iGEM 2022 — Team toulouse-insa-ups

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## Notebook

January
February
March
April
May
June
July
August
September
October

Week 50
Week 51
Week 52

Week 1
Week 2
Week 3
Week 4

Week 5
Week 6
Week 7
Week 8

Week 9
Week 10
Week 11
Week 12
Week 13

Week 14
Week 15
Week 16
Week 17

Week 18
Week 19
Week 20
Week 21

Week 22
Week 23
Week 24
Week 25
Week 26

Week 27
Week 28
Week 29
Week 30

Week 31
Week 32
Week 33
Week 34

Week 35
Week 36
Week 37
Week 38

Week 40
Week 41

## Week of January 3rd to 9th

On 07/01/2022 was our first team meeting. The advisors and the students of the 2021 edition met the student team of the 2022 edition. The competition was presented and each student and advisor was designated to be responsible for an aspect of the project: Communication (Charline), Wiki (Morgane), Experimentation (Guillaume), Entrepreneurship (Samy), Human Practices (Juliette), Modelisation (Raphaël), Finances (Thomas) and Organization (Laure).

![](https://static.igem.wiki/teams/4197/wiki/notebook/image2.jpg)

During the week, the team brainstormed to find as many project ideas as possible to discuss during the next meeting.

## Week of January 10th to 16th

This week we continued to brainstorm on the many ideas we had. Each student worked on 2 to 4 different projects. Of all our ideas, here are the one we have retained at this time:

* - Sound and bacterias, can microorganisms hear?
* - Production of antivenom
* - Treat a disease of cassava by decreasing the viral load of an insect
* - Create a biofilter for used water
* - Looking for the weaknesses of slime mold
* - Treatment of melon downy mildew
* - Production of a microbial deodorant
* - Treatment of sclerosis
* - Production of fabric with microbial cellulose
* - Valorization of the byproducts of wine fermentation
* - Detection and treatment of allergies

## Week of January 17th to 23th

Morgane, Charline and Juliette attended the first meeting about the Exposcience Occitanie 2022 event. On 16/01, Thomas flew to Thailand for 4 months to study for an exchange but he kept working with us from there. On 19/01/2022, the very first online meeting took place with students only. This week, we brainstormed on how to use aptamers to detect IgE antibodies. We also talked about using a biofilm as a cream for desensitization. On January the 14th, Raphaël came back from his Erasmus in Barcelona.

## Week of January 24th to 30th

The brainstorming continued. This week, only 4 project ideas were remaining:

* - Valorization of CO2 in wine fermentation
* - Detection of odors with bacterias
* - Creating microorganisms that can hear
* - Detecting and treating allergies.

This week, on January the 20th, Laure came back from her Erasmus semester in Athens.

## Week of January 31th to February 6th

Brainstorming continued. During the weekly meeting, we talked about using DARPin in our project but we did not know yet for which purpose.

## Week of February 07th to 13th

Brainstorming continued. During the weekly meeting, we had the idea of organizing an event at INSA as a mini-Jamboree with all the French iGEM teams and in partnership with the school EUR Bioeco.
We discovered the basic principles of HTML with Younes, a former iGEMer who kindly took the time to explain how the wiki works.

## Week of February 14th to 20th

We participated in the school opening day to present the iGEM competition and the results of the INSA-UPS 2021 iGEM team to secondary school’s students with Margaux Haon, a member of the 2021’s team.

We organized our first team building evening in real life around a raclette cheese.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-7.png)

## Week of February 21th to 27th

During a brainstorming session, we talked about converting the CO2 of wine fermentation into methanol by electrolysis. We also started to discuss detecting allergies by agglutination of two microorganisms around IgE molecules. Even with a lot of research, we had trouble finding a promising idea for the two other projects (Detect odors with bacterias/Make microorganisms hear).

## Week of February 28th to March 6th

We had the first meeting with Luana, Saber and Lucie, three doctoral students who helped us for the rest of the project.

We decided during the weekly meeting to focus only on two ideas: valorization of CO2 from wine fermentation and detection and treatment of allergies.

## Week of March 07th to 13th

This week we focused on detecting all the challenges and difficulties we could encounter with each project (CO2 valorization or allergy detection and treatment). It seemed there were more difficulties with CO2 valorization so we tried to get more help from experts during the next week.

## Week of March 14th to 20th

We met several professionals to discuss our ideas for the competition and ask questions about our projects. Two specialists in electrochemistry explained to us how difficult it is to use electrochemistry to convert CO2 in methanol. On the other hand, Chloé Beuraud, a researcher in immunology, seemed very enthusiastic about our allergy project idea. That is why, among other reasons, we chose **allergy detection and treatment** as our official project for 2022 edition of iGEM with a vote.

## Week of March 21th to 27th

Now that we have chosen our project, each student met their referent instructor to talk more precisely about their responsibility in the project (modeling, communication, HP, entrepreneurship, experimentations, wiki).

## Week of March 28th to 03rd

We learnt that La Fondation Catalyse granted us 10, 000€ to sponsor our project. This represents a quarter of our whole estimated budget so it was very helpful to get started and register our iGEM team.
We found a name for our project: DAISY for DARPin-Allergen-IgE Screening for immunotherapY. We also did two logo drafts.
![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-13.png)

## Week of April 04th to 10th

This week, we began the design of the project. The first step was to determine which allergens we would produce and to imagine which biobricks would be needed.

## Week of April 11th to 17th

On April the 14th, we met Dr. Gabrielle Potocki-Veronese (Research Director at TBI) to talk about how to use microdroplets to screen hundreds of allergens for IgE detection.
We met members of the EUR Bioeco school to present our idea of organizing a French Meet-Up as a mini-Jamboree with them. They liked the idea and agreed to collaborate with us and to sponsor the event.

![](https://static.igem.wiki/teams/4197/wiki/homepage/20220414-103033-1.jpg)

## Week of April 18th to 24th

On this week, we registered the team for the competition and paid the fee so the adventure officially begun! Now we knew which experiments would have to be conducted for the project during the summer, we had to design the main biobricks to be created and started to design them on Benchling. As we will do our experiments in INSA’s laboratory this summer, Samy and Guillaume, who are UPS students, came to visit the lab.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-16.png)

## Week of April 25th to May 01st

Among several logo ideas, we voted for the one that will represent us during all the season.
The design continued, we brainstormed to define our experiments and the needed DNA sequences and started a list of all we needed to order for the summer internship.

![](https://static.igem.wiki/teams/4197/wiki/notebook/logo-final-sans-fond-1.png)

## Week of May 02nd to 08th

This week, we imagined the first design of our mascot Daisy, a cheerleader dressed as an IgE and wearing our colors.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-18.png)

We met a lot of experts to talk about our project and try to detect issues as soon as possible to have the time to find some solution before the beginning of the experiments. We also ordered our sequences on IDT so that we could receive them at the beginning of the internship.

## Week of May 09th to 15th

This week we met Dr. Alain Liné who works on aggregation models and brainstormed on the model we could make and how to adapt the general model to our aggregation system. We went to the lab to check what material was left from the previous year, what we could use for our project and what we would miss if we did not order it.

## Week of May 16th to 22th

We met members of the EUR bioeco school, our sponsor for the meetup to organize the event and prepare the communication around it. We also met a few days later to continue the preparation and imagine an first schedule for the three-day meetup. As our exams are in a few days we haven't worked as much as the previous weeks on the design.

## Week of May 23th to 29th

On Tuesday, Thomas came back to Toulouse from his exchange semester in Thailand so he was able to begin the experiment the next week. On the weekend, Juliette and Charline built models of our bacterias displaying allergens or DARPin. They were very useful for presentation videos and to explain our project playfully to the public during science popularization events. We were passing our respective exams and could not wait to begin the experiments.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-21.png)

## Week of May 30th to June 05th

We began our internship! During the next four months we will work on iGEM projects all day in the lab. It was a very intense week. We received a lot of material, conducted a few training experiments and worked hard on HP, modeling draft, sponsor research, meet-up organization and communication.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-22.png)

## Week of June 06th to 12th

On the weekend, we attended Exposcience, an event to popularize science to children. It was a great occasion to talk about microbiology, synthetic biology, iGEM and our project Daisy but also to discover a lot of scientific projects conducted by children from 8 to 18 years old in France, Germany and Luxembourg.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-23.png)

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-23.jpg)

On Monday we received our sequences from IDT! So we began the PCR for sequences and plasmid so that we could clone them in our bacterias the next week, hopefully.

Here is a scheme of the evolution of the experiments. Each week, another scheme will be presented to track our progress. We first started with a biomolecular experiment to construct our plasmid and then we performed biochemistry and aggregation experiments. The steps in blue are not finished yet while the green ones are.

## Week of June 13th to 19th

On Monday we met Dr Gabrielle Potocki-Veronese to talk again about microfluidics for our project. She explained to us in detail the patented technology she worked on and we discussed how to use it to screen aggregate. If we have good results in August, we will use their microfluidic device in September.

This week we received the sequencing result of our first construction and we can affirm we successfully constructed a pET21 plasmid with OmpA\_DARPin\_sfGFP insert.

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-24.jpg)

## Week of June 20th to 26th

On Monday, we transformed *E. coli* Tuner (DE3), our strain for expression, for the first time!

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-25.jpg)

## Week of June 27th to July 03st

On the 1st to 3rd of July, was the European Meet-Up in Hambourg. We took a plane to Frankfurt but never managed to reach Hamburg because our flight was canceled. After 48h traveling, we came back to Toulouse tired and frustrated because we could not attend the meetup. However, we sent our poster some days ago and earnt the Best Poster Prize!

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-26.png)

We had our first fluo results! It means we successfully expressed the OmpA-DARPin-sfGFP fusion protein. We will have to check if it is on the membrane. We started Interlab experiments! The first cloning results were unsuccessful but we will try again next week.

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-26.jpg)

## Week of July 04th to 10th

The 7-8-9 of July was the French Meet-Up on our campus. Thanks to Charline, Juliette and our sponsor EUR Bioeco, all went well. It was amazing to discover all the french projects and exchange on our experience through the iGEM adventure! We did less experiments this week to make the most of the Meet-Up.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-27.png)

We designed the primer to suppress sfGFP and added fluorescent genes to our plasmids. We are on the way for our final constructions. After discussing with our advisor Alain Liné, it seemed our aggregation model will be harder to create than predicted. We will be the first to modelize the aggregation of two different bacteria so it was a big challenge.

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-27.jpg)

## Week of July 11th to 17th

For the first time, we ran our aggregation model this week. We also had the proof that our protein of interest is exported on the membrane thanks to the Ompa-DARPin-sfGFP construction.

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-28.jpg)

We started filming our presentation video. Here is a vue of the back stage with Thomas explaining how our bacteria will aggregate.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-28.png)

## Week of July 18th to 24th

We made a photoshoot for our wiki and social media but we were not always very focused.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-29.png)

We finally constructed OmpA-DARPin and OmpA-arah2 plasmids! In the next week we will use them for aggregation experiments!

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-29.jpg)

## Week of July 25th to 31st

On this week, we performed our first agregation experiment! However, the results do not allow us to conclude on the presence of specific aggregates. We also successfully constructed the OmpA-Derp1 sequence and checked that it did not kill the cell.

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-30.jpg)

## Week of August 01st to 07th

We started the filming of the popular science series named “Cracking Allergies”. The aim of these 10 videos is to provide content about allergies, to understand them and live with them. On this occasion, our HP leader Juliette created a new model in polystyrene.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-31.png)

We continued our experiment to check the exportation of our protein of interest, tried to aggregate our strains of *E. coli* and tried to insert a gene for fluorescence in the plasmid we previously created. However, we had a lot of difficulties and did not obtain successful results.

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-31.jpg)

This week, we encountered the iGEM Thessaloniki team from Greece. We discussed how we could helped each other in our projects and to collaborate until the Giant Jamboree.

## Week of August 08th to 14th

We found out the DARPin sequence we have been working on for 3 months was not optimal! This gene was essential for our project so we will have to correct all our plasmid. This will probably make us fall behind schedule but it likely explains why we had poor results the past weeks.

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-32.jpg)

We started the operation "Saving Daisy!".

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-32.png)

As we wait for the delivery of our new sequences, we work hard on Modeling, Entrepreneurship, and Human Practices aspects of the project and the wiki.

A journalist and a cameraman came at the lab to make a TV report about our project and team.

## Week of August 15th to 21th

The writing work has started for the wiki with the Description, Collaboration, Attribution and Team pages. At the same time, the strategy to synthesize the missing sequence of the DARPin was designed and primers were ordered. We worked all summer in the school’s lab and as the classes will start next week, we made some space in the classrooms to let the laboratory staff prepare the first school day.

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-33.jpg)

## Week of August 22th to 28th

We observed blue fluorescence of our strains expressing BFP, so we now know the gene is correctly expressed and that the promoter ihfB800 is working. A journalist from “20 minutes”, a French newspapers, spent time in our lab for an interview and took photos of our lab. Our PI have chosen the speakers for the presentation. Laure, Charline, Juliette and Thomas will present the project at the Jamboree. Juliette has finished making the majority of the video of the serie Cracking Allergies, and Charline sent our 2 min video. The DARPin was rebuilt and showed good results on PCR screening. This is the last week of experiments for Samy and Guillaume, then they will go back to university.

![](https://static.igem.wiki/teams/4197/wiki/notebook/manip-34.jpg)

## Week of August 29th to September 04th

To finish experiments, there was only two weeks left and we were reduced to 5 now in lab, so we decided to change the lab organization:

* - Raphaël and Thomas would make surface controls of bacteria
* - Laure would make with Sophie Lajus aggregation on droplets
* - Charline was responsible to insert the mScarlet and conduct assays on FACS
* - Juliette would make last aggregation assays with news parameters.

A special attention was given to the wiki redaction, the wiki freeze being in one month and half. Thomas has paid the inscription to the Jamboree.

## Week of September 05th to 11th

This week, we worked hard to finish our experiments. Unfortunately, FACS and droplets did not give significant results, there was still no aggregation or there was aggregation on negative controls.
We organized a trip to the Atlantic Ocean to relax before working hard for the final run toward the Wikifreeze in the following weeks. We slept and ate a lot, the rest of the time we were relaxing on the beach and playing with waves. We had a good time even though our car’s battery broke down and we came back with a suspicion of COVID.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-36.png)

## Week of September 12th to 18th

Here is the last week for lab work, and this week we stayed in the lab later than usual to try to finish the draft of the majority of the wiki redaction. The first fake wiki freeze was going to take place at the end of the week so we spent a full Saturday rushing to write the wiki at Laure’s family home and without Juliette, because she was sick.

![](https://static.igem.wiki/teams/4197/wiki/notebook/semaine-37.png)

## Week of September 19th to 25th

For all students class has started, so it became harder to meet all the deadlines but we still managed it so far. Laure, Thomas and Charline went to a high school to explain synthetic biology to the students, talk about the biologies in superior studies and run and experience at the high school laboratory. On the weekend, we met again to work together on the wiki. We started to add most of the content on Gitlab.

## Week of September 26th to 02nd

This was a stressful week! The wiki freeze is very close now and we want to give our best to have the perfect website. We also worked hard to complete all our parts in the registry. We gathered almost everyday this week to work together and stay motivated but the most efficient day was on saturday when we almost finished all the pages.

## Week of October 03rd to 09nd

This was a stressful week! The wiki freeze is very close now and we want to give our best to have the perfect website. We also worked hard to complete all our parts in the registry. We gathered almost everyday this week to work together and stay motivated but the most efficient day was on saturday, the day we called a fake wiki freeze when we almost finished all the pages.

![](https://static.igem.wiki/teams/4197/wiki/notebook/wiki-freeze-3.png)

## Week of October 10th to 16th

We checked one last time every detail of the wiki and the registry to be ready for the wiki freeze and started to prepare the final presentation.
Thank you for reading the entire Notebook. As a reward, here are two memes from Raphaël.

![](https://static.igem.wiki/teams/4197/wiki/notebook/meme1.png)

![](https://static.igem.wiki/teams/4197/wiki/notebook/meme2.png)

[![](https://static.igem.wiki/teams/4197/wiki/fleches/previous.png)](https://2022.igem.wiki/toulouse-insa-ups/engineering)

[![](https://static.igem.wiki/teams/4197/wiki/boutton-up.png)](#top)

[![](https://static.igem.wiki/teams/4197/wiki/fleches/next.png)](https://2022.igem.wiki/toulouse-insa-ups/implementation)

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![Menu aside](https://static.igem.wiki/teams/4197/wiki/description/sort-descending.svg)

* Expression of the DARPin protein in strains D
  + Build: DARPin cloning
  + Test: First aggregation assay
  + Learn: Structural study of the strain D construction
  + Design: DARPin reconstruction
  + Build: Corrected version of the DARPin
  + Test: New aggregation assays
  + Learn: Results of new aggregation assays
  + Design : Integrating modeling and membrane proteins quantification design to improve our idea

* Red fluorescence with mScarlet-I in strains A
  + Design: Choice of fluorescent protein
  + Build: mRFP1 cloning
  + Test: Microscope observation of fluorescence
  + Learn: Conclusions on protein expression and possible cause of the problem
  + Design: Replacement of mRFP1 by mScarlet-I fluorescent protein
  + Build: mScarlet-I cloning
  + Test: Macroscopic observation of fluorescence
  + Learn : Conclusion on protein expression

* Overall conclusions

## Engineering Success

### Overview

The engineering process is never a linear process. In the case of synthetic biology, bench experiments do not always work as planned, despite all the attention we would have paid to their design. In order to question ourselves and solve the problems on what would not have worked as expected, we would have to follow the DBTL cycle (Design, Build, Test, Learn) to create a successful biological device. This has happened to us a few times, of which the most significant example follows.

![Figure 1](https://static.igem.wiki/teams/4197/wiki/engineering-success/fig-1.png "Figure 1")***Figure 1: Iterative design-build-test-learn (DBTL) cycle in Synthetic Biology (Waldby et al. 2018).***

Expression of the DARPin protein in strains D

## Build: DARPin cloning

Two elements are required to detect the IgE responsible for an allergy: strain A displaying the required allergen and strain D displaying the DARPin, a synthetic protein able to bind to the constant part of IgE. If the patient’s blood contains IgE against the tested allergen, the strains aggregate as a means for detection. The first step consisted in building a strain displaying a fusion between OmpA, the DARPin, and a fluorescent reporter (sfGFP)(Fig.2). We wanted to verify the expression and check the subcellular localization of the fusion, expecting the DARPin and sfGFP parts to be displayed on the outer surface of the bacterium.

![Figure 2](https://static.igem.wiki/teams/4197/wiki/engineering-success/fig-2.png "Figure 2")***Figure 2: DARPin-sfGFP construction.** The sfGFP served as a marker to prove the correct expression of the protein and to study its subcellular localization.*

The OmpA-TEV-DARPin-sfGFP-6xHis-tag encoding gene was successfully assembled in a plasmid under the control of the strong inducible pT7 promoter. The plasmid was then transformed into E. coli Tuner (DE3) to identify suitable conditions for expression. Despite high cytosolic concentrations of the OmpA-TEV-DARPin-sfGFP-6xHis-tag fusion, we could confirm the presence of our chimeric protein in the outer membrane. The sfGFP encoding part was then removed from the plasmid by PCR to use the OmpA-TEV-DARPin for IgE detection, creating strain D.

## Test: First aggregation assay

We conducted aggregation assays after constructing the required strains A and D. The idea was to mix strains A and D with a recombinant IgE against the allergen displayed by strains A. Our experiments to check aggregation were designed to test different conditions: the concentration of IgE, the agitation and the incubation temperature. One condition initially showed aggregation in presence of IgE, but we did not manage to repeat the results multiple times independently. In the end, we concluded that there was no difference between the sample containing all the elements required for aggregation (strains A, D and IgE) compared to the negative controls (e.g. strains A and D without any IgE). Furthermore, other results obtained showed no difference between our assays and the negative control. These results led us to assess the correct folding of the construction.

## Learn: Structural study of the strain D construction

After our first aggregation attempts failed, we wondered whether strain D behaved as initially thought during the design part. More precisely, we wanted to investigate whether the OmpA-TEV-DARPin(-sfGFP-6xHis-tag) fusion would fold properly to display the DARPin without altering its secondary structure. An AlphaFold 2 simulation was done to build a structural model of the OmpA-TEV-DARPin-sfGFP-6xHis-tag fusion (Figure 3).

![Figure 3](https://static.igem.wiki/teams/4197/wiki/engineering-success/fig-3.png "Figure 3")***Figure 3: AlphaFold 2 prediction of the OmpA-TEV-DARPin-sfGFP-6xHis-tag structure.**The construction prediction was recolored with PyMol. The OmpA is shown in orange, in yellow the DARPin, and in green the sfGFP construction.*

At first, the complex looked correctly folded. We also checked a previously published DARPin structure obtained by crystallography for comparison (Figure 4).

![Figure 4](https://static.igem.wiki/teams/4197/wiki/engineering-success/fig-4.png "Figure 4")***Figure 4: DARPin structure extracted from the crystal structure 4GRG (Kim et al. 2012).**The extraction of the DARPin structure was made on PyMol.*

We noticed that the AlphaFold 2 model displayed 6 α-helices in the DARPin while the crystallographic structure presented 8 α-helices. This was confirmed by the analysis of another X-ray crystallographic structure of DARPin (PDB: 7MXI). We immediately checked the amino sequence of the DARPin we used on PDB (https://www.uniprot.org/uniprotkb/L7MTK7/history). We realized that the sequence was incomplete and inappropriately referenced so our genetic construction was possibly wrong. This experience made us realize that the sequences published on databases are not always correct and that we have to double-check every piece of information we use for our project. We replaced the partial DARPin sequence with the full one in our OmpA-TEV-DARPin-sfGFP-6xHis-tag fusion and rebuilt a structural model with AlphaFold 2 (Figure 5).

![Figure 5](https://static.igem.wiki/teams/4197/wiki/engineering-success/fig-5.png "Figure 5")***Figure 5: Reconstruction of the OmpA-TEV-DARPin-sfGFP-6xHis-tag fusion protein with AlphaFold 2.** The construction prediction was recolored with PyMol. The OmpA is shown in orange, in yellow the previous DARPin with in clear green the correction of the DARPin sequence, and in green the sfGFP construction.*

The AlphaFold 2 model of the corrected construct showed the expected structure and a proper folding of each element, including the DARPin with its 8 α-helices. We therefore decided to build a corrected version of the plasmid, with the full-length DARPin, before trying again the aggregation assays.

## Design: DARPin reconstruction

To close the DBTL loop, we had to design a strategy for rebuilding the DARPin. Due to strong time constraints, we chose to use a Polymerase Chain Assembly (PCA) strategy to add the missing sequence. This consists in using three 60 bp primers (1\_PCA60, 2\_PCA60 and 3\_PCA60) to build the missing sequence into two steps. First, 1\_PCA60 and 2\_PCA60 have to be assembled and elongated by PCR. Then, the hybridization and the elongation of 3\_PCA60 should enable us to obtain the complete missing sequence (Figure 6).

![Figure 1: gal d 2 fragment](https://static.igem.wiki/teams/4197/wiki/engineering-success/fig-6.png "Figure 1: gal d 2 fragment ")***Figure 6: Principle of PCA to build the missing part of the DARPin.** Three primers 1\_PCA60, 2\_PCA60 and 3\_PCA60 were used to build the complete missing sequence. These three primers were mixed on the same PCR mix, after a first elongation with 1\_PCA60 and 2\_PCA60, 3\_PCA60 binds with the 2\_PCA60 reverse to end up the elongation.*

## Build: Corrected version of the DARPin

The missing part of the gene coding for DARPin was amplified by PCA and inserted at the same time on the OmpA-TEV-DARPin-sfGFP-6xHis-tag and OmpA-TEV-DARPin-6xHis-tag plasmids by In-Fusion. Using the corrected and right plasmids, we were able to repeat aggregation assays less than one week after we realized the initial design flaw.

## Test: New aggregation assays

We followed the same protocol as previously described to conduct new aggregation experiments, with the complete sequence of the DARPin this time. Again, different conditions were tested: the IgE concentration, the temperature of incubation and the temperature. Sadly, we could not observe any aggregation in any of the tested conditions.

## Learn: Results of new aggregation assays

The aggregation tests did not yield any result corresponding to our expectations but brought a lot of relevant information for further troubleshooting. First, we performed better aggregation experiments allowing us to reduce non-specific aggregation with a cleaner negative control sample, and to have a good dispersion of bacteria (Figures 7 and 8). We acquired some “know-how” about this protocol.

![Figure 7](https://static.igem.wiki/teams/4197/wiki/engineering-success/fig-7.png "Figure 7")***Figure 7: aggregation assay before correction of the DARPin with strains A and D mixed with different concentrations of anti-Ara h 2 IgE: 1:100 (A), 1:250 (B), and 1:1000 (C).** This experiment was conducted before the DARPin analysis by AlphaFold 2 and sequence correction.*

![Figure 8](https://static.igem.wiki/teams/4197/wiki/engineering-success/fig-8.png "Figure 8")***Figure 8: aggregation assay with the DARPin corrected with strains A and D mixed with different concentrations of anti-Ara h 2 IgE: 1:100 (A), 1:250 (B), and 1:1000 (C). The same mix with strains A and D without IgE was used as negative control (D).** The complete DARPin on the strain D was used.*

The difference between observations shows the improvement of our aggregation assay design and a better experience of manipulation in the wet lab. This demonstrates that our team can design, build, test, and improve by learning from our errors.

## Design : Integrating modeling and surface controls design to improve our idea

Despite the correction of the DARPin DNA sequence, the aggregation experiments did not work as expected. We had to question ourselves one more time to try to understand what could impede the aggregation of the bacteria. In parallel to these experiments, our modeling efforts allowed us to formulate additional hypotheses. The complexation model showed that the ideal cell concentrations and number of proteins exposed on the surface depends intimately on the affinity of the IgE for the corresponding allergen. The complexation model can now be used to guide further experimental designs as shown on Figure 9. Modeling has brought us a great wealth of information to improve aggregation assays by directing the set-up.

![Figure 9](https://static.igem.wiki/teams/4197/wiki/model/fig-5a-sans-a.png "Figure 9")***Figure 9: Evolution of the aggregation potential for different initial concentrations of A and D free molecules, for the allergen Ara h 2 (KD=1\*10-11 M) tested during aggregation assays for 100 000 displays of allergen and DARPin on each strain.** The A and D molecule concentrations range from 1e-14 M to 1e-7 M. Knowing the number of DARPin and allergens molecules displayed on the surface of each strain, concentrations of each strain can be estimated for aggregation assays.*

Here the complexation model indicates the concentration of DARPin and allergens molecules necessary for aggregation. So to obtain the concentration of strains A and D, the number of DARPin and Allergen molecules displayed on the surface of each strain are needed. That is why membrane proteins quantification experiments have to be performed. These experiments involve checking the presence of DARPin or allergens on the membrane and measuring its concentration by protein extraction. First experiments have pointed out the presence of our OmpA-TEV-DARPin-sfGFP-6xHis-tag fusion protein on both the membrane and the cytoplasm. This result is hopeful and allows to deepen the design to show if the DARPin is correctly displayed on the exterior of the bacterial membrane. Then this strategy can be applied to our allergens strains. Due to the lack of time, we could not continue further with the DBTL cycle despite some promising new ideas for improvement. News tools such as modeling, expression regulation, and the study of proteins on the membrane, will bring together more relevant information to take our project to the proof of functionality.

Red fluorescence with mScarlet-I in strains A

## Design: choice of fluorescent protein

We needed for our FACS experiments to express fluorescence in our strains A
and D. Manon Barthe from TWB has provided the mTagBFP for showing blue
fluorescence for strain D, and mRFP1 showing red fluorescence for strain A
(Figure 10). The sequence coding for both reporters was under the control of
the same constitutive ihfB800 promoter.
While the blue fluorescence of the mTagBFP was successfully shown with only one DBTL loop, the strain with the red
fluorescence has required two loops as explained hereafter.

![Figure 10](https://static.igem.wiki/teams/4197/wiki/engineering-success/arah2rfp.png "Figure 10")***Figure 10: allergen construction with mScarlet-I, example for Ara h 2 allergen.** Promoter used for the fluorescent protein is the constitutive ihfB800.*

## Build: mRFP cloning

The gene coding for mRFP1 was amplified by PCR and inserted at the same
time on three plasmids (OmpA-TEV-Gal d 2-6xHis-tag, OmpA-TEV-Ara h
2-6xHis-tag, and OmpA-TEV-Ana o 3-6xHis-tag) by In-Fusion. Digestions by
restriction enzymes confirmed the correct addition of the gene to the three
plasmids (Figure 11). They were finally transformed into E. coli  Tuner (DE3)
cells to form three different strains A, each with a different allergen.

![Figure 11](https://static.igem.wiki/teams/4197/wiki/engineering-success/digestion-rfp.png "Figure 11")
***Figure 11: digestion by NotI of Ara h 2 with mRFP1 insertion (A), of Ana o 3 with mRFP1 insertion(B) and of Gal d 2 with mRFP1 insertion (C).** The digestion product was checked with 0.6% agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel). Colonies 1, 2 and 3 present the correct size for Ara h 2; colonies 21 and 23 present the correct size for Ana o 3; colonies 7 and 13 present the correct size for Gal d 2.*

## Test: Microscope observation of fluorescence

Expression of mRFP1 was assessed on an epifluorescence microscope. There
was no fluorescence in either of the strains nor in the negative control (data
not shown). In parallel, fluorescence of mTagBFP was successfully observed in
strain D. The original plasmid containing the mRFP1 and our plasmids with the
mRFP1 were sent to sequencing.

## Learn: Conclusions on protein expression and possible cause of the problem

As no fluorescence of mRFP1 was detected, the protein was certainly not
expressed in strains A. As fluorescence of mTagBFP in strain D was observed,
the ihfB800 promoter used for both protein expressions was surely not involved
in the failure of mRFP1 expression. Moreover, sequencing of the mRFP1 gene
showed some mutations, so we hypothesized that the initial mRFP1 sequence
could be incorrect. We therefore decided to choose another fluorescent protein
for our strains A.

## Design: Replacement of mRFP1 by mScarlet-I fluorescent protein

We contacted Maxence Holtz (former iGEM Toulouse member and intern at
Toulouse White Biotechnology). He advised us to use his mScarlet-I protein, as
it was red like the mRFP1 and was supposed to be expressed correctly in E. coli  cells.
We then decided to switch the mRFP1 gene for the mScarlet-I gene,
while keeping in place the constitutive ihfB800 promoter since it was
efficient for the mTagBFP. The final construction for Ara h 2 is shown on
Figure 12.

![Figure 12](https://static.igem.wiki/teams/4197/wiki/engineering-success/mscarl.png "Figure 12")***Figure 12: allergen construction with mScarlet-I, example for Ara h 2 allergen.**  Promoter used for the fluorescent protein is the constitutive ihfB800.*

## Build: mScarlet-I cloning

The gene coding for mScarlet-I was amplified by PCR and inserted instead of
mRFP1 in the plasmids containing OmpA-TEV-Gal d 2-6xHis-tag, OmpA-TEV-Ara h
2-6xHis-tag, and OmpA-TEV-Ana o 3-6xHis-tag by In-Fusion, still under the
ihfB800 promoter control. Digestions by restriction enzymes confirmed the
correct addition of the gene for the three plasmids. They were first
transformed into Stellar competent cells in order to amplify them. They were
finally transformed into E. coli Tuner (DE3) cells to form three different
strains A, each with a different allergen.

## Test: macroscopic observation of fluorescence

As shown on Figure 13, red fluorescence was observed with the naked eye as
soon as Stellar cells were plated on Petri dishes, giving a strong first idea
of the correct expression of the mScarlet-I.

![Figure 13](https://static.igem.wiki/teams/4197/wiki/engineering-success/boiterose.jpg "Figure 13")***Figure 13: Second plating of pET-21 b (+)\_Ara h 2\_OmpA\_mScarlet-I transformed cells and pET-21 b (+)\_Ana o 3\_OmpA\_mScarlet-I
transformed Stellar cells.** The colonies were selected on LB-ampicillin plates. Pink coloring indicates the expression
of the mScarlet-I.*

After transformation into E. coli  Tuner cells, expression of mScarlet-I
was also assessed on an epifluorescence microscope. Vivid red fluorescence
was observed while the negative control was not fluorescent at all (see
Figure 14 for the strain A displaying Ana o 3).

![Figure 14](https://static.igem.wiki/teams/4197/wiki/engineering-success/mscarlmicro.png "Figure 14")***Figure 14: fluorescence of pET-21 b (+)\_Ana o 3\_OmpA\_mScarlet-I transformed Tuner cells.**  Cells transformed with
plasmids not containing mScarlet-I were used as negative controls. Observations were made at the microscope parameters for
mScarlet-I.*

## Learn: Conclusion on protein expression

As fluorescence was observed the mScarlet-I protein was expressed
successfully in our three strains A. It confirmed that the ihfB800 promoter
was indeed functional and that the mRFP1 was surely defective. Having strains
with functional fluorescence allowed us to start aggregation experiments with
FACS.

---

### Overall conclusions

Despite the experimenter's full dedication, experiments do not always work as
expected. In the Daisy project, the incomplete DNA sequence of the DARPin as well as the
defective mRFP1 were really good examples of design issues. These mistakes were very
instructive and allowed us to prove that we can be resilient to find solutions quickly.
The DBTL cycle is a great strategy to understand what could have gone wrong, emit new
hypotheses and test them incrementally to reach the initial purpose. Failure is nothing
but an occasion to learn and become better, finding new exciting ways for the research to
continue.

[![](https://static.igem.wiki/teams/4197/wiki/fleches/previous.png)](https://2022.igem.wiki/toulouse-insa-ups/model)

[![](https://static.igem.wiki/teams/4197/wiki/boutton-up.png)](#top)

[![](https://static.igem.wiki/teams/4197/wiki/fleches/next.png)](https://2022.igem.wiki/toulouse-insa-ups/notebook)

## References

* [Waldby C, Gray P, Griffiths P, Vickers C, Meek S, Small I. 2018. Synthetic Biology in Australia: an outlook to 2030. Australian Council of Learned Academies Expert Working Group Horizon Scanning Project. https://acola.org/wp-content/uploads/2018/12/2018Sep-ACOLA-Synthetic-Biology\_Report.pdf](https://acola.org/wp-content/uploads/2018/12/2018Sep-ACOLA-Synthetic-Biology_Report.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

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

![Menu aside](https://static.igem.wiki/teams/4197/wiki/description/sort-descending.svg)

* First tries of aggregation
  + Collection of allergens (strains A)
  + Construction of strain D
  + First aggregation experiments

* Troubleshooting

* New aggregation experiments
  + Correction of strain D
  + Detecting aggregates using microscopy

* Compatibility of our strategy with high-throughput approaches
  + Microdroplets
  + FACS

* Overall conclusions

## Results

### Overview

This section describes the experimental work performed during our 4 months internship in the lab. A first part details the construction and characterization of the allergen-expressing strains and the DARPin-expressing strain, as well as our first tries to detect **aggregation between the two strains in presence of IgE**. A second part presents the **troubleshooting** of these preliminary experiments, and a third explains the **correction of the DARPin strain** resulting from this troubleshooting, as well as the **new attempts of aggregation**. Finally, we present the development of two high throughput screening methodologies (**droplets and cytometry**) as a first step towards the detection of IgE presence in patient blood.

First tries of aggregation

In this section, we describe the set up of a collection of allergen-expressing *E. coli* strains, of the DARPin expressing strain and their combination in our first aggregation assays.

## Collection of allergens (strains A)

The leading idea of our project is to screen hundreds of allergens. As a proof of concept, we chose to focus on engineering a small number of allergens: Gal d 2, Ara h 2, Der p 1 and Ana o 3 (see [Design](https://2022.igem.wiki/toulouse-insa-ups/design)).

---

### Gal d 2 strain

![Figure 1: gal d 2 fragment](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/gal-d-2-parts.png "Figure 1: gal d 2 fragment ") ***Figure 1: OmpA\_Gal d 2 fragment.**The expression of OmpA\_Gal d 2 construction is controlled by the T7 promoter. OmpA was linked to TEV+Gal d 2 by a GISS linker. Gal d 2 was linked to the HISTag by the same linker.*

OmpA\_Gal d 2 fragment from IDT gblock (shown above in Figure 1) was amplified by [PCR](https://2022.igem.wiki/toulouse-insa-ups/protocols#PCR) using the high fidelity Phusion polymerase with primers IF3\_allergen-F and IF4\_Gal D2/DARPin-R (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). Expected size of the amplicon was 1675 bp.

pET-21 b (+) vector was linearized by PCR using the high fidelity Phusion polymerase with primers IF1\_GalD2/DARPin-F and IF2\_plasmid-R (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). Expected size of the amplicon was 5442 bp.

Amplification product sizes were checked on EtBr stained agarose [electrophoresis gel](https://2022.igem.wiki/toulouse-insa-ups/protocols#electro) (see [Electrophoresis](https://2022.igem.wiki/toulouse-insa-ups/protocols#Electrophoresis)) (Figure 2).

![Figure 2: pet lin and gal fragment](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/pet-21-b-linearized-and-gal-d-2-fragment.png "Figure 2: pet lin and gal fragment")***Figure 2: pET-21 b (+) linearized (A) and Gal d 2 amplified fragment (B). Expected sizes of the amplicons were 5442 bp (A) and 1675 bp (B).** PCR amplicon sizes of pET-21 b (+) (A) and Gal d 2 (B) were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels. (note that a different ladder is presented on the theoretical gel).*

Amplification products matched the expected size, they were further purified from the gel (see [Gel extraction](https://2022.igem.wiki/toulouse-insa-ups/protocols#extraction)).

The Gal d 2-OmpA construction was then inserted into pET-21 b (+) by [In-Fusion®](https://2022.igem.wiki/toulouse-insa-ups/protocols#fusion). The resulting products were [transformed](https://2022.igem.wiki/toulouse-insa-ups/protocols#Transformation) into Stellar competent cells. Transformants were selected on [LB-agar-ampicillin plates](https://2022.igem.wiki/toulouse-insa-ups/protocols#LB) (see [Cultures](https://2022.igem.wiki/toulouse-insa-ups/protocols#Cultures)). 20 transformants were screened by colony PCR with primer pairs flanking the insertion zone ( screening\_inserts-F and screening\_inserts-R, expected size of the amplicon : 2092 bp) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). 2 positive transformants were detected (Figure 3).

![Figure 3: gal screening](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/gal-d-2-screening.png "Figure 3: gal screening")***Figure 3: identifying fragments that bear pET21 b (+)\_Ompa\_Gal d 2 by colony PCR. Expected size of the amplicon was 2092 bp. The positive clones were colonies 17 and 24.** PCR amplicon sizes of colonies with Gal d 2 plasmid were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

These transformants had their plasmid extracted by [Miniprep](https://2022.igem.wiki/toulouse-insa-ups/protocols#Miniprep) and [digested](https://2022.igem.wiki/toulouse-insa-ups/protocols#restriction) by EcoRV to assess the assembly (expected fragments at 4332 bp and 2785 bp, see Figure 4).

![Figure 4: gal digestion](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/gal-d-2-digestion.png "Figure 4: gal digestion")***Figure 4: restriction profile of pET-21 b (+)\_OmpA\_Gal d 2 final construction. Enzyme used was EcoRV. Expected sizes of the amplicons were 4332 bp and 2785 bp.** Plasmids were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

The correct restriction maps were obtained and the insert sequence was further validated by sequencing. The plasmid was named **pET-21 b (+)\_OmpA\_Gal d 2**. The plasmids were eventually used to transform *E. coli* Tuner cells in order to express the OmpA\_Gal d 2 construction at the cell membrane (see [Preparation of chemically competent cells *E. coli*](https://2022.igem.wiki/toulouse-insa-ups/protocols#prepa)).

After cloning this first allergen, the plasmid obtained was used as a basis to build the other allergen constructions of our bank: Ara h 2, Der p 1 and Ana o 3.

---

### Ara h 2 strain

![Figure 5: ara parts](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ara-h-2-part.png "Figure 5: ara parts")***Figure 5: OmpA\_Ara h 2 fragment.** The expression of OmpA\_Ara h 2 construction is controlled by the T7 promoter. OmpA was linked to TEV+Ara h 2 by a GISS linker. Ara h 2 was linked to the HISTag by the same linker.*

Ara h 2 gene ordered on IDT (shown above in Figure 5) was amplified by PCR using the high fidelity Phusion polymerase with primers IF3\_allergen F and IF4\_Ara h 2 (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). Expected size of the amplicon was 567 bp.

In parallel, pET-21 b (+)\_Gal d 2\_OmpA vector was linearized, excluding the Gal d 2 fragment by PCR. The primers used were IF1\_allergen and IF2\_plasmid (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). Expected size of the amplicon was 5924 bp.

Amplification product sizes were checked on EtBr stained agarose gel (Figure 6).

![Figure 6: pet lin and ara fragment](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/pet-21-b-linearized-and-ara-h-2-fragment.png "Figure 6: pet lin and ara fragment")***Figure 6: pET-21 b (+)\_OmpA linearized with Gal d 2 exclusion (A) and Ara h 2 amplified fragment (B). Expected sizes of the amplicons were 5924 bp (A) and 567 bp (B).** PCR amplicon sizes of pET-21 b (+)\_OmpA (A) and Ara h 2 (B) were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

Amplification products matched expected sizes, they were further purified from the gel.

The Ara h 2 fragment was then inserted into pET-21 b (+)\_OmpA by In-Fusion. The In-Fusion assemby reaction was transformed into Stellar competent cells. Transformants were selected on LB-ampicillin plates. 15 transformants were then screened by colony PCR with primer pairs flanking the insertion zone (primers used: screening\_inserts-F and screening\_inserts-R, expected size of the amplicons: 1450 bp) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). 13 positive transformants were detected (Figure 7).

![Figure 7: ara screening](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ara-h-2-screening.png "Figure 7: ara screening")***Figure 7: Identifying strains that bear pET21 b (+)\_OmpA\_Ara h 2 by colony PCR. The expected size of the amplicons was 1450 bp. 13 positive transformants were detected.** PCR amplicon sizes of colonies with Ara h 2 plasmid were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

Four of these transformants (colonies 21, 22, 23, 24) had their plasmid extracted by Miniprep. Sequences were all validated by Sanger sequencing. The plasmid was named **pET-21 b (+)\_OmpA\_Ara h 2**.

The plasmid was finally used to transform *E. coli* Tuner cells to express OmpA\_Ara h 2 at the cell membrane.

---

### Der p 1 strain

![Figure 8: der parts](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/der-p-1-part.png "Figure 8: der parts")***Figure 8: OmpA\_Der p 1 fragment.** The expression of OmpA\_Der p 1 construction was controlled by the T7 promoter. OmpA was linked to TEV+Der p 1 by a GISS linker. Der p 1 was linked to the HISTag by the same linker.*

Der p 1 construction from IDT gblock (shown above in Figure 8) was amplified by PCR using the high fidelity Phusion polymerase with the primers IF3\_allergen F and IF4\_Der p 1 (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). Expected size of the amplicon was 994 bp.

Amplification product sizes were checked on EtBr stained agarose gel (Figure 9).

![Figure 9: der fragment](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/der-p-1-fragment.png "Figure 9: der fragment")***Figure 9: Der p 1 amplified fragment. Expected size of the amplicons was 994 bp.** PCR amplicon sizes Der p 1 were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

PCR products matched expected sizes and amplicons were further purified from the gel. The Der p 1 construction was inserted into linearized pET-21 b (+)\_OmpA (see Figure 6) by In-Fusion.

The In-Fusion assemby reaction was transformed into Stellar competent cells. Transformants were selected on LB-ampicillin plates. 15 transformants were screened by colony PCR with primer pairs flanking the insertion zone ( screening\_inserts-F and screening\_inserts-R; expected size of the amplicons: 1894 bp) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). 12 positive transformants were detected (Figure 10).

![Figure 10: der screening](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/der-p-1-screening.png "Figure 10: der screening")***Figure 10: identifying strains that bear pET21 b (+)\_OmpA\_Der p 1 by colony PCR. The expected size of the amplicons was 1894 bp. 12 positive clones were detected.** PCR amplicon sizes of colonies with Der p 1 plasmid were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

Four of these transformants (colonies 2, 6, 11 and 13) had their plasmid extracted by Miniprep and digested by SalI (expected size of the fragments: 6 kb and 1 kb) or double-digested by HindIII and EcoRI-HF (expected size of the fragments: 5.5 kb and 1.5 kb) to assess the assembly (Figure 11).

![Figure 11: der digestion](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/der-p-1-digestion.png "Figure 11: der digestion")***Figure 11: restriction profile of pET-21 b (+)\_OmpA\_Der p 1 for one of the clones. Enzymes were SalI for the simple digestion (expected sizes of the fragments were 6.0 kb and 1.0 kb) and EcoRI and HindIII for the double digestion (expected sizes of the fragments were 5.5 kb and 1.5 kb).** Plasmids were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

The correct restriction maps were obtained for the clones which were further validated by sequencing. The plasmid was named **pET-21 b (+)\_OmpA\_Der p 1**.

The plasmids were finally used to transform *E. coli* Tuner cells to express the Ompa\_Der p 1 construction at the cell membrane.

---

### Ana o 3 strain

![Figure 12: ana parts](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-part.png "Figure 12: ana parts")***Figure 12: OmpA\_Ana o 3 fragment.** The expression of OmpA\_Ana o 3 construction was controlled by the T7 promoter. OmpA was linked to TEV+Ana o 3 by a GISS linker. Ana o 3 was linked to the HISTag by the same linker.*

Ana o 3 construction from IDT gblock (shown above in Figure 12) was amplified by PCR using the high fidelity Phusion polymerase with the primers IF3\_allergen F and IF4 Ana o 3 (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). Expected size of the amplicon was 479 bp.

Amplification product sizes were checked on EtBr stained agarose gel (Figure 13).

![Figure 13: ana fragment](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-fragment.png "Figure 13: ana fragment")***Figure 13: Ana o 3 amplified fragment. Expected size of the amplicons was 479 bp.** PCR amplicon sizes Ana o 3 were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

The products matched expected sizes and amplicons were further purified from the gel. The Ana o 3 construction was inserted into pET-21 b (+)\_OmpA linearized (see Figure 6) by In-Fusion.

In-Fusion assemby reaction was transformed into Stellar competent cells. Transformants were selected on LB-ampicillin plates. 15 transformants were screened by colony PCR with primer pairs flanking the insertion zone (primers used: screening\_inserts-F and screening\_inserts-R) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). 4 positive transformants were detected (Figure 14).

![Figure 14: ana screening](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-screening.png "Figure 14: ana screening")***Figure 14: pET21 b (+)\_OmpA\_Ana o 3 construction fragments from colony PCR.** PCR amplicon sizes of colonies with Ana o 3 plasmid were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

These transformants (colonies 1, 3 and 9) had their plasmid extracted by Miniprep and digested by Eco-RI and Eco-RV (expected size of the fragments: 5052 bp and 3211 pb) to assess the assembly (Figure 15).

![Figure 15: ana digestion](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-digestion.png "Figure 15: ana digestion")***Figure 15: restriction profile of pET-21 b (+)\_Ana o 3 final construction. Enzymes were EcoRI and EcoRV.** Plasmids were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

The correct restriction maps were observed and these clones were further validated by sequencing. The plasmid was named **pET-21 b (+)\_OmpA\_Ana o 3**.

The plasmid was finally used to transform *E. coli* Tuner cells to express the OmpA\_Ana o 3 construction at the cell membrane.

Achievements so far: we managed to have all our allergen construction correctly cloned.

---

### Controls of correct protein expression

We first assessed the expression of the allergens at the outer surface of the bacteria, an important control experiment prior to the aggregation experiments. As only anti-Ara h2 and anti-Der p 1 IgE were available, we prioritized the verification of these allergen expressions first. Two experiments were conducted to do so: one with a fluorescent anti-HIS-tag IgG and one with an IgE coupled to a fluorescent anti-IgE IgG.

#### Test of Ara h 2 protein expression by HIS-tag detection

As the protein was HIS-tagged, we first tried to detect it by binding a fluorescent anti-HIS-tag IgG to the cell membrane and measuring fluorescence (see [HIS-tag detection by immunofluorescence](https://2022.igem.wiki/toulouse-insa-ups/protocols#HIS-tag detection by immunofluorescence)). Cells without IgG were observed as negative control. Microscope observations are shown in Figure 16. The microscope parameters to observe GFP were the following: excitation filter 360/40, dichroic beamsplitter 400 nm, emission filter 470/40.

![Figure 16: histag](https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ara-h-2-histag-detection.png "Figure 16: histag")***Figure 16: fluorescence of*** E. coli *Tuner cells containing the pET-21 b (+)\_OmpA\_Ara h 2 vector mixed with 1:100 anti-HIS-tag IgG (A), or without IgG for negative control (B). Each image represents an observation in phase contrast (on the left) and in fluorescence (on the right). Observations were made at the microscope parameters for GFP.*

No difference in fluorescence was visible between the control and the sample. The only fluorescence observed was due to autofluorescence of cells, so we could not conclude if the protein was expressed at the membrane, especially without a positive control (like binding the protein to a HIS-tag resin and observing fluorescence).

#### Test of the allergens functionality by double antibody recognition (ELISA test)

We assessed the functionality of the allergens by binding specific IgE to them (anti-Ara h 2 and anti-Der p 1), and then binding a fluorescent anti-IgE IgG to the complex (see [Allergen detection and recognition on the surface by immunofluorescence](https://2022.igem.wiki/toulouse-insa-ups/protocols#Allergen detection and recognition on the surface by immunofluorescence)). No fluorescence was observed in either case (data not shown). Without a positive control, we could not conclude between an incorrect exposition of the protein (not facing the outer surface), or a mere problem during the experimental setup.

## Construction of strain D

To be able to screen all these allergens, we also needed to construct our strain D, displaying DARPin proteins on its surface. The first step was the construction of a DARPin-sfGFP strain to prove the correct expression of our protein and to evaluate its subcellular localization. The sfGFP sequence was later deleted to obtain the final strain D.

---

### DARPin-sfGFP strain

A second strain was generated with the OmpA\_DARPin-sfGFP construction in order to evaluate the outer exposition of the fusion protein at the cell membrane. The aim was to later delete the sfGFP sequence from the plasmid to obtain the DARPin strain.

#### Vector construction

![Figure 17](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/dgfp-part.png "Figure 17")***Figure 17: DARPin-sfGFP construction.** The sfGFP served as a marker to prove the correct expression of the protein.*

OmpA\_DARPin-sfGFP fragment from IDT (shown above in Figure 17) was amplified by PCR using the high fidelity Phusion DNA polymerase with primers IF3\_allergen-F and IF4\_Gal d 2/DARPin-R (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). The expected size of the amplicon was 1468 bp.

The pET-21 b (+) vector was linearized by PCR using the high fidelity Phusion DNA polymerase with primers IF1\_Gal d 2/DARPin-F and IF2\_plasmid-R (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). The expected size of the amplicon was 5442 bp.

Amplification product sizes were checked on EtBr stained agarose gel. Amplicons were further purified from the gel. Figure 18 shows the amplification product of the OmpA\_DARPin-sfGFP construct matching the expected size. For the plasmid related amplicon, see Figure 2.

![Figure 18](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/dgfp-fragment.png "Figure 18")***Figure 18: OmpA\_DARPin-sfGFP fragment amplified by PCR. The expected size of the amplicon was 1468 bp.** The PCR amplicon size was checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the left and the NEB 1 kb DNA ladder was employed for the experimental gel. (note that a different ladder is presented on the theoretical gel).*

The DARPin-sfGFP construction was then inserted into our linearized pET-21 b (+) by In-Fusion to assemble the **pET-21 b (+)\_OmpA\_DARPin-sfGFP** plasmid.

The In-Fusion mixture was first transformed into chemically competent *E. coli* Stellar cells. Transformants were selected on LB-ampicillin plates. 42 transformants by colony PCR with a primer pair flanking the insertion zone (primers used: screening\_inserts-F and screening\_inserts-R, expected size of the amplicon : 1885 bp) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). 18 positive transformants were detected. Figure 19 shows an example of such positive clones.

![Figure 19](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/dgfp-screening.png "Figure 19")***Figure 19: identifying strains that bear pET-21 b (+)\_Ompa\_DARPin-sfGFP by colony PCR. The expected size of the amplicon was 1885 bp. The positive clones were colonies 13, 14, 15 and 16.** The PCR amplicon size was checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the left and the NEB 1 kb DNA ladder was employed for the experimental gels (note that a different ladder is presented on the theoretical gel).*

Five of those transformants had their plasmid extracted by Miniprep and digested with the restriction enzyme SalI to assess the assembly. The expected fragments were expected at 5574 bp and 1336 bp. Figure 20 shows correct restriction maps for all clones. The assembled plasmids were further validated by Sanger sequencing.

![Figure 20](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/dgfp-digestion.png "Figure 20")***Figure 20: restriction profile of the pET-21 b (+)\_OmpA\_DARPin-sfGFP final construction. The enzyme used was SalI. The expected sizes of the fragments were 5574 bp and 1336 bp. All clones had correct restriction profiles.** Plasmids were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the left and the NEB 1 kb DNA ladder is used for the experimental gel (note that a different ladder is presented on the theoretical gel).*

The plasmids were finally transformed into chemically competent *E. coli* Tuner cells that were used to verify the OmpA\_DARPin-sfGFP construction. It was then necessary to identify the best induction conditions and to assess whether our fusion protein was well presented onto the outer surface of the cell membrane.

#### Controls to verify the correct protein expression

As the presence of the OmpA\_DARPin(-sfGFP) fusion protein at the outer surface was key for our aggregation experiments, we tried to assess its cellular localization through several experiments. First of all, we needed to verify whether the protein was well expressed by the bacteria. To do so, we performed observations under an epifluorescent microscope. The goal was then to verify precisely the OmpA\_DARPin-sfGFP localization , which we did by fractionating the bacterial cells and monitoring fluorescence emission with a microplate reader. These experiments are detailed hereafter.

##### *• Validation of the protein expression by microscopy*

The expression of the OmpA\_DARPin-sfGFP encoding gene in *E. coli* Tuner cells was induced with a concentration of 50 µM of IPTG. The empty pET-21 b (+) plasmid was used as a negative control here. After 4 hours of incubation at 37°C, fluorescence was observed on an epifluorescence microscope as shown on Fig. 21.

![Figure 21](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/fig-21-micro.png "Figure 21")***Figure 21: fluorescence of*** E. coli *Tuner cells containing the pET-21 b (+)\_OmpA\_DARPin-sfGFP vector (A) or the empty pET-21 b (+) plasmid (B) as a negative control. Each image represents an observation in phase contrast (on the left) and in the GFP fluorescence channel (microscope parameters for GFP).*

Some fluorescence emission was clearly observed with pET-21 b (+)\_OmpA\_DARPin-sfGFP whereas none was seen with the empty plasmid. This suggests that the construction indeed allowed expressing the OmpA\_DARPin-sfGFP fusion. The fluorescence seemed to be localized in the cytoplasm as it colored the entire cell. However, the resolution of the microscope could not allow us to determine if the membrane was fluorescent as well.

To determine the optimal concentration of IPTG to express our construction, we next assessed the expression after induction with different concentrations: 10 µM, 25 µM and 100 µM (see [Induction with IPTG on liquid medium](https://2022.igem.wiki/toulouse-insa-ups/protocols#induction)). After 4 hours of incubation at 37°C, fluorescence was observed on an epifluorescence microscope. We also added an assay with 10 µM of IPTG at 22°C to test the influence of temperature. All microscope observations are shown in Figure 22.

![Figure 22](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/fig-22-micro.png "Figure 22")***Figure 22: fluorescence of*** E. coli *Tuner cells containing the pET-21 b (+)\_OmpA\_DARPin-sfGFP vector, with induction at different concentrations of IPTG: 10 µM (A), 25 µM (B), and 100 µM (C). Another assay was induced at 10 µM of IPTG and incubated 4 hours at 22°C (D). Each image represents an observation in bright field (on the left) and in the GFP fluorescence channel (microscope parameters for GFP). All observations were compared to an empty plasmid which showed no fluorescence (data not shown).*

With 10 µM of IPTG at 37°C, fluorescence was low and a phenomenon of bleaching quickly occured, so it was difficult to take pictures. With 25 µM of IPTG at 37°C, fluorescence intensity was higher and it was easier to take pictures. With 100 µM of IPTG at 37°C, lots of inclusion bodies were visible, indicating that the expression levels were too high for proper protein folding.

With 10 µM of IPTG at 22°C, some bacteria had a filamentous shape, maybe due to the low temperature that caused some forms of abiotic stress.

Based on our results, we concluded that the best condition was an induction with 25 µM of IPTG at 37°C as the corresponding colonies displayed very few inclusion bodies but high levels of fluorescence. These conditions were chosen for the other experiments.

##### *• Validation of the expression at the membrane by cell fractionation*

To determine where the OmpA\_DARPin-sfGFP fusion proteins were situated in the *E. coli* Tuner cells, we designed a fractionation protocol that allowed the separation of the different parts of the cells (cytoplasm and periplasm versus membrane). Briefly, after induction with 25 µM of IPTG at 37°C, we used sonication to break up the cells, resuspension in separating buffers and differential centrifugation steps (see [Cell fractionation and surface control](https://2022.igem.wiki/toulouse-insa-ups/protocols#Cell fractionation and surface control)). We then measured the fluorescence emission from each fraction on a microplate reader. A strain with an empty plasmid was induced at 50 µM of IPTG as a negative control. Curves of fluorescence depending on the dilution factor were established as shown on Figure 23.

![Figure 23](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/fig-23-microplaque.png "Figure 23")***Figure 23: fluorescence of the cytoplasm/periplasm and membrane fractions.** The negative control (empty plasmid induced at 50 µM of IPTG) is represented in blue and the actual assay (construction induced at 25 µM of IPTG) in orange. The excitation wavelength of the microplate reader was 485 nm. Emission was observed at a wavelength of 528 nm.*

As expected, the control fractions were considerably less fluorescent than the samples (approximately a hundred times). This indicates that the OmpA\_DARPin-sfGFP fusion protein was present in both the cytoplasm and surrounding membranes. The fluorescence in the membranes was twice lower than in the cytoplasm.

We concluded that the protein was mainly present in the cytoplasm, but also in the membranes as we wanted. We hypothesized that our pET-21 b (+)\_OmpA\_DARPin-sfGFP plasmid led to too high expression levels, leading to saturation of the membrane and expression in the cytoplasm as well.

Even if we confirmed the presence of at least some OmpA\_DARPin-sfGFP protein in the membrane fraction, we could not be sure if the protein was exposed at the very surface of the bacteria or inside the membrane (facing inwards). We conducted another experiment to investigate this last point.

##### *• Validation of the outer expression at the membrane by cell fractionation and TEV treatment*

To make sure that the OmpA\_DARPin-sfGFP was exposed at the surface of the cells and to quantify more precisely the fluorescence emission from each fraction, the experiment was later repeated with the addition of a TEV treatment after breaking the cells. The TEV protease should allow releasing the DARPin-sfGFP fusion from OmpA (Fig.17), meaning that after the TEV treatment, the fluorescence emission from the membrane fraction should decrease. Results are shown in Figure 24. This time again we included an empty vector control.

![Figure 24](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/fig-24-microplaque.png "Figure 24")***Figure 24: fluorescence of the cytoplasm/periplasm and membrane fractions.** The negative control (empty plasmid induced at 50 µM of IPTG) is represented in blue and the assay (construction induced at 25 µM of IPTG) in orange. Each fraction was treated with TEV protease and compared with a non-treated sample. The excitation wavelength of the microplate reader was 485 nm. Emission was observed at a wavelength of 528 nm.*

Similarly to the previous experiment, the control fractions were considerably less fluorescent than the samples (approximately a hundred times), indicating that GFP was well present in both the cytoplasm and membrane fractions of the induced sample. The fluorescence in the membrane appeared to be 2 to 3 times lower than in the cytoplasm, corroborating results from the previous experiment.

There was no difference between samples whether any TEV treatment was included or not. This could have been expected since we treated here the proteins alone and not bound to the membrane, meaning that we compared DARPin-sfGFP alone isolated after TEV treatment versus OmpA\_DARPin-sfGFP, i.e, the same quantity of sfGFP in both. A more meaningful experiment would have been to purify the membrane before or after TEV treatment and to compare sfGFP fluorescence.

---

### DARPin strain

After successful assembly of the OmpA\_DARPin-sfGFP construct and assessment of the fusion protein localization, the next step was to delete the sfGFP encoding part. The final OmpA\_DARPin construction (shown below in Figure 25) would later be used for the captation of IgE and aggregation experiments.

![Figure 25](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/darpin-parts.png "Figure 25")***Figure 25: OmpA\_DARPin construction.** This construction was used to transform the final strain D, used for aggregation experiments.*

#### Vector construction

To delete the sfGFP fragment, pET-21 b (+)\_OmpA\_DARPin-sfGFP was linearized by PCR using the high fidelity Phusion DNA polymerase with primers “exclusion sfGFP by In-Fusion (F)” and “exclusion sfGFP by In-Fusion (R)”, which excluded the sfGFP sequence (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). The expected size of the amplicon was 6202 bp.

Amplification product sizes were checked on EtBr stained agarose gel. Amplicons were further purified from the gel. Figure 26 shows an amplification product matching the expected size.

![Figure 26](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/darpin-plasmid-linearized.png "Figure 26")***Figure 26: pET-21 b (+)\_OmpA\_DARPin linearized. The expected size of the amplicons was 6202 bp.** PCR amplicon sizes were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the left and the NEB 1 kb DNA ladder is used for the experimental gels. (note that a different ladder is presented on the theoretical gel).*

The linearized plasmid was then recircularized by In-Fusion to form the **pET-21 b (+)\_OmpA\_DARPin** vector.

pET-21 b (+)\_OmpA\_DARPin was transformed into *E. coli* Stellar competent cells. Transformants were selected on LB-ampicillin plates. We screened 12 transformants by colony PCR with a primer pair flanking the zone where the sfGFP encoding fragment was deleted (primers used: screening\_inserts-F and screening\_inserts-R, expected size of the amplicon: 1162 bp) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). We detected 10 positive transformants. Figure 27 shows an example of such positive clones.

![Figure 27](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/darpin-screening.png "Figure 27")***Figure 27: identifying the strains that bear pET21 b (+)\_Ompa\_DARPin by colony PCR. The expected size of the amplicon was 1162 bp. Only two clones were not positive (D.3 and D.6). All clones were compared to a control with a linearized plasmid still containing the sfGFP fragment.** PCR amplicon sizes were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the left and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

Four of those transformants had their plasmid extracted by Miniprep and digested by the restriction enzymes EcoRV and SalI to verify the assembly. The expected sizes of the fragments were 4853 bp and 1334 bp. Figure 28 shows correct restriction profiles for all the clones. The plasmids from these clones were further confirmed by Sanger Sequencing.

![Figure 28](https://static.igem.wiki/teams/4197/wiki/results/construction-of-strain-d/darpin-digestion.png "Figure 28")***Figure 28: restriction profile of the pET-21 b (+)\_OmpA\_DARPin final construction. The enzymes used were EcoRV and SalI. The expected sizes of the fragments were 4853 bp and 1334 bp. All clones had correct restriction maps.** Plasmids were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the left and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

#### Controls of correct protein expression

We then tried to assess the expression and functionality of the DARPin, i.e. its capacity to bind IgE. Two experiments were conducted for this purpose. As the DARPin protein was HIS-tagged, we first tried to detect it by binding a fluorescent anti-HIS-tag IgG to the cell membrane and measuring fluorescence. No fluorescence was observed (data not shown), so we could not conclude if the protein was expressed at the membrane, especially without a positive control.

We then tried to assess the functionality of the DARPin by binding an IgE to it (the DARPin being able to link to the constant part of IgE), and then binding a fluorescent anti-IgE IgG to the complex. No fluorescence was observed (data not shown). Without a positive control, we cannot conclude between an incorrect exposition of the protein (not facing the outer surface), or a mere problem during the setup of the experiment.

## First aggregation experiments

The required *E. coli* A and D strains having been constructed, a first set of aggregation experiments was conducted. We aimed at capturing specific IgE in order to detect them via the formation of bacterial aggregates (see [Aggregation](https://2022.igem.wiki/toulouse-insa-ups/protocols#Aggregation)).

Here, our A strain produced the Ara h 2 allergen. It was mixed with the D strain designed to display DARPin proteins on its surface. Finally, we added an anti-Ara h 2 IgE to the mixture. We conducted several experiments to determine the optimal parameters (cellular concentration of each strain, strain ratio, IgE concentration, influence of agitation, incubation temperature…).

##### *• Influence of IgE concentration*

The first parameter tested was the concentration of IgE in our reaction mix. Three different IgE dilutions were chosen: 1:100 (final IgE concentration: 10 µg/mL), 1:250 (4 µg/mL) and 1:1000 (1 µg/mL). Equal quantities of strains A and D were mixed, reaching an OD600 of 3.2 after induction. Incubation was made overnight at room temperature. Three replicates were included to evaluate repeatability, as shown in Figure 29.

![Figure 29](https://static.igem.wiki/teams/4197/wiki/results/first-aggregation-experiments/fig-29-micro.png "Figure 29")***Figure 29: test of aggregation with strains A and D mixed with different concentrations of anti-Ara h 2 IgE: 1:100 (A), 1:250 (B), and 1:1000 (C).** A mixture comprising strains A and D without any added IgE was used as negative control (D). Three replicates were made. Observations were made in phase contrast for replicates 1 and 2, and in bright field for replicate 3.*

The first replicate presented bacterial aggregates for the three tested IgE dilutions, while the negative control showed no aggregates. There was no visible difference in the proportion or size of the aggregates between the three dilutions, so the IgE concentration did not have a strong impact. While these first observations looked promising, two other replicates performed later did not show any aggregate neither for the samples nor for the negative controls. As the experiment did not seem reproducible, we wanted to determine if the observed aggregates in replicate 1 could have stemmed from non-specific interactions between the bacterial cells, without any contribution from the IgE.

##### *• Influence of cell concentration*

We hypothesized that a high cell density may lead to non-specific aggregates. We therefore repeated the experiment but with a tenth of the previous cellular concentration. After adding the anti Ara h 2 IgE, incubation was made overnight at room temperature. Results for the 1:100 IgE dilution are shown in Figure 30.

![Figure 30](https://static.igem.wiki/teams/4197/wiki/results/first-aggregation-experiments/fig-30-micro.png "Figure 30")***Figure 30: test of aggregation with strains A and D mixed with 1:100 anti-Ara h 2 IgE, with cells at OD600 = 3.2 (A) or ten times more diluted (B).** A mix with strains A and D without IgE was used as negative control (C). Observations were made in bright field.*

As expected, cell density was considerably lower. We did not observe any aggregate formation, neither in the samples nor in the negative control. There was thus no obvious link between cell density and aggregation state.

##### *• Influence of the reaction mix dilutions*

For the next test, we decided to keep the same proportion of cells and IgE as in the first experiment but with a greater dilution of the total reaction mix to avoid non-specific aggregation. We compared 1:1, 1:2 and 1:10 mix dilutions. Incubation was made overnight at room temperature. Results for the 1:100 IgE dilution are shown in Figure 31.

![Figure 31](https://static.igem.wiki/teams/4197/wiki/results/first-aggregation-experiments/fig-31-micro.png "Figure 31")***Figure 31: test of aggregation with strains A and D mixed with 1:100 anti-Ara h 2 IgE, at different dilutions of the reaction mix: 1:1 (A), 1:2 (B), and 1:10 (C).** A mix with strains A and D without IgE was used as negative control (D). Observations were made in phase contrast.*

This time again we did not observe any aggregate, neither in the samples nor in the negative control. Our results overall indicate that no aggregate formation occured when mixing strain A, strain D and the corresponding IgE in these conditions.

##### *• Influence of agitation*

We hypothesized that adding some agitation during the incubation may increase interactions between the strains and IgE. The experiment was repeated but this time the mixture of strain A, strain D and the IgE was shaken with a benchwaver type agitator at 80 rpm. Incubation was made overnight at room temperature. Results for the 1:100 IgE dilution are shown in Figure 32.

![Figure 32](https://static.igem.wiki/teams/4197/wiki/results/first-aggregation-experiments/fig-32-micro.png "Figure 32")***Figure 32: test of aggregation with strains A and D mixed with 1:100 anti-Ara h 2 IgE, without agitation (A) or with agitation with a benchwaver type agitator at 80 rpm (B).** Mixtures with strains A and D without IgE were used as negative controls, non-agitated (C) and agitated (D). Observations were made in phase contrast.*

There were aggregates in the non-agitated sample as well as in its negative control but none in both agitated sample and negative control, so it seemed that the agitation prevented aggregation. These aggregates were certainly nonspecific as they appeared in the non-agitated negative control as well.

To conclude, the aggregation experiment may have succeeded once (first replicate of the test of IgE concentration) but could not be repeated. All the other experiments showed either nonspecific aggregates or no aggregates at all. Some troubleshooting had to be done to analyze the interaction between proteins and IgE on a molecular scale and draw hypotheses as to why the aggregation failed. The lack of positive controls was also a problem to identify what went wrong between the design and the experimental conditions. This also highlighted the plethora of parameters that could impact the aggregation phenomenon. This prompted us to build a model to optimize *in silico* these parameters and help in rationalizing the conditions to be assessed (see [Modeling](https://2022.igem.wiki/toulouse-insa-ups/model)).

Troubleshooting

We performed several attempts to aggregate bacteria or even to detect the presence of DARPins by immunofluorescence, without any success. These failures have led us to reconsider the literature. The use of Lpp-OmpA for surface display was clearly demonstrated, but we still ended up questioning a potential issue regarding the 3D structure of the DARPin protein coded from our biobrick. Therefore, we used AlphaFold 2, an innovative AI based software used in research to solve the structure of proteins.

Thus, the amino acid sequence of OmpA-DARPin-sfGFP-6xHis-tag fusion protein was submitted to AlphaFold, the resulting structure being compared to the crystallographic structure of a multidomain protein containing DARPin (Figure 33).

![Figure 33](https://static.igem.wiki/teams/4197/wiki/results/troubleshooting/fig-33-alphafold.png "Figure 33")***Figure 33: Comparison between the predicted DARPin structure (extracted from the OmpA-DARPin-sfGFP-6xHis-tag fusion protein) (A) and the crystallographic structure (PDB: 4GRG) of DARPin E2\_79 (B).** Figure A displays two different viewpoints of the DARPin E2\_79 (PDB: 4GRG), showing 8 α-helix. Figure B shows the entire complex (OmpA in orange, DARPin in yellow, and sfGFP in green). On the DARPin 6 α-helix can be counted.*

In the crystallographic structure (Figure 33A), E2\_79 DARPin displayed 4 pairs of antiparallel α-helices linked by a short β-turn, whereas the AlphaFold prediction of our original fusion protein highlighted an unexpected structure for the DARPin E2\_79 (Figure 33B), with 3 pairs of antiparallel α-helices. Clearly, the model revealed an obvious mistake in our DNA sequence.

Actually, Kim and colleagues (Kim et al., 2012) published an incorrect DNA sequence of DARPin E2\_79, even if the amino acid sequence described in their crystallographic structure (PDB: 4GRG) is right. We think that this error may have gone unnoticed because the sequence published on Uniprot starts with methionine, which is very often the first amino acid of proteins.

We applied a correction to our genetic sequence by complementing the gene coding DARPin with 45 N-terminal amino acids coming from a multi-sequences alignment based on two the crystallographic structures of DARPin (PDB 4GRG and 7MXI). As no nucleotide sequence was available, the 45 amino acids were reverse translated into nucleotides using the Reverse Translate tool from the Sequence Manipulation Suite (Stothard P. 2000). We then optimized the DNA sequence for *E. coli* translation using Benchling. The DARPin containing DNA sequence was rewriten accordingly on our *in silico* Benchling construction. A second simulation on AlphaFold confirmed the accuracy of the correction, recovering the full 4 pairs of α-helix (Figure 34).

![Figure 34](https://static.igem.wiki/teams/4197/wiki/results/troubleshooting/fig-34-alphafold.png "Figure 34")***Figure 34: Reconstruction of the sequence of OmpA-TEV-DARPin-sfGFP-6xHis-tag with AlphaFold.** The construction prediction was recolored usingPyMol. The OmpA is shown in orange, in yellow the previous DARPin with in clear green the correction of the DARPin sequence, and in green the sfGFP construction.*

The corrected plasmid coding for the full OmpA-DARPin-sfGFP-6xHis-tag fusion protein
was then PCR produced as quickly as possible in order to continue the project.

New aggregation experiments

As AlphaFold 2 highlighted a truncation in the sequence coding for our DARPin, a reconstruction strategy was decided to add the missing gene fragment. This corrected version of the DARPin was thereafter named DARPIN\*

## Correction of strain D

#### Vector construction

A PCR Assembly was conducted to assemble the missing fragment. Primers 1\_PCA60, 2\_PCA60, and 3\_PCA60 (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)) and the high fidelity Phusion polymerase were used, with two elongation cycles to enable complete synthesis of the fragment (Figure 35).

![Figure 35](https://static.igem.wiki/teams/4197/wiki/results/correction-of-darpin-construction/fig-35-pca.png "Figure 35")***Figure 35: Principle of PCR Assembly to build the missing part of the DARPin.** Three primers (1\_PCA60, 2\_PCA60 and 3\_PCA60) were used to build the complete missing sequence. These three primers were mixed in the same PCR mix. After a first elongation with 1\_PCA60 and 2\_PCA60, 3\_PCA60 binds to the generated amplicon to end up the elongation.*

The fragment was then amplified by PCR using the high fidelity Phusion polymerase with the primers IF3\_ampli\_DARPin (F) and IF4\_ampli\_DARPin (R) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). The expected size of the amplicon was 135 bp. Amplification product size was checked on EtBr stained agarose gel. Figure 36 shows the amplification product matching the expected size.

![Figure 36](https://static.igem.wiki/teams/4197/wiki/results/correction-of-darpin-construction/fig-36-fragment-ampli.png "Figure 36")***Figure 36: DARPin\* missing fragment amplified. Expected size of the amplicon was 135 bp.** PCR amplicon size was checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the left and the NEB 1 kb DNA ladder is used for the experimental gel (note that a different ladder is presented on the theoretical gel).*

Next, the pET-21 b (+)\_OmpA\_DARPin-sfGFP and pET-21 b (+)\_OmpA\_DARPin plasmids were linearized by PCR using the high fidelity Phusion polymerase with primers IF1\_plasmid\_DARPin (F) and IF2\_plasmid\_DARPin (R) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). The expected sizes of the amplicons were 6910 bp and 6187 bp, respectively. Amplification product sizes were checked on EtBr stained agarose gel. Figure 37 shows amplification products matching expected sizes.

![Figure 37](https://static.igem.wiki/teams/4197/wiki/results/correction-of-darpin-construction/fig-37-darpin-and-dgfp-lin.png "Figure 37")***Figure 37: pET-21 b (+)\_OmpA\_DARPin and pET-21 b (+)\_OmpA\_DARPin-sfGFP linearized fragments. Expected sizes of the amplicons were respectively 6187 bp and 6910 bp.** PCR amplicon sizes were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

The missing DARPin fragment was then inserted into each of these plasmids by In-Fusion. The constructions obtained were first transformed into Stellar competent cells. Transformants were selected on LB-ampicillin plates, and were further screened by colony PCR with primer pairs flanking the insertion zone (primers used: screening\_inserts-F and screening\_inserts-R) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)).

Plasmid from positive transformants were extracted by Miniprep and digested to assess the assembly (data not shown). Sequences of all the clones were validated by sequencing, completing the **pET-21 b (+)\_OmpA\_DARPin\*** and **pET-21 b (+)\_OmpA\_DARPin\*-sfGFP** constructions.

The plasmids were finally used to transform *E. coli* Tuner cells to hopefully express the DARPin\* and DARPin\*-sfGFP constructions at the cell membrane.

#### Controls to verify correct protein expression

As the insertion of the missing fragment could have changed the expression of the protein, it was necessary to verify it again. To assess the subcellular localization of the new fusion protein *in vivo*, we repeated the fractionation of the cells and fluorescence intensity measurement with a microplate reader, coupled to TEV treatment. This experiment, done with the DARPin\*-sfGFP strain, is detailed hereafter.

To determine where the DARPin\*-sfGFP fusion proteins were expressed in the E. coli Tuner cells, we conducted the same fractionation protocol as earlier. We measured the fluorescence of each fraction on a microplate reader (Figure 38). The DARPin\* strain without sfGFP was induced with 25 µM of IPTG as a negative control.

![Figure 38](https://static.igem.wiki/teams/4197/wiki/results/correction-of-darpin-construction/fig-38-microplaque.png "Figure 38")***Figure 38: fluorescence of cytoplasm/periplasm and membrane fractions.** The negative control (DARPin\* strain inducted at 25 µM of IPTG) is represented in blue and the assay (construction inducted at 25 µM of IPTG) in orange. Each fraction was treated with TEV protease and compared with a non-treated sample. The excitation wavelength of the microplate reader was 485 nm. Emission was observed at a wavelength of 528 nm.*

The results were the same as before the insertion of the missing fragment: the control fractions were considerably less fluorescent than the samples, indicating the accumulation of the fusion protein in both the cytoplasm and membrane. The fluorescence in the membrane still appeared to be 2 to 3 times lower than in the cytoplasm. There still was no difference in samples whether the TEV treatment was applied or not, so we could not conclude whether the protein was exposed at the outer surface of the cell.

In conclusion, the integration of the missing DARPin fragment did not alter the expression of the protein, which was still expressed in the membrane as wanted. We still could not determine if it was exposed correctly at the surface or inside the membrane.

Now that the corrected strain D (expressing the DARPin\* protein) was constructed, we conducted another set of aggregation experiments with that strain.

## Detecting aggregates using microscopy

As the OmpA\_DARPin\* construction was corrected, new aggregation assays were conducted to determine if the new fusion protein was able to form aggregates to capture IgE.
The process was similar as in “First tries of aggregation”, with strain A displaying the Ara h 2 allergen for aggregation. The reaction mix included this strain A, along with strain D with the corrected DARPin gene and anti-Ara h 2 IgE. Three parameters were tested: the influence of IgE concentration, agitation and temperature of incubation, over aggregation.

##### *• Influence of IgE concentration*

Likewise to the first aggregation experiments, we tested three different IgE dilutions ( overnight incubation at room temperature with a benchwaver type agitator at 80 rpm; Figure 39).

![Figure 39](https://static.igem.wiki/teams/4197/wiki/results/new-aggregation-experiments/fig-39-micro.png "Figure 39")***Figure 39: test of aggregation with strains A and D mixed with different concentrations of anti-Ara h 2 IgE: 1:100 (A), 1:250 (B), and 1:1000 (C).** A mix with strains A and D without IgE was used as negative control (D). The correct DARPin\* on the strain D was used.*

This aggregation assay did not show any difference between each sample and the negative control.

##### *• Influence of incubation temperature*

To compare the influence of temperature for two overnight incubation conditions tested (4°C and room temperature), for reminder, the incubation is the time spent between adding IgE and the observation on a microscope. The assay with 1:250 dilution of IgE anti Ara h 2 was shown in Figure 40.

![Figure 40](https://static.igem.wiki/teams/4197/wiki/results/new-aggregation-experiments/fig-40-micro.png "Figure 40")***Figure 40: test of aggregation with strains A and D incubated overnight with a dilution of IgE anti Ara h 2 of 1: 250 at different temperatures**: 4°C (A) and Room temperature (B), negative control without anti Ara h 2 IgE incubated at 4°C (C). The correct DARPin\* on the strain D was used.*

The comparison does not show any incidence of the tested temperatures on the behavior of bacteria for an IgE dilution of 1:250. Therefore, the temperature of incubation did not trigger unpecific aggregates.

##### *• Influence of stirring*

Then the agitation condition was tested with a benchwaver type agitator at 80 rpm at room temperature for overnight incubation. Results are shown for 1:250 IgE anti-Ara h 2 dilution in Figure 41.

![Figure 41](https://static.igem.wiki/teams/4197/wiki/results/new-aggregation-experiments/fig-41-micro.png "Figure 41")***Figure 41: test of aggregation with strains A and D incubated overnight with a dilution of IgE anti Ara h 2 of 1: 250 at room temperature: With stirring (A) Without stirring (B) Negative control without IgE Anti-Ara h 2 with stirring (C).** The correct DARPin\* on the strain D was used. The agitation was made at room temperature with a benchwaver type agitator.*

Agitation did not show any incidence on the aggregation capacity.

Despite the troubleshooting and the reconstruction of the DARPin, we could not detect any phenomenon of aggregation. As it happens, a new loop of the DBTL cycle (see [Engineering success](https://2022.igem.wiki/toulouse-insa-ups/engineering)) has to be implemented to test a larger range of conditions (temperature, stirring, IgE concentrations, medium, other types of IgE and corresponding strain A, etc). For that, we can prospect two outlooks of the DBTL cycle. First we can deepen our efforts on surface control experiments to try to understand how our complexes are positioned on the membrane of strains A and D, and modify it if necessary. Second, our modeling effort (see [Modeling](https://2022.igem.wiki/toulouse-insa-ups/model)) has been achieved at this step of the project and could eventually be used to optimize our set-up of aggregation assays, with a better control of parameters. The model will allow us to save a lot of time in the wet lab and resources for experiments.

The lack of time did not allow us to put into practice this new loop of the DBTL cycle in the wet lab, but this new approach of aggregation assay with the new DARPin and the mastering of the aggregation protocol should lead to a better control of assays and more accurate results by limiting non-specific aggregation.

Compatibility of our strategy with high-throughput approaches

Our final goal is to detect and identify IgE in patient blood. To do so, we need to ensure the compatibility of our strategy with high-throughput approaches. Thus, we assessed the possibility to use DAISY with microdroplets and with FACS analysis.

## Microdroplets

Microdroplets is a state-of-the-art microfluidics approach to parallelize a multiplicity of tests. For DAISY, it offers the possibility to test a multitude of strain A/strain D/IgE combinations without having any aggregates containing a mix of only strains A. It is then possible to select microdroplets presenting aggregation and hence, highlight the presence of allergenic IgE which could be further identified by sequencing the allergen sequence of the strain A. Even if the aggregation in microtubes did not work as expected, we decided to continue so that we could discover this microfluidics method.
The strategy was to encapsulate strains A, strain D and IgE in 18 µm diameter microdroplets. This way, each droplet acts like an individual reactor in which cells can aggregate independently. To do so, we force two orthogonal flows to cross each other. One of aqueous medium containing the cells and antibody, the other of oil. When they meet, the oil “squeezes” the aqueous medium and so, generates tiny droplets (see [Design](https://2022.igem.wiki/toulouse-insa-ups/design)).

As we aim later to display a bank of allergen on the surface of strain A and isolate each cell in a different droplet, we chose to use a very diluted suspension of strain A (OD600= 0.01). On the contrary, with the aim of maximizing the size of the aggregates, we worked with a higher density of strain D (OD600= 4) to have 10 cells per droplet on average.

Strains A (displaying Ara h 2 allergen) and D were grown, induced for the expression and display of the allergen and DARPin, resuspended in PBS and encapsulated in microdroplets. After incubation overnight at room temperature, the droplets were observed with a microscope, as shown on Figure 42.

![Figure 42](https://static.igem.wiki/teams/4197/wiki/results/microdroplets/fig-42-droplets.png "Figure 42")***Figure 42: first aggregation assay in microdroplets.** Observation in bright field, after overnight incubation in PBS at room temperature of droplets containing either (A) strain D with 10 cells on average, (B) strain D with 10 cells on average and maximum one cell of strain A ara h 2, (C) strain D with 10 cells on average, maximum one cell of strain A ara h 2 and IgE anti Ara h 2 at 1 µg/mL, (D) strain D with 10 cells on average, maximum one cell of strain A ara h 2 and IgE anti Ara h 2 at 10 µg/mL. The bacteria were mixed before encapsulation and the IgE were added during the droplet generation.*

Aggregates were visible in most droplets, no matter the conditions considered. It means the aggregates were not due to the presence of IgE. The cells were not very mobiles. As the bacteria were resuspended in PBS 48h before the observation, we assumed some bacteria lysed because of the lack of substrate and released their DNA which glued the other bacteria together.

To avoid the lysis of the bacteria during incubation in PBS, we performed another experiment using LB medium instead of PBS. Plus, we calculated that a concentration of 1µg/mL of IgE corresponded to 43 000 IgE per droplet. As there are 10-11 bacteria per droplet on average, this number of IgE may saturate all the binding sites and inhibit the aggregation so we chose a concentration of 10 ng/mL. Finally, as we were running low on anti-Ara h 2 IgE, we used strain A Der p 1 and anti-Der p 1 IgE. Microscope observations of these microdroplets are shown in Figure 43.

![Figure 43](https://static.igem.wiki/teams/4197/wiki/results/microdroplets/fig-43-droplets.png "Figure 43")***Figure 43: second aggregation assay in microdroplets.** Observation in phase contrast, after overnight incubation in LB at room temperature of droplets containing (A) strain A Der p 1 with maximum 1 cell, (B) strain D with 5 cells on average and maximum one cell of strain A Der p 1, (C) strain D with 5 cells on average, maximum one cell of strain A Der p 1 and anti-Der p 1 IgE at 10 ng/mL, (D) strain D with 5 cells on average, (E) strain D with 5 cells on average and IgE anti Der p 1 at 10 ng/mL. Strain D was mixed IgE before encapsulation and strain A Der p 1 was added during the droplet generation.*

There were a lot more than 5 bacteria per droplet, as there should have been just after the encapsulation. This is likely due to the chosen rich medium, allowing the growth of the bacteria overnight. Aggregates were again visible in most droplets, no matter the conditions considered. As the bacteria were moving fastly, it is unlikely that some bacteria lyzed and glued the other ones. The nonspecific aggregation may be due to the high cell density of the physical medium properties.

With more time, it would have been valuable to optimize the cell concentration or physical parameter of the medium. Then the next step would have been to train the IA to identify aggregation in the droplets. The final step would have been running the experiment with labeling of the IA chosen droplets, sorting of the labeled droplet by FACS, and subsequent detection of the allergen expressed by strain A.

## FACS

The second high-throughput approach we wanted to try was the FACS (Fluorescence-Activated Cell Sorting). The FACS platform was chosen for its capacity to sort cells by discriminating them according to their fluorescence.

---

### Creating fluorescent strains for FACS application

To identify cellular aggregates including both strain D and strain A with FACS, we needed to discriminate between both strains. In this scope, we chose to render cells fluorescent with the constitutive expression of blue mTagBFP for strain D and red mRFP1 for strains A (expressing either allergens Ana o 3, Der p 1, Gal d 2 and Ara h 2). The fluorescent reporters were obtained from Manon Barthe (Toulouse White Biotechnology and former iGEM Toulouse 2016 member) who used the followings Parts:

* - [BBa\_J06504](http://parts.igem.org/Part%3ABBa_J06504) for mRFP1
* - [BBa\_K592100](http://parts.igem.org/Part%3ABBa_K592100) for mTagBFP

From her experience, both fluorescent reporters can be used without signal overlapping in cytometric approaches. She also advised us to use the *E. coli ihfB* promoter with 800 bp before the translation start site for optimal expression (Zhou et al., 2011 ; Barthe et al., 2020).

#### Construction of strain D expressing blue fluorescence

The ihfb800-mTagBFP construction provided by Manon Barthe was amplified by PCR with a high-fidelity Phusion polymerase using BFP/RFP IF+BglII R site R and BFP/RFP IF+BglII R site F primers (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). The expected size of the amplicon was 1595 bp which matches with the obtained fragment (Figure 44).

![Figure 44](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-44-bfp-fragment.png "Figure 44")***Figure 44: PCR amplification of ihfb800-BFP from the psb1c3 plasmid.** ihfb800-BFP PCR amplicon size of 1595 bp was checked with 0.6% agarose electrophoresis gel and revealed with EtBr. The elongation time used is 2’ and the hybridization temperature was tested from 55°C to 62°C. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels. (note that a different ladder is presented on the theoretical gel).*

To insert the fragment obtained in the DARPin-expressing plasmid, a linearization has been conducted with primers: planB\_FPIF\_pET21b(+)\_R, planB\_FPIF\_pET21b(+)\_F (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). Expected size was 6317 bp which matches with the obtained result (Figure 45).

![Figure 45](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-45-linearization-darpin.png "Figure 45")***Figure 45: PCR linearization of pET-21 b (+)\_OmpA\_DARPin.** The PCR product of linearization was checked with 0.8% agarose electrophoresis gel and revealed with EtBr. The hybridization temperature was 58°C and the elongation time was 7 min. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

The linearized pET-21 b (+)\_OmpA\_DARPin plasmid and the ihfb800-BFP fragment were extracted from gel and purified with the [PCR clean-up](https://2022.igem.wiki/toulouse-insa-ups/protocols#clean) protocol, then assembled by In-Fusion. The product was transformed in Stellar cells and transformants were selected on Ampicillin. Plasmids from the resulting colonies were extracted by Miniprep. The presence of BFP was checked by PCR screening with screening\_insert-R and screening\_insert-F primers (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). The PCR product containing both BFP and DARPin displayed the correct size at 2892 bp only for colony 2 (Figure 46).

![Figure 46](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-46-darpin-bfp-screening.png "Figure 46")***Figure 46: PCR screening of pET-21 b (+)\_OmpA\_DARPin.** The electrophoresis gel was revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel). Colony 2 presented the correct profile.*

Colony 2 was selected and an overnight preculture was performed to control the blue fluorescence on a microscope (Figure 47). A colony containing the pET-21 b (+)\_OmpA\_DARPin without ihfb800-BFP was used as a negative control. The microscope parameters to observe BFP were the following: excitation filter 360/40, dichroic beamsplitter 400 nm, emission filter 470/40.

![Figure 47](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-47-bfp-micro.png "Figure 47")***Figure 47: Observation on microscope of pET-21 b (+)\_OmpA\_DARPin strain with ihfb800-BFP (A) pET-21 b (+)\_OmpA\_DARPin strain with ihfb800-BFP were used a negative control (B).** Colonies showed significant blue fluorescence. Observations were made in phase contrast (left of each panel) and at the microscope parameters for BFP (right of each panel).*

Sequencing revealed a mutation in the promoter but it did not seem to impact mTagBFP expression. This validated the blue-fluorescent strain D.

The construction, however, was obtained with the first DARPin sequence. We therefore had to insert the missing fragment of the protein. The method was the same as followed for the non-fluorescent protein. A PCR Assembly was conducted to assemble the missing fragment. The fragment obtained for the non-fluorescent DARPin construction was used again for this construction (see figure 36). Briefly, pET-21 b (+)\_OmpA\_DARPin\_BFP plasmid was linearized by PCR using the high fidelity Phusion polymerase with the primers IF1\_plasmid\_DARPin (F) and IF2\_plasmid\_DARPin (R) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). Expected size of the amplicons was 7812 bp. Amplification product sizes were checked on EtBr stained agarose gel (figure 48).

![Figure 48](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-48-linearization-darpin-bfp.png "Figure 48")***Figure 48: pET-21 b (+)\_OmpA\_DARPin\_BFP linearized fragments. Expected size of the amplicon was 7812 bp.** PCR amplicon size was checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels. (note that a different ladder is presented on the theoretical gel).*

Since it was validated, the missing DARPin fragment was then inserted by In-Fusion,The resulting products were first transformed into Stellar competent cells. Transformants were selected on LB-ampicillin plates. 11 colonies were further screened by colony PCR with primer pairs flanking the insertion zone (primers used: exclusion sfGFP (F) and IF4 ampli DARPin, expected size : 384 pb) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). 8 PCR products were matching the expected sizes (Figure 49).

![Figure 49](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-49-new-darpin-bfp-screening.png "Figure 49")***Figure 49: PCR screening of pET-21 b (+)\_OmpA\_DARPin\_BFP. Expected size was 384 bp.** The electrophoresis gel was revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

Plasmid from positive transformants were extracted by Miniprep and digested to assess the assembly (data not shown). Sequences were validated by sequencing, thus completing the **pET-21 b (+)\_OmpA\_DARPin\*\_BFP** construction.

The plasmids were finally used to transform E. coli Tuner cells to express the pET-21 b (+)\_OmpA\_DARPin\_BFP correct construction at the cell membrane. The construction was finally ready to be used for the FACS.

#### Construction of strain A expressing red fluorescence with mRFP

The ihfb800-mRFP1 construction from Manon Barthe was amplified by PCR with the high-fidelity Phusion polymerase using BFP/RFP IF+BglII R site R and BFP/RFP IF+BglII R site F primers (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). The expected size of the amplicon is 1622 bp and this was watching the results on the gel (Figure 50).

![Figure 50](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-50-rfp-ampli.png "Figure 50")***Figure 50: PCR amplification of ihfb800-RFP from the psb1c3 plasmid.** ihfb800-RFP PCR amplicon size of 1622 bp was checked with 0.6% agarose electrophoresis gel and revealed with EtBr. The elongation time used is 2’ and the hybridization temperature was 57°C. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels. (note that a different ladder is presented on the theoretical gel).*

After [DpnI digestion](https://2022.igem.wiki/toulouse-insa-ups/protocols#dpnI), the fragment was recovered by PCR clean-up.
To insert the fragment on allergen-expressing plasmids, linearization of these plasmids has been conducted in parallel (Gal d 2, Ana o 3, Der p 1 and Ara h 2 expressing plasmids) with primers: planB\_FPIF\_pET21b(+)\_R and planB\_FPIF\_pET21b(+)\_F (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)), as shown in Figure 51. For each linearization, the expected sizes were:

* - pET-21 b (+)\_OmpA\_Gal d 2: 7117 bp
* - pET-21 b (+)\_OmpA\_Ana o 3: 6475 bp
* - pET-21 b (+)\_OmpA\_Der p 1: 6919 bp
* - pET-21 b (+)\_OmpA\_Ara h 2: 6475 bp

![Figure 51](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-51-allergens-linearization.png "Figure 51")***Figure 51: PCR linearization of Gal d 2, Ana o 3, Der p 1 and Ara h 2.** The PCR product of linearization was checked with 0.6% agarose electrophoresis gel and revealed with EtBr. The hybridization temperature was 58°C and the elongation time was 7 min. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).*

Except for Der p 1, the analytical gel showed two bands for each PCR product with one for each sample corresponding to the expected size. Since we aimed to obtain at least two constructions with at least one compatible with commercial antibodies we owned, we carried on without Der p 1.

In-Fusion was performed to insert the ihfB800-mRFP1 fragment in the linearized Gal d 2, Ana o 3 and Ara h 2 expressing plasmids. The resulting products were transformed in Stellar cells and transformants were selected on ampicillin. Colonies were screened by PCR using screening\_insert\_R and screening\_insert\_F primers (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). The expected sizes of amplicons were: for Ara h 2, 3046 bp with RFP and 1454 bp without; for Ana o 3, 2944 bp with RFP and 1453 bp without; for Gal d 2, 3688 bp with RFP and 2096 bpwithout. Figure 52 shows the corresponding gels. We obtained correct sizes for the three allergen constructions.

![Figure 52](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-52-allergens-rfp-screening.png "Figure 52")***Figure 52: verification of the insertion of RFP fragment: Ara h 2 gel (A), Ana o 3 gel (B), Gal d 2 (C).** The PCR screening was checked with 0.6% agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel). Colonies 1, 2 and 3 present the correct size for Ara h 2 ; colony 8 for Ana o 3; colonies 9, 10, 11 for Gal d 2.*

Successful colonies were further tested by digestion with NotI which cut both in the plasmid and in the mRFP1 gene (Figure 53). Correct sizes were expected to be 1280 and 6791 bp for Ara h 2, 1178 and 6791 bp for Ana o 3 and 1922 and 6791 bp for Gal d 2.

![Figure 53](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-53-allergen-rfp-digestion.png "Figure 53")***Figure 53: digestion by NotI of Ara h 2 with mRFP1 insertion (A), of Ana o 3 with mRFP1 insertion(B) and of Gal d 2 with mRFP1 insertion (C).** The digestion product was checked with 0.6% agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel). Colonies 1, 2 and 3 present the correct size for Ara h 2; colonies 21 and 23 present the correct size for Ana o 3; colonies 7 and 13 present the correct size for Gal d 2.*

Sequencing of each sample showed the same mutation in the ihfb800 promoter than for the DARPin-mTagBFP construction. This mutation seemed generic and was likely present in the initial sequence. As it did not appear to impact expression, we validated the sequences.

We then assessed the mRFP1 fluorescence on a fluorescent microscope. Very unfortunately, we did not succeed to obtain more fluorescence than with a negative control without the ihfB800-mRFP1 construction (figure not shown). Consequently, we decided to replace the mRFP1 sequence by those of the mScarlet-I fluorescent reporter, whose excitation and emission characteristics are similar.

#### Construction of strain A expressing red fluorescence with mScarlet-I

As we could not determine if the mRFP1 was indeed expressed in our strain A, we replaced its gene by that of the mScarlet-I (see Figure 54). The mScarlet-I gene was supplied by Maxence Holtz (TWB and former iGEM Toulouse 2021).

![Figure 54](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-54-arah2-construction.png "Figure 54")***Figure 54: allergen construction with mScarlet-I, example for Ara h 2 allergen.** Promoter used for the fluorescent protein is the constitutive ihfB800.*

We designed primers to replace the mRFP1 coding sequence with those of the mScarlet-I gene. PCR amplification was done using Phusion polymerase and IF3\_mSCARLET-I (F) and IF4\_mSCARLET-I (R) primers (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). Expected size of the amplicon was 699 bp. The correct size was obtained (Figure 55A).

We had antibodies against Ara h 2 so we decided to clone mScarlet-I in the pET-21 b (+)\_OmpA\_Ara h 2 strain for the experiment.
We did not have antibodies against Ana o 3 so we decided to use the pET-21 b (+)\_OmpA\_Ana o 3 strain for the negative control.
To amplify these vectors excluding mRFP1 sequence, we used primers IF1\_exclusion\_RFP (R) and IF2\_exclusion\_RFP (F) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)). Expected size of the amplicon were 7365 bp for Ara h 2 and 7263 bp for Ana o 3. The correct size was obtained (Figure 55B). Amplicons were further purified from the gel.

![Figure 55](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-55-mscarlet-fragment-and-allergen-linearizations.png "Figure 55")***Figure 55: mScarlet-I amplified fragment (A), pET-21 b (+)\_Ara h 2\_OmpA and pET-21 b (+)\_Ana o 3\_OmpA linearized (B). Expected sizes of the amplicons were 699 bp (A) and 7365 bp and 7263 bp (B).** PCR amplicon sizes were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels. (note that a different ladder is presented on the theoretical gel).*

The mScarlet-I gene was inserted into pET-21 b (+)\_OmpA\_Ara h 2 and pET-21 b (+)\_OmpA\_Ana o 3 linearized by In-Fusion. In-Fusion assemby reaction was transformed into Stellar competent cells and transformants were selected on LB-ampicillin plates. Two transformants clones were red directly on the plate, straightly indicating the expression of mScarlet-I (Figure 56).

![Figure 56](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-56-mscarlet-petri.jpg "Figure 56")***Figure 56: Second plating of pET-21 b (+)\_Ara h 2\_OmpA\_mScarlet-I transformed cells and pET-21 b (+)\_Ana o 3\_OmpA\_mScarlet-I transformed Stellar cells.** The colonies were selected on LB-ampicillin plates. Pink coloring indicates the expression of the mScarlet-I.*

The plasmids were extracted and used to transform E. coli Tuner cells. They also turned red (data not shown) indicating that the ihfB800 promoter is well functional as well in Stellar as in Tuner *E. coli* backgrounds.

The colonies containing the pET-21 b (+)\_OmpA\_Ara h 2\_mScarlet-I and pET-21 b (+)\_OmpA\_Ana o 3\_mScarlet-I plasmids were observed under the fluorescence microscope to detect the mScarlet-I fluorescence (Figure 57). The microscope parameters to observe mScarlet-I fluorescence were the following: excitation filter 545/30x, dichroic beamsplitter 570 nm LP, emission filter 610/75.

![Figure 57](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-57-mscarlet-micro.png "Figure 57")***Figure 57: fluorescence of pET-21 b (+)\_Ana o 3\_OmpA\_mScarlet-I transformed Tuner cells.** Cells transformed with plasmids not containing mScarlet-I were used as negative controls. Observations were made at the microscope parameters for mScarlet-I.*

---

### FACS experiments

As for the microfluidic experiment, we wanted to go through the whole cytometric approach since we had achieved to build the strains and had the material to do so, and even if we did not have enough time to perform all the needed controls to assess allergen and DARPin functions.

#### Cytometric validation of the fluorescent strains

The first step was to check if the blue and red signals were detectable using a Spectral FACS from Cytek Biosciences. Liquid cultures of DARPin with and without BFP or of both allergens with and without mScarlet-I strains were performed and used to calibrate the FACS machine. (Figure 58).

![Figure 58](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-58-facs-graph.png "Figure 58")***Figure 58: mTagBFP and mScarlet-I detection with the Spectral FACS.** PBS medium was tested on (A), strain A Ana o 3 with constitutive expression of mScarlet-I on (B), Strain A Ara h 2 expressing Ara h 2 with constitutive expression of mScarlet-I on (C), strain A Ara h 2 without expression of mScarlet-I on (D), strain D with constitutive expression of mTagBFP1 on (E) and strain D without expression of mTagBFP1 on (F). 10,000 events were recorded. The population was selected according to the area of their shadow under the laser and their granulometry before selectioning particles corresponding to cell and cellular aggregates. Laser channel V3-A was used for blue signals and YG3-A for red signals.*

Nonfluorescent signals from the PBS could be seen on Figure 58A while the specificity of the DARPin/mTagBFP and Allergen/mScarlet-I fluorescent cells was validated on respectively figure 58E, and 58B and 58C, with lost of the signals without the fluorescent reporters (58D and 58F).

#### Observing aggregation through FACS

We then performed a first preliminary aggregation assay by mixing fluorescent strain A and strain D with or without the IgE corresponding to strain A (Figure 59).

![Figure 59](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-59-facs-graph-aggreg.png "Figure 59")***Figure 59: aggregation assay and cytometric analysis.** Strain A Ara h 2 with constitutive expression of mScarlet-I was mixed with strain D with constitutive expression of mTagBFP1 in presence of either no IgE (A), non-specific anti-Der p 1 IgE (B) or specific anti-Ara h 2 IgE (C). 100,000 events were recorded for (A) and (C) and 10,000 events were recorded for (B). The population was selected according to the area of their shadow under the laser and their granulometry before selectioning particles corresponding to cell and cellular aggregates. Laser channel V3-A was used for blue signals and YG3-A for red signals.*

While it was possible to observe aggregation (double blue-red signals in the upper right sections of the figures 59ABC), these aggregates were not specific to the presence of the corresponding IgE since the same occurrences were obtained without IgE, with a non-specific IgE or with a specific IgE (respectively figures 59A, 59B and 59C). We could only conclude this assay remains to be optimized.

#### Detecting allergen enrichment

Even if the previous assay pointed out a lack of specificity of the aggregates for the IgE, we chose to go for the final experiment of the project by assessing a putative enrichment in strain A Ara h 2 in the aggregates when in presence of Ara h 2 IgE. To do so, we isolated 96 aggregates on a LB-ampicillin filled microtitration plate using the sorting function of the Spectral FACS. As control, 96 aggregates from the control without IgE were isolated too. The aggregates were sorted in LB medium with ampicillin and left for growth during 72 hours.
Analytical PCR were then performed on 7 aggregates to assess our capacity to identify the allergen carried by strain A for each aggregate (Figure 60). Primers used were screening\_insert F/ IF4\_Ana o 3 (expected size: 584 bp) and screening\_insert F/IF4\_Ara h 2 (expected size: 748 bp) (see [Primers](https://2022.igem.wiki/toulouse-insa-ups/materials#slide1-chapter4)).

![Figure 60](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-60-facs-pcr-aggreg.png "Figure 60")***Figure 60: analytical PCR of the aggregates.** Strains A with Ara h 2 and Ana o 3 with constitutive expression of mScarlet-I was mixed with strain D with constitutive expression of mTagBFP1 in presence of either specific Ara h 2 IgE (A) or no IgE (B), and sorted by FACS on LB-ampicillin filled microtitration plates. 7 colony PCR were performed using primers screening\_insert F and IF4\_Ara h 2 to investigate Ara h 2 presence (expected size 584 bp) and primers screening\_insert F and IF4\_Ana o 3 to investigate Ana o 3 (expected size 748 bp) presence in the aggregate. (A) gel of the PCR product from the assay. (B) gel of the PCR product from the negative control without IgE.*

As could have been expected from the cytometric profiles of Figure 59, no specific enrichment was obtained for Ara h 2. Ara h 2 strains were present in 5 over 7 aggregates of the assay (Figure 60A). It would have been of interest to assess if Ana o 3 was present in the 2 other aggregates but the PCR targeting Ana o 3 did not work and there was unfortunately no time left to try it again. Besides, Ara h 2 strains were present in all the negative control aggregates (without the corresponding IgE). We concluded that our sorted aggregates were not specific, and further statistical analysis would be pointless. This is not surprising since the sorting step previously showed no increase in the aggregate number when adding the antibody. However, we were able to prove that our idea was feasible as it is possible to sort aggregates out to identify which strain composed them.

#### Conclusion

We concluded that the cytometric approach is technically feasible since we were able to create the strains, to detect aggregates with a cytometric approach and to assess which allergen is present in these aggregates. We did not have enough time in the course of the iGEM competition to perform troubleshooting and reproduce the experiment with a variety of conditions. Many controls would have been necessary here, starting with the validation of DARPin and allergen expression at the cellular membrane before the FACS, which could not be achieved precedently (see “Controls to verify correct protein expression” in “Correction of strain D”). Optimization of the clumping conditions according to our modeling results will also be mandatory to improve the assay.

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### Overall conclusions

We knew from the start we were engaging in a very ambitious project. Positive results would have meant this project to have major consequences well above iGEM. We also knew from the start that four months for such an accomplishment would have been miraculous. In September, we had to make a choice between troubleshooting the mis-functioning controls or pushing toward high-throughput approaches (a summary of our activities is shown in Figure 61). We opted for the latter because we were eager to learn about these state-of-the art approaches.

![Figure 61](https://static.igem.wiki/teams/4197/wiki/results/facs/fig-61-gantt.png "Figure 61")***Figure 61: GANTT of our experimental activities.** First correct constructions were obtained in the beginning of July allowing controls and microscopy activities. Last constructions with mScarlet-I reporter were performed in September. The improved version of the DARPin was built and tested by the end of August. Controls took a lot of our time for a somewhat meager result and high-throughput technologies were investigated in the last two weeks of the project.*

All in all, we are proud to have done so much over the summer, from cloning to modeling, from cell culture and structural biochemistry to microscopy, cytometry and microfluidic. Thanks iGEM!

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## References

* [Kim, B., Eggel, A., Tarchevskaya, S. S., Vogel, M., Prinz, H., & Jardetzky, T. S. (2012). Accelerated disassembly of IgE–receptor complexes by a disruptive macromolecular inhibitor. *Nature*, *491*(7425), 613–617. https://doi.org/10.1038/nature11546](https://doi.org/10.1038/nature11546)
* [*The Sequence Manipulation Suite*. Home - Bioinformatics.org. https://www.bioinformatics.org/sms2/](https://www.bioinformatics.org/sms2/)
* [Zhou, K., Zhou, L., Lim, Q., Zou, R., Stephanopoulos, G., & Too, H.-P. (2011). Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. *BMC Molecular Biology*, *12*(1), 18. https://doi.org/10.1186/1471-2199-12-18](https://doi.org/10.1186/1471-2199-12-18)
* [Barthe, M., Tchouanti, J., Gomes, P. H., Bideaux, C., Lestrade, D., Graham, C., Steyer, J.-P., Meleard, S., Harmand, J., Gorret, N., Cocaign-Bousquet, M., & Enjalbert, B. (2020). Availability of the Molecular Switch XylR Controls Phenotypic Heterogeneity and Lag Duration during Escherichia coli Adaptation from Glucose to Xylose. *mBio*, *11*(6), Article e02938-20. https://doi.org/10.1128/mbio.02938-20](https://doi.org/10.1128/mBio.02938-20)

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