Our PI Heli Viskari helped us to start the iGEM registration process this week, on 5th April. Team members then
self-registered by 9th April.

We were happy to hear positive feedback about our proposed project topic from our PIs in general, and were told by Ville
Paavilainen that DARPins are a good option as they are small enough to pass through the biofilms polysaccharide matrix.
PIs advised us to now find a simple starting point for the potential research and to break it down into small steps.

During this week's team meeting, held on 7th, different members were assigned different research tasks to further refine
the project idea. These pressing research topics included:

* how to construct a DARPin library, on the dry-lab side
* researching the protocol for ribosome display, for which we might need to contact Brendan Battersby, who has expertise
  on sequencing ribosome-bound mRNA
* how the biosensor for measuring successful auto-inducer peptide inhibition can be built
* how different types of AIPs work and whether they are suitable for inhibition by DARPins (for example, how do the AIP
  and AI2 systems differ, as both are used by gram-positive bacteria)
* what safety level are the bacteria that utilise the AIP and AI2 quorum sensing systems and can a level 1 bacterium be
  found
* any other molecule we could either as a target or as a potential inhibitor?

Week 15

Made our 2022 Instagram debut by introducing the two team leaders on 12th and 14th April.

Week 16

On 20th April, as suggested by our PI Markus Linder, we contacted Christopher Jonkergouw to discuss the biosensor that
he previously built to detect AHL molecules (used in gram negative quorum sensing). On this same day, we also contacted
Thermofisher regarding fundraising.

On the 21st, much of our team meeting was dedicated to making a Human Practices roadmap. We came up with a list of
potential HP routes to go down, including contacting and interviewing clinicians and expert researchers, starting an
education and human practice-focused podcast. The podcast could be done in collaboration with the Aalto Entrepreneurship
Society (AaltoES), which has a podcast-dedicated recording room on campus.

We were happy to receive a positive reply from Thermofisher on the 22nd stating their interest in sponsoring us.

Week 17

On the 25th, we had two important meetings. The first one was with TEK, in the morning. In the afternoon, we met with
Jouni Lounasmaa, former CEO of the Startup Foundation in Finland, at the Terkko Health Hub of the University of Helsinki
Faculty of Medicine. On the same day, we sent a sponsorship proposal to Synbio Powerhouse, which operates under VTT, one
of Finland's leading state-owned research innovation institutions.
On 28th, we met with the University of Helsinki's Faculty of Agriculture and Forestry. We were also happy to receive a
positive response from Synbio Powerhouse on this day, specifically from their BioGarage branch. We were offered a 1500€
sponsorship, the opportunity to use labspaces for free at the brand new GMO lab, as well as hold an event in
collaboration with them.

## May

Week 18

On the 3rd we met with Sesilja and Ville in order to discuss the DARPin design. We decided to rather use a ribosome
display than a phage display for the affinity selection of the DARPins and we discussed the potential aims we want to
focus on for our DARPin including either the AgrC receptor or the AIP itself. However, we were recommended to only focus
on one target to simplify the project.
Two days later on the 5th we were able to meet with our PIs and advisors and discuss the project in more detail. We
decided that we want to establish our bioreporter in only one plasmid and that we will add a positive control to our
ribosome display by utilising a known GFP targeting DARPin. After an intense meeting we had our social monthly event
watching a movie. On the next day we met with the Faculty of Science at the University of Helsinki to discuss possible
funding. We received feedback and worked towards a mutually beneficial collaboration between our team and the Faculty of
Science.
On the 7th the iGEM opening day took place and most of our team attended that event to learn what iGEM is all about.

Week 19

On the 12th we had a meeting with Heureka. Heureka is a science centre in the Helsinki metropolitan area and we
discussed our collaboration in further detail. Heureka gave us ideas of what they would anticipate the workshop that we
would prepare to look like and we were able to grasp ideas from that to prepare an ideal workshop for several age
groups.
In our weekly meeting we finalised our funding and decided the salaries of the team members. Furthermore, we confirmed
future meetings that everyone is able to attend. We continued working on our DARPin sequences and finalised our
bioreporter.

Week 20

This week we focused on our podcast starting the week with a podcast meeting on the 17th and podcast recording on the
20th interviewing our co-leaders. In between, we had a meeting with our advisors Sesilja and Ville to discuss the
potential size of our DARPin library. We were recommended to rather order our DARPin library than assemble it in order
to save time during our project.
On the 19th we prepared our talk for the synthetic biology course in Aalto University. In our weekly meeting, we started
to suggest names for our product and started to assemble lab protocols as well as reach out to other teams for potential
collaborations. Additionally, the social media team started to be more active since the team introduction was finished.
On the 21st we had our May social event, which involved going to Heureka, so that we could explore the facilities that
would be available to us in order to prepare our workshop.

Week 21

We started the week on the 23rd by presenting our project to the synthetic biology course at Aalto University. On the
24th we had a meeting with the Microbiology and Microbial Biotechnology (MMB) Master's programme social media team of
the University of Helsinki. In this meeting, we decided which content the MMB programme would require for our
collaboration.
We had a funding meeting with Kemianteollisuus. In our weekly meeting, we decided upon the date to start the wet lab and
have an introductory safety course to be allowed to access the Aalto School of Chemical Engineering building. We planned
further meetings for the future and focused more on the collaborations again. On the 30th we were able to finalise
QBlock as our project name.

## June

Week 22

This week, kicked off our first week of full-time iGEM work, which will continue over the whole summer period. Both our
designated office space and our lab are located in the Aalto University School of Chemical Engineering, and all team
members completed three compulsory safety training courses before beginning. We were kindly given a comprehensive lab
tour by the department's two technicians, Mikael Hytti and Ulla Ahman, who gave clear instructions on where to find
chemicals and supplies as well as how to notify staff when stocks need to be replenished. On the 2nd, we met with our
PhD mentors as well as Esko Kankuri, regenerative medicine professor and wound healing researcher, to seek advice on our
project workflow, methods and goals. At the end of the week, on 3rd, we met with GeneArt to discuss prices for our
potential orders, though we did not end up using their services.

Week 23

On 7th, 2021 iGEM team member Quique showed us around the laboratory a bit more. On the 8th, the whole team met at the
Helsinki Conference Centre (Messukeskus) for the ChemBio event. Here, we spoke to many different companies, including
Eppendorf, Thermofisher, and Immunodiagnostics Oy. For the latter, we were able to see the CODEX DNA synthesis machine
in real life and negotiate our temporary use of the equipment in the summer. On 9th June, the team presented an overview
of our project in the School of Chemical Engineering's lecture theatre to keep the other researchers and groups housed
in the same building abreast of what we are hoping to achieve. We also tested the vinyl press available for printing our
logo on team shirts at Helsinki central library, Oodi. On 10th, we published an educational Instagram post on what
biofilms are.

Week 24

On the 13th we met with the HUS Wound and Burn Care staff for the first time, explaining our project to them and seeking
feedback on whether they'd be interested in our solution in the hospital setting - it was positively received. On 14th
we began our first day in the lab with amplification of the pET28a-sfGFP plasmid, which we continued for the next two
days with transformation and purification protocols. The expression and extraction of the pET28a-sfGFP plasmids were
successful, and the highest yield, as well as purity, was achieved for pET28a-sfGFP plasmid 1. On the 15th, our project
was also publicly revealed on Instagram. We also published the first, June edition of our newsletter. The mailing list
includes our sponsors, PIs, and faculty members, as well as any other interested collaborators such as clinical staff
and start-up founders. We

Week 25

On 20th, we met with the Dresden team to discuss our shared wet-lab plan, primarily the introduction of our GFP DARPin
into their hydrogel, and how we could send or transport our DARPin to them.

On the 21st, met with Anja, postdoc and research coordinator at the University of Helsinki institute for biotechnology.
We also sent our application for the Impact Grant, in which we detailed the tremendous health economics as well as the
quality of life costs of chronic wounds and how and why our solution plays into relieving society from some of this
burden. On this day we also started our transformation of the pET28a-sfGFP plasmid into E. coli. As the pET28a-sfGFP
plasmid contains GFP, our aim is for the plasmid to be expressed in BL21 E. coli cells amplified and the GFP expression
induced. We continued this experiment using the Transformation protocol also on the following day.
Also on the 22nd, we met via Zoom with the Latvia team - the first iGEM participants from the country - and presented
our projects to each other while ideating how we could create a regional Baltic Collaboration. Also on the 22nd, we
published the first episode of our podcast on Spotify and spoke about the upcoming Nordic Conference meet-up with the
Linkoping, Sweden team.

On 23rd, we began with colony expansion and expression of GFP in the lab using our protein expression protocol. We tried
to record the second episode of our podcast but due to equipment malfunction, recording stopped and data was wiped from
our SD card when we were about to conclude the episode. Since there was no way to repair the machine within the next
week, we decided to delay recording to early July. We also met with the CODEX and Immunodiagnostics Oy, who will be
providing us with their BioXP DNA synthesis machine for a trial run in August, to synthesise 32 DARPins. On 24th we met
with our sponsor, BioGarage.

Week 26

On 27th, the team gathered at the University of Helsinki Viikki Campus, where the BioCentres are located. Here we met
with Lars Paulin, NGS expert to advise us on how we can work together for our sequencing steps. On 28th, we wrote the
1-minute elevator pitch required for the European meet-up at the end of the week, met with our PI Heli Viskari, and with
our two collaborators/sponsors CODEX DNA and BioGarage. Between 28 and 30th June we started GFP extraction and
purification in the lab. After GFP was expressed and extracted it needed to be purified in order to be utilised as a
target for the ribosome display. It was checked with an SDS-PAGE that mainly GFP was expressed. Additionally, buffers
were prepared for the extraction of GFP.

On 29th, we had a meeting with Heidi Salonen from Aalto's engineering school, who was interested in hearing about our
project progress. On 30th, several team members embarked on a trip to Hamburg for the European iGEM meetup.

## July

Week 27

In the lab, we repeated the SDS gel for the expressed GFP after adapting the protocol and successfully showed that we
expressed GFP. Additionally, part of our team returned from the European iGEM Meetup and we had a meeting with Ville and
Kah Ying Ng to discuss our ribosome display and arrange the possibility to perform the first in vitro transcription and
translation in Ville's lab under the supervision of Kah Ying.
On the next day, we had a meeting with IDT, because we needed to discuss the synthesis of our bioreporter parts. Our
bioreporter showed issues in the synthesis, therefore, we met with IDT to adjust and discuss the synthesis. We also
prepared buffers for the extraction of our expressed GFP and continued with the GFP purification via Ni-NTA spin
purification for the next two days. Additionally, we started our promotion video preparation and had a meeting to
discuss the script on the 7th and finished our second podcast episode recording on the 8th.

Week 28

This week we started with the amplification of the second part of our bioreporter containing the AgrC and AgrA gene.
However, due to false positives, we repeated the PCR several times replacing water and primers to identify the
contaminant. The primers were successfully identified as the contaminating source of the PCR and the bioreporter part 2
was extracted. On Friday we measured the DNA concentration of both the NGFP plasmid and the bioreporter part 2 and
digested them both with XbaI and PaeI.

Week 29

This week we struggled with a successful ligation of our digests and realised at the end of the week that we assumed the
plasmid size wrong and needed to recalculate the correct ratio of plasmid and digest in order to achieve a successful
ligation. Additionally, we focused heavily on our dry-lab collab with Dresden and researched data points that are needed
for the collaboration. Furthermore, we also set-up a wet-lab collaboration with Dresden, which involves transporting a
GFP targeting DARPin from Helsinki to Dresden. The Dresden team will analyse the diffusion of the DARPin through their
hydrogel. On Tuesday the 19th we had our July social event and went to Nuuksio and on Thursday the 21st we went to Turku
to meet the Aboa Team. At this meeting, we exchanged the experience of our project work and also fundraising issues. The
meeting was beneficial for both of us and we planned activities that we wanted to do in Heureka for our workshop
targeting several age groups.

Week 30

We repeated digestion and ligation twice and incubated the second ligation reaction longer than the first one.
Additionally, we realised that we were not supposed to run the finished ligation product on an agarose gel for gel
extraction due to supercoiling and transformed it into E. coli and observed three colonies on Friday that we put in the
fridge over the weekend. On Tuesday, we also performed in vitro transcription and translation under Kah Ying’s
supervision in Ville's lab, where we also stored our products until Ville returns.

## August

Week 31

We started our week with the weekly meeting and finalising our trip details for the Nordic iGEM conferences. On the 2nd
of August we also had a meeting with our PI Heli to inform her about our progress and we had a meeting with the
ImmunoDiagnostics Oy marketing team to come up with a social media strategy. On the 1st and 2nd August, amplification of
the cloned plasmid was performed. We grew three of the four colonies overnight and made a PCR colony from these before
performing a plasmid purification. The concentration of each plasmid was determined using the nanodrop. Unfortunately,
no bands were observed in the colony PCR in which just colonies were picked from the plates indicating unsuccessful
transformation. The colony PCR might have not worked due to inhibition of other components present in the *E. coli*, so we
repeated a general PCR with the purified plasmid on the following day.

For this repeat, on the 3rd of August, the agarose gel showed three clear bands indicating that successful
transformation and ligation of the plasmid took place. On the 3rd of August, we also published the 3rd episode of our
podcast on Spotify, during which Zsofia spoke with last year's team leader Sally about the challenges and successes of
the 2021 project. In the evening, 6 of our team members embarked on an overnight ferry adventure from Helsinki to
Stockholm to attend the Nordic iGEM Conference in Linköping, Sweden. The conference lasted from Friday 5th to Sunday 7th
and gave us the opportunity to practice our presentation skills for the Grand Jamboree.

Week 32

After coming back from Linköping, we had a meeting with the Aboa team to discuss our workshop for Heureka. On the 10th
of August we gave a presentation to the Finnish IBO team about our project and iGEM itself. We also had a meeting with
our mentors to inform them on our progress. We also retrieved the plasmid for expressing the GFP-targeting DARPin for
the TU Dresden Team. The next day the BioXPTM machine from Codex DNA was transported to our lab and we set it up. On the
same day we had a meeting with the TU Dresden team discussing the modelling approaches of the DARPin passing through the
biofilm.

In the lab we were busy with troubleshooting the amplification of bioreporter part 1 and also digested, ligated and
transformed it into TOP10 E. coli.

Week 33

On Monday 15th of August, our videographers Mari, Joose and Hanna met with the clinical staff at the HUS Wound and Burn
Centre to film interviews and hospital shots for the promotion video. We also performed the ribosome display in Viikki
on Monday with the help of Anja. Later on Tuesday we were supposed to continue with the RNA purification at Aalto, but
we realised we were missing the RNA purification kit. We continued with the ligation and transformation of bioreporter
part 1 during the week, and went through the feedback from the Nordic iGEM Conference with the whole team. On the 23rd,
we had a zoom call with Turku and Heureka to introduce them and to prepare a plan for the workshop, and made a list of
all needed equipment for the workshops. Additionally, we had a meeting with Codex DNA to discuss marketing.

We also started to amplify, digest, ligate and transform the GFP-targeting DARPin into the plasmid provided by Sami.

Week 34

We started our week with the weekly meeting updating everyone on what has been going on in the previous weeks. On the
23rd, a few of the team went to Heureka to try out some of the materials we were going to use in the workshops, and in
the lab we had an exciting day as we finally did the DARPin DNA synthesising run with the Codex DNA BioXp machine. We
got the Immunodiagnostic Oy representative Anni Kivinen also with us onsite for the run and Codex DNA representative
Andrew Jones online with us. The run went well and we stored the DNA fragments in the freezer until we were ready to
start the next ribosome display run. On the 24th we had a call with Turku about the Science Basement presentation, where
we planned the presentation slides and went through the content. On the 25th we also gathered and filmed a few team
shots for the promotion video, which was fun. During the weekend 26-28th of August, one of our team members Zsófia also
flew to Germany for the JuniorJam meetup in Münster, where she met our partnership iGEm team Dresden.

In the same week, we also continued with the purification and dry-freezing of the GFP-targeting DARPin. However, we were
not able to see a band on the SDS-PAGE and realised then that the GFP-targeting DARPin presented the wrong start codon,
which probably prevented expression. Therefore, we re-ordered the GFP-targeting DARPin with the correct start codon and
restriction sites, because we received a different plasmid from our mentor Sami then expected.

Week 35

During our last week working full time with iGEM, we had a small recap of what both wet-lab and dry-lab had done so far,
to make sure that everyone was up to date and could explain both parts of the project if needed. We also took during the
30th new profile pictures and team photos for the wiki. We faced some struggles still with the transformation of
bioreporter part 1 in the lab, and we tested a few different ratios of DNA to plasmid concentration to see if that would
help get successful colonies after transformation. We ended the week with a movie night where we watched the Hamilton
musical. During the week we also finalised our promotion video and sent it to iGEM. Additionally, we received the new
GFP-targeting DARPin and cloned it into Sami's plasmid, grew and extracted the DARPin.

## September

Week 36

The week started with a meeting with Heidi Salonen. We updated her about our progress on the project and promised to
send her our results, so that she can share them with the faculty for further internal communication about iGEM in Aalto
University. On the same day, we had our weekly meeting in which we discussed mainly the presentation that we will
present to the Science Basement and everyone checked that they were aware of their writing tasks. On Thursday we had our
meeting with the Science Basement and the Aboa Team. We presented our presentation together and received a lot of
helpful feedback from Noemi Gutierrez-Valdes and Noemi Turpin from the Science Basement. Furthermore, we met with the TU
Dresden to discuss our dry-lab collaboration. The TU Dresden Team informed us that the DARPins would be only present for
30 minutes at the wound site and that the AIP concentration would change over time. On Saturday we were able to give our
two workshops with the Aboa team in Heureka. The workshops were a lot of fun and the children, parents and we enjoyed it
very much.

We also progressed in our wet-lab work. On Monday we both in vitro transcribed and translated our DARPins that we
previously synthesised in the BioXPTM machine. We furthermore immobilised the AIP on the 96-well plate. However, the AIP
showed trouble dissolving due to probably the increased hydrophobicity of the peptide. Therefore, we repeated the
immobilisation on the next day and were able to affinity select on Wednesday followed by reverse transcription, and
amplification of the DARPins. The affinity-selected DARPins were submitted for sequencing on Thursday at the Institute
of Biotechnology.
Additionally, we further worked on expressing the GFP targeting DARPin for Dresden by running the extracted and purified
protein on an SDS-PAGE. The same steps without purification were repeated again at different temperatures Friday to
Sunday to improve the DARPin expression, because most of the DARPin was present in the pellet after extraction.

Week 37

This week we started with our weekly meeting and we focused mainly on writing tasks, the upcoming science basement
presentation, and sitsit preparation. The alumni sitsit was held on Saturday and was a great success. We had fun games
and were able to let the alumni relive iGEM memories and share our experiences.
We also progressed further in the lab, because we were finally able to successfully ligate bioreporter part 1 into the
plasmid containing GFP and bioreporter part 2. Furthermore, the ligate was successfully transformed into E. coli NEB
alpha-5. The ligate was extracted and digested in order to be run on the gel to confirm successful cloning.
Furthermore, the work on the GFP targeting DARPin expression did not stop. We changed IPTG concentration and further
temperatures and settled that an overnight induction at 20 °C seemed to be most successful. Furthermore, we purified and
deep-freezed all the GFP-targeting DARPin we had to at least have some DARPin to send to TU Dresden as soon as possible.

Week 38

The week started with the weekly meeting and we discussed writing tasks for the wiki, science basement and the science
basement presentation, which were able to successfully present on Saturday. On Wednesday Lilith presented our iGEM
project and iGEM to the microbial biotechnology course in the University of Helsinki.
Additionally, we changed the approach for the expression of the GFP-targeting DARPin and in vitro transcribed and
translated it, because we realised that the previous expression was not successful. The successful protein synthesis
could not been proven, because the gel was not successful, however due to observing white powder we deemed the synthesis
as successful and informed TU Dresden and they decided that they were able to receive this GFP-targeting DARPin in this
form without the final confirmation. We were able to transport it to Germany and send it via mail to Dresden. We also
progressed in our bioreporter and successfully expressed the plasmid in BL21 E. coli. This allows us to express GFP more
successfully upon sensing the AIP.

Week 39

We started our week by participating in a workshop of Olle Bergman that was organised by Lund university, where we had
the opportunity to listen to many iGEM presentations, but also present our own presentation. Amna presented for the
entire team and received a lot of helpful feedback for both slide design and body language. We also had our weekly
meeting on Monday, where we focused again on the wiki writing tasks and started planning our grand jamboree presentation
for which we had an additional meeting on Wednesday.
In the lab, we also progressed and finally tested our bioreporter in the presence of AIP. We also had controls with BL21
E. coli expressing the GFP-targeting DARPin, but no GFP to and no AIP presence to ensure that the fluorescent signal is
only being expressed in the combination of the bioreporter and AIP.

## October

Week 40

During the last week before the wiki freeze we focused mainly on finishing the last writing tasks for the wiki. In
wet-lab we analysed the data from the bioreporter measurements and concluded that there is some problem with the P2
promoter, either that it is leaky and active even if it is not induced or then our strong constitutive promoter is too
strong and also starts transcription in the other direction. Further testing would be necessary if we would have the
time for that. Otherwise the cell growth wasn't affected by the bioreporter and only our bioreporter cells emitted the
GFP signal and not the controls, which was positive to see. The dry-lab continued to work on the sequencing data
analysis and finalised our wiki pages. We also had a meeting on 4th to plan and discuss the Jamboree presentation and
started working on the slides for that.

February

March

April

May

June

July

August

September

October

* [February](#feb)
* [March](#mar)
* [April](#apr)
* [May](#may)
* [June](#june)
* [July](#july)
* [August](#aug)
* [September](#sep)
* [October](#oct)

{% endblock %}
{% block script\_content %}

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

# Engineering

Here you can read about the engineering cycles our project and project parts have been through.

## Engineering Cycle for Project Design

### 1. Design

We have made clear plans for the project and divided our project into three parts that we would focus on. Library
construction, Ribosome Display and Bioreporter building. For each part we prepared by reading literature of what has
already been done, and started planning on what we could do differently. We also asked a lot of help from our
instructors and mentors who have the best knowledge in each subject, to ensure that we avoid any unnecessary mistakes.

### 2. Build

After designing the plan for each part, we will begin composing the actual plasmid, or DARPin design for the testing
phase. We ordered all designed fragment parts and on the dry lab side, we built the algorithms needed for the selection
of DARPins. We based our construction on the help we had gotten in the design phase, and got inspired for the building
phase from what we had been reading from the literature ( Stumpp et al., 2008,
[Team IISER, Kolkata](https://2021.igem.org/Team%3AIISER_Kolkata/Parts)).

### 3. Test

During testing we will do most of the lab work, where we will test how well our designed and built fragments actually
works. We will both do drylab and wet lab work during this time to optimise the time spent. We worked simultaneously on
the different parts of the project.

### 4. Learn

From the results in the testing phase, we will then reconsider our constructs and if needed go back to the designing
phase to see if we need to change something in the construct or if the issues could possibly be fixed with outer
changes, especially in the wet lab work.

## Our Project in a Nutshell

Our project is based on chronic wounds and the build-up of biofilm, which is a both local and a global issue our
iGEM
team wants to tackle. Our solution is based on protein-peptide binding modelling and execution and for that, we also
need tools to confirm that our design actually works. For this, we needed to design both the binding protein
(DARPin) as
well as a bioreporter for detection.
We needed a bioreporter that would give us something to measure the activity of specific genes when our target
peptide
was both present and absent. For this, we planned based on the research and based on the past iGEM team's success to
do
similar parts that were found in the parts registry. We thought that measuring GFP would be the most manageable
signal
to read for us, so we planned to have one inducible promoter that would start transcription of the fluorescent
proteins
only if the target peptide was present. We would also have the required signalling pathways proteins expressed at
all
times. For ordering all parts, we thought we would order as big sequence fragments as possible to minimise the time
needed for cloning. We planned to use basic restriction cloning for both parts.
In the following sections we describe how we used the engineering cycles to work on these parts.

## Cycle 1

### 1. Design

We needed a bioreporter that would give us something to measure the activity of specific genes when our target peptide
was both present and absent. For this, we planned based on the research and based on the past iGEM team's success to do
similar parts that were found in the parts registry. We thought that measuring GFP would be the most manageable signal
to read for us, so we planned to have one inducible promoter that would start transcription of the fluorescent proteins
only if the target peptide was present. We would also have the required signalling pathways proteins expressed at all
times. For ordering all parts, we thought we would order as big sequence fragments as possible to minimise the time
needed for cloning. We planned to use basic restriction cloning for both parts.

So, for our bioreporter, we needed to make 2 composite parts which included all the needed parts to complete our
bioreporter. To learn more about the individual parts and constructs, see Parts (link) page. Our part1 for the
bioreporter consists of two promoters in two different directions. The idea of the bidirectional promoters was from past
year's iGEM team, especially team IISER Kolkata, who had done a similar construct in 2021. They had proved that the
bidirectional model of the two promoters worked better than if the promoters were to be placed after each other. After
both promoters, there is the RBS sequence.

### 2. Build

We tried to order this part1 (fig.1) from IDT, however, in the production phase, we ran into the problem of the part not
passing the quality control, even though the part was accepted in the initial check before ordering.

![Sequence for first draft of bioreporter.](https://static.igem.wiki/teams/4159/wiki/figures/biorep-first-draft.png)

Figure 1. First draft of Part1 for bioreporter.

We got in contact with our sales representative Silvija Jovic from IDT, with whom we had a meeting about our project. We
discussed the issues with her and agreed on sending the fragment to the IDT specialists who made some changes to the
beginning of the sequence. The issue with the fragment sequence was the T nucleotide heavy beginning which made the
sequence probably very unstable and that is why it failed the quality control tests. In fig. 2, the change to the first
sequence is shown in red colour.

![Sequence for second draft of bioreporter.](https://static.igem.wiki/teams/4159/wiki/figures/biorep2nd-draft.png)

Figure 2. Second draft of Part1 for bioreporter.

### 3. Test

We started working first with the second draft of the fragment. In the testing phase for the second draft of part1, we
succeeded up to the transformation phase. In Figure 3. Is the successfully amplified part1 (second draft).

![Gel picture](https://static.igem.wiki/teams/4159/wiki/figures/eng-fig3.png)

Figure 3. The DNA concentration of the band from the annealing temperature 62 °C and 63 °C was measured at 17.5 ng/µL with a
purity at A260/280 of 1.95 and the the band from the annealing temperature 68 °C was measured at 10.5 ng/µL with a
purity at A260/280 of 1.42.

After this we continued the digestion and ligation. The digestion gel picture is presented in Figure 4.

![Gel picture](https://static.igem.wiki/teams/4159/wiki/figures/eng-fig4.png)

Figure 4. The digest of part1 and pET28a-sfGFP plasmid containing BBa\_K4159009. The DNA concentrations were measured at 11.8 ng/µL
for part1 and 3.1 ng/µL for pET28a-sfGFP plasmid containing BBa\_K4159009.

However, for some reason, the transformation of the fragment did not work. We tried different cells for the
transformation, both alpha5 and top10 E.coli cells for the transformation, but with zero success in getting colonies. We
also tried to prolong the ligation time in case the issue was there, and different ratios (3:1 and 5:1) of DNA to vector
ratio. We also went back to the first step of PCR, and tried different annealing temperatures to optimise it to have as
high of a concentration of the fragment for digestion and ligation as possible.

### 4. Learn

During the time we were doing the lab work on this, we were also taking part in the Nordic iGEM Conference in Sweden.
During the presentations at the conference, we got some feedback about our parts for the bioreporter. The concern that
there was too little space between the promoters was raised, and it made us think that the small space in between could
result in neither of the promoters working. We hypothesised that the lack of space could disrupt both promoters as there
was not enough room, specifically for the P2 promoter to be activated. We discussed this also with our mentors and got
the advice from Sami Jalil to try the Second draft part1 first, and in case it doesn't work, design a third with about
20-50bp in between the promoters.

## Cycle 2

### 1. Design

After, struggling and troubleshooting in the lab with the second draft, we went back to the design phase again.

For our third design we removed the 6bp spacer from the second draft and replaced it with a random sequence of 50bp. The
random sequence was generated from the
 [University of California's Random DNA generator](http://www.faculty.ucr.edu/~mmaduro/random.htm).
The GC content for the randomization was put as 0.5.

![Sequence for third draft of bioreporter.](https://static.igem.wiki/teams/4159/wiki/figures/biorep3rd-draft.png)

Figure 5. Third draft of Part1 for bioreporter.

### 2. Build

We hoped that the change in spacer length would be the fix to our problems. The difference from the earlier fragment was
also the supplier. As our IDT account had reached its limit, we ordered now from Twist Bioscience, however, this should
not change anything in the protocols.

### 3. Test

For the testing phase we followed the same protocol as before in the beginning. We had some troubles in the beginning
with the PCR and got very low concentration after the amplification, however, at the end we succeeded again all the way
to the transformation phase. After a few trials and failure of the transformation with the new third draft of the part1
fragment, we decided to change the protocol a bit and lowered the temperature and decreased the time of the heat shock
from 45C in 45 sec to 42C for 30sec.

### 4. Learn

This change in protocol gave a positive result and the plasmid was successfully transformed into the cells. We assume
that the change in protocol had in this case the bigger effect on the successful outcome. However, we think it was still
a good idea to change the length of the spacer in between of the promoters to avoid that they would somehow falsely
start transcribing the signal even if there is no signalling molecule binding to the cell. The temperature change in the
transformation protocol was also a good lesson to learn, that it might not always be good to have the temperature so
high, even if the cells used in the transformation are supposed to handle the temperature in the transformation.

## References

1. Stumpp, M. T., Binz, H. K., & Amstutz, P. (2008)

   DARPins: a new generation of protein therapeutics

   *Drug discovery today, 13(15-16), 695-701*

   [Google Scholar](https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=Stumpp%2C+M.+T.%2C+Binz%2C+H.+K.%2C+%26+Amstutz%2C+P.+%282008%29.+DARPins%3A+a+new+generation+of+protein+therapeutics.+Drug+discovery+today%2C+13%2815-16%29%2C+695-701&btnG=)

Project Design

Project in a Nutshell

Cycle 1

Cycle 2

* [Project Design](#general)
* [Project in a Nutshell](#recap)
* [Cycle 1](#cycle1)
* [Cycle 2](#cycle2)

{% endblock %}
{% block script\_content %}

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

# Experiments

Here you can find all the experiments we did.

On this page, we have gathered the list of experiments and our goals we have aimed for in our wet-lab work. We divided
our wet-lab work into three sections, focusing on ribosome display, bioreporter cloning, and GFP-DARPin expression
separately. To see more detailed information about all the labnotes from the experiments check out our labbook and
[protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).

### Labbooks

* [Ribosome Display](https://static.igem.wiki/teams/4159/wiki/exp-pdf/ribosome-display.pdf)
* [Generation of a bioreporter](https://static.igem.wiki/teams/4159/wiki/exp-pdf/generation-of-a-bioreporter.pdf)
* [GFP expression](https://static.igem.wiki/teams/4159/wiki/exp-pdf/gfp-expression.pdf)
* [GFP targeting Darpin expression](https://static.igem.wiki/teams/4159/wiki/exp-pdf/gfp-targeting-darpin-expression.pdf)

## Goals of the Experiments

1. Ribosome Display for DARPin affinity selection
   * Expression of GFP
   * Synthesis of own DARPin library
   * Affinity Selection of DARPins
2. Bioreporter
   * Bioreporter cloning
   * Bioreporter testing
3. GFP-binding DARPin synthesis
   * Diffusion of DARPin through a hydrogel

## Ribosome Display

### Idea

We chose ribosome display as the method for the binding selection, as it has been described as a good method for
protein-peptide binding assays (Chen et al. 2021). The idea behind the method is to do in vitro transcription and
translation which will result in the targeting peptide being “stuck” on the ribosome. It is crucial that the targeting
protein does not have a stop codon and a long enough spacer after the protein sequence, to result in the peptide being
far out from the ribosome but still attached to the ribosome, see Figure 1 (Lipovsek and Plückthun, 2004 ).

![Ribosome Display](https://static.igem.wiki/teams/4159/wiki/figures/exp-fig1.png)

Figure 1. Schematic overview of the ribosome display (created with Biorender.com)

### Aim

As we wanted to prove that the selection method could be successful for our AIP binding DARPins, we firstly wanted to
prove the method with a known protein-protein binding. We chose GFP binding DARPins as our test targeting protein, and
naturally GFP as the target protein. For this, we aimed to produce our own GFP protein by expressing superfold-GFP in E.
coli expression cells. We also aimed to test our own AIP binding DARPins, which meant we built a small library of
potential AIP binding DARPins. To read more about the background of the selection of DARPin sequences, please visit our
modelling page. For the experiments, we aimed to test both our own AIP binding DARPins, as well as our known GFP binding
DARPin. To prove the quality of the binding affinity, we also placed our AIP binding DARPin in the same reaction with
the GFP binding DARPin. This will prove that the most suitable DARPin will always bind to the specific target.

### Testing

We ordered the needed GFP-binding DARPin from IDT. The construction of the fragment can be seen below in Figure 2.

![Sequence of GFP-binding DARPin](https://static.igem.wiki/teams/4159/wiki/figures/exp-fig2.png)

Figure 2. Ordered GFP-binding DARPin composite part for ribosome display

After we received the DNA fragment, we proceeded with the in vitro transcription and translation according to the
[protocol.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D)

After in vitro transcription and translation, elution of RNA from the mRNA-ribosome-protein-complex is needed to isolate
mRNA from the selection of binders during the ribosome display, for sequencing and identifying, which DARPin bound the
best.

The mRNA then needs to be purified and reverse transcribed, for these steps we used ready-made kits, see protocols for
the exact methods and tools used.

The last step before sequencing and finding the best suitable DARPin, we needed to prepare the cDNA for sequencing by
adding on specific overhangs needed for the sequencing. The forward and reverse overhang can be seen in Figure 3, the
same overhangs were added to all DARPin cDNA fragments. After this the cDNA DARPin fragments with correct overhangs were sent for sequencing.
We acknowledge DNA Sequencing and Genomics Laboratory, Institute
of Biotechnology, University of Helsinki for sequencing.

![Sequence of primer overhangs](https://static.igem.wiki/teams/4159/wiki/figures/exp-fig3.png)

Figure 3. Overhangs for sequencing

### Results

We performed in total three ribosome display experiments. Two of them were affinity selecting our 42 DARPins against the
biotinylated AIP1 target, while one of them was affinity selecting our GFP-targeting DARPin against GFP. For the
ribosome display containing the GFP-targeting DARPin we added additionally a small aliquot of DARPin #12. This would
confirm that the GFP-targeting DARPin would bind the GFP target in higher quantities.

Based on the results from the sequencing, we were successful in the ribosome display with our GFP binding DARPin. The
sequence logo for our GFP DARPin and the retrieved sequence from the binding assay can be seen in the Figure 4. It was
shown that the sequence found to be bound was actually the GFP DARPin and not the added DARPin #12.

![Sequencing logo comparing template GFP-DARPin and results of ribosome display.](https://static.igem.wiki/teams/4159/wiki/figures/exp-fig4.png)

Figure 4. Data sequencing logos of the GFP-binding DARPin template (left) and the sequenced results of the ribosome display
(right).

From our results in the wet-lab we could demonstrate that sequencing was successful. Specifically, the N-cap for the
GFP-targeting DARPin was completely identified, while slight changes of the sequence occurred later. The majority of the
sequence presented, however, was the GFP-targeting DARPin. Therefore, we were able to demonstrate that the ribosome
display can be used for future experiments, and could be used as an easy tool to find the right DARPin sequence for the
target signalling molecule.

We also saw some affinity of sequencing of the AIP binding DARPins. We could retrieve from the sequencing data the same
N-cap and beginning of the DARPin sequence. However, unfortunately, due to time restrictions, we couldn't show any
specific DARPin that showed great affinity against our AIP. More time for testing and re-runs of the ribosome display
would be needed to confirm the exact DARPin sequence that bound the best.

## Bioreporter

### Idea

The idea behind our bioreporter is to prove the impact that the DARPins have on the signalling molecules. We wanted a
bioreporter that was able to produce the needed membrane proteins and internal signalling proteins, to bind the
signalling molecules (AIP's) and then give an output to show that the cell has received the information of the binding.
For this part of the project we took inspiration from a last year's iGEM team ([Team IISER, Kolkata](https://2021.igem.org/Team%3AIISER_Kolkata/Parts), 2021 iGEM team), who had
built a bioreporter with the parts aimed to sense signalling molecules released from S. aureus bacteria. They had
successful results, which made us confident that we can build a similar bioreporter but aimed for *S. epidermidis*.

### Aim

We aimed to clone two composite parts that we ordered through IDT for the bioreporter. The first part will have both
promoters that we need, one for transcription of GFP, and one for transcription of AgrC and AgrA genes. As proven by the
IISER Kolkata iGEM team, it is crucial to have the promoters in opposite directions to avoid any leaking of expression
from the strong constitutive promoter. The other composite part will include the needed membrane protein AgrC and the
internal signalling protein AgrA. After cloning, we will measure the output signal (GFP fluorescence) when the
bioreporter has AIP's in its surroundings, and if possible test the bioreporter when both AIP's and our AIP binding
DARPin are in the surroundings of the cell.

### Testing

The two composite parts needed for the bioreporter were ordered directly from IDT. The final used and modified parts can
be seen in Figure 5.

![Sequences of part 1 and part 2 of the bioreporter](https://static.igem.wiki/teams/4159/wiki/figures/exp-fig5.png)

Figure 5. PART1 and PART2 ordered for bioreporter

Both parts were amplified and checked with gel electrophoresis. We started with PART2 cloning as that part arrived
before PART1 from IDT. This was due to changes that needed to be made, to read more about the changes, see our
[engineering page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D).

Both parts were digested with the correct restriction enzymes to get the right restriction sites and were then ligated
and transformed into BL21 *E. coli* expression cells according to [protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D). The set up of the plasmid and
restriction sites can be seen in Figure 6.

![Schematic circular overview of bioreporter.](https://static.igem.wiki/teams/4159/wiki/figures/exp-fig6.png)

Figure 6. Bioreporter construction

To test the bioreporter, our aim was to test the GFP intensity of the bioreporter cells when they were induced with
AIP's, and then if we would have time, test if the signal would be lower in case DARPins were present in the
environment. Unfortunately, we only had time to test the bioreporter with AIP inducement.

For the testing we wanted to know if different concentrations of AIP's would affect the GFP signal, or if it would be
the same as long as the threshold of inducement had been achieved. The exact amount of AIP molecules needed to activate
the P2 promoter has not been measured as far as we know in literature. However, we had done some estimations of the
needed concentration for our modelling work, and for this we chose to see if high concentration would yield high
intensity. We ordered the AIP's directly from Genscript as purified protein. The AIP's were dissolvable in DMSO,
however, we ran into some trouble of not all protein dissolving in the DMSO when we were preparing the dilutions. Due to
this we had only some type of estimates of the concentrations but renamed them to high and low concentrations of the
AIP's. The lower concentration should be somewhere around 250nM, however, this is unfortunately not tested due to a lack
of equipment available. The amount of each tested AIP's can be seen below in Table 1.

Table 1. Amount of AIP for bioreporter inducement| High concentrated | Low concentrated |
| 5 µL | 50 µL |
| 2 µL | 20 µL |
| 1 µL | 10 µL |
|  | 5 µL |
|  | 2 µL |

The cells were grown up to 1.2 OD600, we added 5uL of culture to 200uL of LB media, which equals 4.8\*106 cells in the
beginning. After that we induced 3 wells with each concentration and measured the OD600 and GFP intensity at both 470
and 511nm wavelength. The measurements were done with the BioTek Synergy H1 Plate Reader, and the measurements were
taken for 10 hour every 20 min.

As blank we had LB media with kanamycin to avoid contamination growth, and as negative control, we had the same
expression cells as for the bioreporter, but these ones included the GFP DARPin expression cassette. The set up on our
96 well plate can be seen in Table 2.

Table 2. 96 well plate set up for GFP and OD600 measuring
![Table showing 96 well plate.](https://static.igem.wiki/teams/4159/wiki/figures/exp-tab2.png)

### Results

We successfully cloned both composite parts into the target plasmid containing our GFP gene. The bioreporter was assumed
to be functional after checking by PCR for the reason of performing restriction cloning, which would align the parts in
the direct direction.

The final test of the functional bioreporter was also somewhat successful. We received the GFP signal from all
bioreporter cells, which had the GFP gene, and no signal from our negative controls which didn't have the GFP gene. We
can conclude that some changes should still be done to the setup of the promoters in the bioreporter. It seems that the
P2 promoter might be active even if it hasn't been induced, or then the strong constitutive pBAD promoter has something
to do with the expression of GFP. However, it seems not likely as it is about 70bp downstream from the GFP encoding
gene, and the pBAD promoter is inserted to start transcription in the opposite direction. Future testing of inserting
just the P2 promoter in the plasmid prior to the GFP gene would be needed to figure out what might be the cause of the
false GFP signal, for those bioreporter cells which gave a GFP signal even thought they were not induced with the AIP
molecules, which is needed for activation of transcription of the GFP gene.

## GFP DARPin synthesis

### Idea

As a part of the proof of concept of our project, we also wanted to prove how we could produce the DARPins in
genetically modified organisms. We wanted to express our test GFP binding DARPin also as a part of our partnership with
the Dresden iGEM team, who wanted to do testing with our protein from their developed hydrogel.

### Aim

Our aim was to clone our composite part which includes the GFP binding DAPRin gene, as well as a His-tag for
purification and an Avi-tag for other downstream processing. We aim to transform this modified pET42b-HF-BE3 plasmid
with our target genes into E.coli expression plasmids and induce the cells with IPTG, extract the proteins from the
cell, and purify the protein.

### Testing

We ordered from IDT the needed DNA fragment, including the T7 promoter and RBS, the needed His-tag and Avi-tag, the
GFP-binding DARPin and the termination of the gene with a stop codon and additional His-tag. To speed up the process of
the cloning we ordered the fragment with the correct needed restriction sites at the end to fit into our cut plasmid.
The ordered fragment can be seen in Figure 7.

![Sequence of GFP-binding DARPin expression construct.](https://static.igem.wiki/teams/4159/wiki/figures/exp-fig7.png)

Figure 7. GFP-binding DARPin expression construct.

After receiving the fragment, we digested both the pET42b-HF-BE3 plasmid and our fragment with XbaI and XhoI restriction
enzymes and did ligation and transformation into BL21 *E. coli* expression cells.

After this we inoculated a few colonies overnight, at different temperatures, and different amounts of time. We then did
the main culture, which we induced with IPTG and did the protein extraction by sonication, all according to the [protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).
The protein was then purified with Ni-NTA columns to the best ability, however, most of the expressed protein
remained in the pellet.

### Results

The GFP-targeting DARPin was mainly present in the pellet and not in the soluble fraction. The best results were present
for induction of the GFP-targeting DARPin expression at 20 °C overnight. However, expression and extraction could not be
improved. Therefore, we decided to proceed with in vitro transcription and translation of the protein due to the time
constraint for our collaboration with TU Dresden.

## References

1. Chen, X., Gentili, M., Hacohen, N. and Regev, A. (2021)

   A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies

   *Nature communications, 12(1), pp.1-14*

   [Google Scholar](https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=A+cell-free+nanobody+engineering+platform+rapidly+generates+SARS-CoV-2+neutralizing+nanobodies&btnG=#d=gs_cit&t=1665333998992&u=%2Fscholar%3Fq%3Dinfo%3A5lbqM-XxDCUJ%3Ascholar.google.com%2F%26output%3Dcite%26scirp%3D0%26hl%3Den)
2. Lipovsek, D. and Plückthun, A. (2004)

   In-vitro protein evolution by ribosome display and mRNA display

   *Journal of immunological methods, 290(1-2)*

   [Google Scholar](https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=In-vitro+protein+evolution+by+ribosome+display+and+mRNA+display&btnG=#d=gs_cit&t=1665333961307&u=%2Fscholar%3Fq%3Dinfo%3AtYiws32xtbsJ%3Ascholar.google.com%2F%26output%3Dcite%26scirp%3D0%26hl%3Den)

Goals

Ribosome Display

Bioreporter

GFP DARPin

* [Goals](#goals)
* [Ribosome Display](#ribosome)
* [Bioreporter](#biorep)
* [GFP DARPin](#gfp)

{% endblock %}
{% block script\_content %}

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

# Results

Read more about the results of our wet-lab and dry-lab work.

## Project Achievements

### Wet-lab Results

We successfully:

1. affinity selected DARPins binding to AIP1 via ribosome display (sequences)
2. expressed GFP
3. expressed DARPins
4. synthesised DARPins *in vitro*
5. designed and cloned a bioreporter for quorum sensing
6. collaborated with Dresden to assess how a DARPin would diffuse through their hydrogel
7. synthesised our own DARPins with the BioXP machine from Codex DNA

### Dry-lab Results

1. Designed successfully DARPins
2. Modelled DARPin-AIP binding *in silico* via CABS-dock
3. Modelled DARPin-AIP binding *in silico*via Mdock-PEP
4. Compared modelling of DARPin-AIP binding
5. Modelled diffusion and binding of DARPin and AIP in biofilm

## GFP AND DARPIN PRODUCTION

### GFP expression

GFP was expressed from the pET28a-sfGFP plasmid provided by Norman Adlung. This GFP was used for the ribosome display in
order to have a positive control and proving that the GFP-targeting DARPin binds the GFP during ribosome display.
Additionally, this experiment confirms that the pET28a-sfGFP plasmid expresses GFP, because this plasmid with a
different promoter will be used for the bioreporter.

![Gel picture](https://static.igem.wiki/teams/4159/wiki/figures/res-fig1.png)

Figure 1. GFP supernatant and pellet of the GFP extraction from *E. coli* BL21 cells with a ladder [M] indicated on the
left.

Mainly the GFP was present in the GFP supernatant deeming the GFP expression as successful. This experiment confirmed
the GFP expression as successful at 28 kDA. The pET28a-sfGFP plasmid will be later used for the bioreporter and
expression of the GFP from the plasmid is necessary to retrieve a positive signal in case of AIP binding. This
experiment confirmed that we can use this plasmid for the establishment of our bioreporter, but we also retrieved GFP,
which we will use as the positive control during our ribosome display.

[Read more about it!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

### GFP-targeting DARPin Expression

We decided to express the GFP-targeting DARPin for our collaboration with TU Dresden. We wanted to assess how our DARPin
would pass through their hydrogel for a combined treatment. We decided to use the GFP-targeting DARPin, because for our
positive control we had a confirmed target. The GFP-targeting DARPin was first aimed to be expressed via cloning in the
GFP-targeting DARPin gene with an additional His- and Avi-tag into the pET42b-HF-BE3 plasmid. The cloning of the gene
into the plasmid was successful and colonies were observed. The GFP-targeting DARPin expression was further induced with
IPTG and purified (Figure 2).

![Gel picture](https://static.igem.wiki/teams/4159/wiki/figures/res-fig2.png)

Figure 2. SDS-page gel image of GFP-DARPin from both pellet and supernatant

The GFP-targeting DARPin was mainly present in the pellet and not in the soluble fraction. The best results were present
for induction of the GFP-targeting DARPin expression at 20 °C overnight. However, expression and extraction could not be
improved. Therefore, we decided to proceed with in vitro transcription and translation of the protein due to the time
constraint for our collaboration with TU Dresden.

[Read more about it!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

### GFP-targeting DARPin *in vitro* synthesis

The GFP-targeting DARPin for the TU Dresden collaboration was *in vitro* transcribed and translated after the extraction
was not successful and due to the time constraint we considered other possibilities. The GFP-targeting DARPin was
synthesised and dry-freezed before it was sent to the TU Dresden team.

[Read more about it!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

## Predicting the Structure of the AIP-binding DARPin

### Design of DARPin Library

Our original DARPin library was planned to have a size of 1024, however due to limited funding it was not possible to
design a library in this size and the DARPin library was limited to a size of 42 DARPins. The N-cap and C-cap were
optimised according to previous publications and fixed (Hansen et al., 2017; Schilling et al., 2014). It was decided to
only randomise eight positions in the internal repeats due to previous publications (Hansen et al., 2017; Schilling et
al., 2014; Seeger et al., 2013). For more information about the design, please refer to the [modelling page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D).

During the modelling and prediction of the structure of AIP and the DARPins the library was confirmed to be very
diverse, because out of the ten predicted DARPins all of them interacted at different bindings indicating variability in
the library. For more information, please refer to the [modelling page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D).

### Structure Prediction & Protein-Peptide Docking

AlphaFold2 was used to predict the structure of our DARPins. Ten of the DARPins were randomly selected and their binding
predicted both via CABS-dock and MDockPeP. These algorithms were used to score the interaction between both and
generally predict high domains of interaction. These algorithms were particularly useful, because they take the input
sequences without previous assumptions of binding sites.
All ten selected DARPins were observed to interact with AIP according to the modelling results and additionally, the
modelling predicted that the DARPins between position 11 and 140 would interact with the AIPs between position 1 to 8.
However, a general trend was observed that predicted that AIP will most likely bind between the last amino acids of the
N-cap until the first amino acids of the C-cap with highest probability of binding between the first and third internal
repeats. For more information upon the modelling results please refer to the [Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D).

## Diffusion Modelling

TU Dresden modelled how their phages and growth factors would pass through their hydrogel of their product Wunderband.
Both of us focused on chronic wounds; therefore, we came together and collaborated on using their model to assess how
DARPins pass through the biofilm and bind AIP. The model was very advantageous in order to discuss the implementation of
our product. According to the model 30 µM of DARPins would be applied and they would have a half-life of 10 hours. This
would give us the information that the DARPins should be applied every ~10 hours. For more information upon the
[diffusion modelling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)  and also the [implementation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D) check the appropriate wiki pages.

## Results from Ribosome Display

We performed in total three ribosome display experiments. Two of them were affinity selecting our 42 DARPins against the
biotinylated AIP1 target, while one of them was affinity selecting our GFP-targeting DARPin against GFP. For the
ribosome display containing the GFP-targeting DARPin we added additionally a small aliquot of DARPin #12. This would
confirm that the GFP-targeting DARPin would bind the GFP target in higher quantities.

Based on the results from the sequencing, we were successful in the ribosome display with our GFP binding DARPin. The
sequence logo for our GFP DARPin and the retrieved sequence from the binding assay can be seen in the figure 3. It shows
that the sequence found to be bound was actually the GFP DARPin and not the added DARPin #12. We show here that the
ribosome display can be used for future experiments, and could be used as an easy tool to find the right DARPin sequence
for the target signalling molecule.

![Sequence logo](https://static.igem.wiki/teams/4159/wiki/figures/res-fig3-2.png)
![Sequence logo](https://static.igem.wiki/teams/4159/wiki/figures/res-fig3-1.png)

Figure 3. GFP-binding DARPin and retrieved sequencing data's sequence logos

We also saw some affinity of sequencing of the AIP binding DARPins. We could retrieve from the sequencing data the same
N-cap and beginning of the DARPin sequence. However, unfortunately, due to time restrictions, we couldn't show any
specific DARPin that showed great affinity against our AIP. More time for testing and re-runs of the ribosome display
would be needed to confirm the exact DARPin sequence that bound the best.

## Synthesis of DARPins

We designed the DARPin library ourselves and also synthesised the DARPins in collaboration with Codex DNA by using their
BioXPTM machine. For this we uploaded 32 of our DARPins and assessed how successful DNA synthesis of these were before
we synthesised them in the BioXP™ machine. The whole machine run for DNA synthesis took around 15 hours (Figure 4).

![Sequencing machine](https://static.igem.wiki/teams/4159/wiki/figures/res-fig4.png)

Figure 4. BioXP™ machine shortly before synthesising all our 32 DARPins.

The synthesised fragments were run on a gel to observe the expected size of 815 bp and after that were gel extracted to
have no additional fragments interfering with the latter ribosome display (Figure 5).

[Read more about it!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

![Sequencing machine](https://static.igem.wiki/teams/4159/wiki/figures/res-fig5.png)

Figure 5. 2 % agarose gel of the DARPins (D1-D32) synthesised with the BioXp machine from Codex DNA. The ladder [L] used
is the 1 kb Plus DNA ladder with the base pairs [bp] indicated on the left side.

The DNA synthesis via the BioXPTM machine was successful and the created DARPin DNA could be successfully used for the
latter *in vitro* transcription and translation for ribosome display.

## Building and Testing the Bioreporter

### Bioreporter Design

The bioreporter was built in BL21 *E. coli* and designed to contain both the AgrA and AgrC genes, which are responsible to
sense AIP in *S. epidermidis*. Both these genes were under expression of pBAD, which is a constitutive promoter and
expression of both of them would be constant. Additionally, the plasmid of the bioreporter contained GFP, which was
under control of the inducible P2 promoter, which would only be expressed in the presence of AIP. This system would
therefore cause a green fluorescent signal in the presence of AIP and meaning that if the DARPin would bind to AIP the
green fluorescent signal would be decreased.

During the assembly of our bioreporter we had initial trouble fitting in the bioreporter part 1, which contained both of
the promoters. Therefore, we decided to elongate the part and add more base pairs between the promoters to also avoid
later steric hindrance of the promoters. After both the parts of the bioreporter were added into the pET28a-sfGFP
plasmid another PCR of both bioreporter parts was performed to confirm the presence of both parts (Figure 6).

![Gel picture](https://static.igem.wiki/teams/4159/wiki/figures/res-fig6.png)

Figure 6. 1 % agarose gel with amplified bioreporter part 1 and bioreporter part 2 that were amplified from the finished
bioreporter in the pET28a-sfGFP plasmid. X indicates empty wells.

The bioreporter was assumed to be functional after this PCR for the reason of performing restriction cloning, which
would align the parts in the direct direction. The final test of the functional bioreporter shall be done with the
addition of AIP.

[Read more about it!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

### Bioreporter Test

To test the bioreporter, our aim was to test the GFP intensity of the bioreporter cells when they were induced with
AIP's, and then if we had time, test if the signal would be lower in case DARPins were present in the environment.
Unfortunately, we only had time to test the bioreporter with AIP inducement.

For the testing we wanted to know if different concentrations of AIP's would affect the GFP signal, or if it would be
the same as long as the threshold of inducement had been achieved. The exact amount of AIP molecules needed to activate
the P2 promoter has not been measured as far as we know in literature. However, we had done some estimations of the
needed concentration for our modelling work, and for this we chose to see if high concentration would yield high
intensity. We ordered the AIP's directly from Genescript as purified protein. The AIP's were dissolvable in DMSO,
however, we ran into some trouble of not all protein dissolving in the DMSO when we were preparing the dilutions. Due to
this we had only some type of estimates of the concentrations but renamed them to high and low concentrations of the
AIP's. The lower concentration should be somewhere around 250nM, however, this is unfortunately not tested due to lack
of equipment available. The amount of each tested AIP's can be seen below in table 1.

Table 1. Amount of AIP for bioreporter inducement| High concentrated | Low concentrated |
| 5 µL | 50 µL |
| 2 µL | 20 µL |
| 1 µL | 10 µL |
|  | 5 µL |
|  | 2 µL |

The cells were grown up to 1.2 OD600, we added 5 µL of culture to 200 µL of LB media, which equals 4.8\*106 cells in the
beginning. After that we induced 3 wells with each concentration and measured the OD600 and GFP intensity at both 470
and 511nm wavelength. The measurements were done with the BioTek Synergy H1 Plate Reader, and the measurements were
taken for 10 hour every 20 min.

As blank we had LB media with kanamycin to avoid contamination growth, and as negative control, we had the same
expression cells as for the bioreporter, but these ones included the GFP DARPin expression cassette. The set up on our
96 well plate can be seen in table 2.

Table 2. 96 well plate set up for GFP and OD600 measuring
![Table showing 96 well plate.](https://static.igem.wiki/teams/4159/wiki/figures/exp-tab2.png)

The results of the growth and fluorescence can be seen below in table 3. All wells started nicely from the same OD600
which was the aim. And the blanks where there are no cells showed low to almost no growth. The small OD600 can be due to
some contamination during the pipetting work or just the reader calibration. It is also good that the growth is the same
for both our bioreporter cells and for the negative control cells, as they were the same strain. And based on these
results, the transformation of the bioreporter into the cells shows no defect in the growth. A closer look at the growth
and fluorescence signal overtime can be seen in graph 1, for one of the wells with our bioreporter cells which was
induced with 50uL of AIP's of the lower concentration. The GFP intensity can be seen in graph 2. It is quite clear that
the concentration of AIP's did not change the GFP output signal. It was also seen that the bioreporter cells which were
not induced with AIP's still showed also some GFP intensity.

Table 3. Synergy reads of max OD600 and GFP fluorescence intensity over 10 hours
![Table showing fluorescence intensity.](https://static.igem.wiki/teams/4159/wiki/figures/res-tab3.png)

![Curve indicationg growth of bioreporter.](https://static.igem.wiki/teams/4159/wiki/figures/res-graph1.png)

Graph 1. Growth of bioreporter cell (F3) over 10 hours

![Boxplots of average GFP signal.](https://static.igem.wiki/teams/4159/wiki/figures/res-graph2.png)

Graph 2. GFP signal intensity over 10 hours

We can conclude that there should still be some changes done to the setup of the promoters in the bioreporter. It seems
that the P2 promoter might be active even if it hasn't been induced, or then the strong constitutive pBAD promoter has
something to do with the expression of GFP. However, it seems not likely as it is about 70 bp downstream from the GFP
encoding gene, and the pBAD promoter is inserted to start transcription in the opposite direction. Future testing of
inserting just the P2 promoter in the plasmid prior to the GFP gene would be needed to figure out what might be the
cause of the false GFP signal.

[Read more about it!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

## Future Tests

Possible future tests definitely involve the fine-tuning of our bioreporter in order to ensure the level of AIP that is
required for our bioreporter to respond. Furthermore, the bioreporter should be used in combination with the best
performing DARPin to ensure that the DARPin blocks the fluorescent signal. This last test could not be performed due to
time limitation, but due to the results from the collaboration with TU Dresden we already have potential testable DARPin
concentrations.

Additionally, potential high affinity DARPins are now known to bind to AIP. However, the library can always be improved.
The ribosome display could be repeated by altering the Tween concentrations to make the selection more harsh and find
the best affinity binder out of the ones we isolated from the first round of ribosome display. In addition to repeating
ribosome display we can specifically model the wet-lab results to retrieve results about the binding *in silico*.

In addition to testing the final DARPin with the bioreporter and analyse it *in silico*, we should also test its behaviour
on an actual *S. epidermidis* biofilm. For this we plan to grow an *S. epidermidis* biofilm and measure its biofilm biomass
with crystal violet. We assume that the biofilm biomass might decrease with the addition of the DARPin, because more
bacteria would stay planktonic (Cruz et al., 2018). We could also analyse the biofilm build-up and in which way it
changes when the DARPin is added. This could be done via FISH (Fluorescence *in situ* hybridization). FISH has previously
been proposed as a tool to diagnose biofilms with and it would be interesting to know in which way the phenotype of the
biofilm changes with the addition of DARPins as well as quantify the biofilm in an exact measurement (Moter and Göbel,
2000).
After the DARPin has been analysed for it's functionality it would be beneficial to analyse it's stability on different
surfaces most importantly skin. Therefore, it would be ideal to move on to *in vivo* experiments with animal models or
utilise organoids.

## References

1. Cruz, C.D., Shah, S. and Tammela, P. (2018)

   Defining conditions for biofilm inhibition and eradication assays for Gram-positive clinical reference strains

   *BMC microbiology, 18(1), pp.1-9*

   [Google Scholar](https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=Cruz%2C+C.D.%2C+Shah%2C+S.+and+Tammela%2C+P.%2C+2018.+Defining+conditions+for+biofilm+inhibition+and+eradication+assays+for+Gram-positive+clinical+reference+strains.&btnG=)
2. Moter, A. and Göbel, U.B. (2000)

   Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms

   *Journal of microbiological methods, 41(2), pp.85-112*

   [Google Scholar](https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=Moter%2C+A.+and+G%C3%B6bel%2C+U.B.%2C+2000.+Fluorescence+in+situ+hybridization+%28FISH%29+for+direct+visualization+of+microorganisms&btnG=)

Achievements

GFP & DARPin

Structure Prediction

Diffusion Modelling

Ribosome Display

DARPin Synthesis

Bioreporter

Future

* [Achievements](#achiev)
* [GFP & DARPin](#prod)
* [Structure Prediction](#pred)
* [Diffusion Modelling](#diff)
* [Ribosome Display](#rib)
* [DARPin Synthesis](#syn)
* [Bioreporter](#bio)
* [Future](#fut)

{% endblock %}
{% block script\_content %}

{% endblock %}