iGEM 2025 — Team wageningenur

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BCoated Notebook
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# NOTEBOOK

---

Contents

* [Team notebook](#team-notebook)
* [Functionalisation notebook](#functionalisation-notebook)
* [Production platform notebook](#production-platform-notebook)
* [Seed coating & application notebook](#seed-coating-and-application-notebook)
* [Dry lab notebook](#dry-lab-notebook)

# Team notebook

Weekly team meetings

**January–February**

Students and Supervisors were welcome to attend weekly iGEM brainstorming sessions held by the Principle Investigators (PIs). Over the course of two months, the ideas for the project were narrowed down and the BCoated team, consisting of eleven students, was formed.

**March–April**

The team entered the Synthetic Biology Project Design course for two months. In the first month, the team was introduced to research methods required to develop a good iGEM project. In the second month, each student developed their own subproject culminating in BCoated’s project as a whole.

**May–October**

**12/05/2025** - In this meeting we decided on our team name – BCoated. We discussed possible special prize nominations for our team and had short updates from every team (design, finance, and HP).

**19/05/2025** - In this meeting we mainly discussed our main storyline. We held a brainstorming session where five different questions were asked, and each team was given three minutes to write down answers and three minutes to discuss in groups of three, before finally sharing the findings with the rest of the team. Through this, we identified the main threads of how we perceive the story and how it could appeal more effectively. Additionally, we began discussing the logo and the technical aspects of our project for the laboratory work.

**26/05/2025** - In this meeting we decided which prizes we wanted to target by voting as a team, and agreed to focus on entrepreneurship and integrated human practices. Additionally, we received feedback on the draft logo from the team.

**16/06/2025** - In this meeting we updated each other on our progress in the lab (scientific updates), generally on how our project was going, whether we were struggling with anything, and what we had accomplished so far. We also discussed the non-scientific aspects, including timelines and a team contract. We considered different options for selecting our village, weighing up the pros and cons, and additionally started sharing ideas for our graphical abstract.

**23/06/2025** - In this meeting we discussed the common medium we should use for *K. sucrofermentans* and also discussed BC weight measurement. We also talked about the protocol deadline and prepared a protocol presentation for the supervisors’ meeting.

**30/06/2025** - In this meeting we had a team coaching session which gave us points to improve upon. We learnt more about how to make decisions and maintain clear expectations within the group. We also decided to hold feedback sessions and appointed someone to support the team’s well-being.

**07/07/2025** - In this meeting each team (design, finance, and HP) updated the group on their progress. We received feedback on the wiki and promotional video. HP had contacted many industry users and planned to contact more end-users.

**14/07/2025** - In this meeting we finalised and signed the team contract. We discussed the script for the promotional video as well as the graphical abstract for our project. We brainstormed slogans and created multiple options, which were later put to a vote. The winning slogan is now featured on our logo.

**21/07/2025** - In this meeting we discussed presentation ideas for the Jamboree. The entrepreneurship team shared their plans after attending a workshop. We also explored different types of seed coatings and the possibility of dyeing the BC.

**04/08/2025** - In this meeting we had further updates from the design and finance teams. This week we began coding our wiki and sourcing vendors for printing our hoodies and other merchandise. The finance team planned to conduct interviews for the business plan. We also started setting deadlines for the wiki writing.

**11/08/2025** - In this meeting we agreed on a deadline for when each of us should stop lab work and begin focusing on writing the wiki. We also discussed which special prizes and project components would contribute to the team’s submissions. By this week we had also decided on the colour of our team hoodie (baby pink!).

**18/08/2025** - In this meeting we decided on the storyline for the home page. We confirmed our special prize choices and divided tasks for the Mini Jamboree in Eindhoven. We set writing rules to support coherent writing for our wiki and agreed to draft it in Overleaf.

**25/08/2025** - In this meeting we finalised the layout for the wet lab section of the wiki, splitting it into three pages to reflect the three parts of our project. The design team reported that hoodies had been ordered and that the script for the presentation video was being prepared. We divided tasks for the short abstracts required for Jamboree presentation slots and decided which topics to sign up for.

**12/05/2025** - In this meeting we decided on our team name – BCoated. We discussed possible special prize nominations for our team and had short updates from every team (design, finance, and HP).

**01/09/2025** - In this meeting we agreed to finish lab work by the second week of September. We discussed several deadlines, including the attributions form, the notebook, and the wiki writing. We also considered how best to incorporate all aspects of our project into the wiki to create a clear storyline. Additionally, we discussed the new iGEM parts registry and the need to upload our parts soon.

**08/09/2025** - In this meeting we revisited deadlines for the wiki project pages. We agreed to seek feedback from both the team and supervisors for each page. We also standardised our plots using Python so that the graph formats would be unified. Additionally, we conducted an anonymous feedback survey for team members.

**15/09/2025** - In this meeting we discussed the team booth safety proposal and live stage talk proposals to be submitted, as well as the team roster freeze, ensuring that all members, supervisors, and PIs were registered. We reviewed the deliverables form and decided who was still permitted to work in the lab. Additionally, we discussed hyperlinking and terminology for the wiki writing.

**22/09/2025** - In this meeting we received our first feedback. We discussed registering parts on the iGEM registry and ensuring that everyone uploaded their contributions. We also identified pages that were still unwritten and assigned them to team members.

Weekly meetings with PIs and instructors

**14/05/2025** - In this meeting, we discussed which special prizes we wanted to aim for.

**21/05/2025** - In this meeting, Niels presented the progress on his use case subproject, *Targeted pest control*, and received feedback on how to proceed further. Furthermore, the design team unveiled the first drafts they developed for the BCoated logo and received feedback on these drafts.

**28/05/2025** - In this meeting, Jochem presented the progress the team made regarding successful Bacterial Cellulose (BC) production. Rick presented his progress on developing the model for the genetic circuit used to regulate external ethanol concentration in the co-culture of *Saccharomyces cerevisiae* and *Komagataeibacter sucrofermentans*, and received feedback.

**04/06/2025** - In this meeting, Varsha presented the progress she made on developing a standardised protocol for BC production, and Tifara presented the challenges she faced with cloning her constructs for the Biodegradability subproject. Following this, a discussion was held regarding which village to select: Biomanufacturing or Agriculture.

**18/06/2025** - In this meeting, Iris and Camilie presented the challenges they faced in developing a co-culture of *S. cerevisiae* and *K. sucrofermentans*, as well as the genetic circuit in *S. cerevisiae* for regulating external ethanol concentration. Following the presentations, after gaining insight on how to progress further, the finalised BCoated logo was revealed.

**25/06/2025** - In this meeting, Andra presented the challenges she faced with cloning her constructs for the Porosity subproject. Subsequently, a discussion was held regarding which medium; Hestrin-Schramm (HS) or Yeast Extract Peptone Dextrose (YPD) to use for experiments with BC.

**09/07/2025** - In this meeting, Anirudhan presented the challenges he faced in developing the model for consortium dynamics, and an update was given on all the standardised BC protocols developed so far. Finally, the team announced that they had selected the Biomanufacturing village.

**16/07/2025** - In this meeting, Rick presented the final version of his model for the genetic circuit in *S. cerevisiae*, and the HP team provided an update on all the interviews conducted so far.

**23/07/2025** - In this meeting, Iris and Daan presented the progress of the experiments they had conducted for their subproject, and the team revealed the finalised slogan: *Tailored coatings for tomorrow's crops*. Furthermore, the team received feedback on how to improve the first draft of the main graphical abstract outlining the entire BCoated project.

**30/07/2025** - In this meeting, a discussion was held regarding the different methods to coat seeds.

**06/08/2025** - In this meeting, Yoerik presented the seed germination and coating protocol he developed, and the Entrepreneurship team presented the progress they had made in developing the business plan.

**13/08/2025** - In this meeting, the design team presented the first draft of the homepage and received feedback on the layout.

**27/08/2025** - In this meeting, Camilie and Iris presented the progress they had made with their subprojects.

**01/09/2025** - All meetings held in September were dedicated to providing feedback on design and text written for the wiki, and the final presentation video.

Entrepreneurship

**27/07/2025 – 09/08/2025**

We identified the market size (TAM, SAM, SOM) and performed competitor analysis.

**10/08/2025 – 16/08/2025**

We compared bacterial cellulose with other materials.

**17/08/2025 – 30/08/2025**

We conducted a cost analysis and risk analysis.

**31/08/2025 – 06/09/2025**

We performed a financial analysis.

**07/09/2025 – 13/09/2025**

We worked on acquiring funding and developing a timeline.

**14/09/2025 – 20/09/2025**

We worked on acquiring funding and defining the minimum viable product.

**21/09/2025 – 27/09/2025**

We outlined a Business Model Canvas, explored our first customers, and discussed the process of patenting.

**28/09/2025 – 04/10/2025**

We reviewed the team's capabilities and the long-term effects of BCoated and we reviewed and answered the judging criteria.

Integrated human practises

**26/03/2025** - Camile and Jochem interviewed Dr. Shanmugam Thiyagarajan, a polymer expert from Wageningen University & Research (WUR).

**04/04/2025** - Nico Classens contacted and kept in touch with Prof. Tom Ellis from Imperial College London (ICL), working in the Department of Bioengineering, via e-mail. Yoerik interviewed Prof. Dr. Ir. Jonne Rodenburg, an Associate Professor of Weed Ecology from WUR, working at the Centre for Crop Systems Analysis.

**10/04/2025** - Jochem, Iris, Varsha, Yoerik and Daan interviewed Dr. Amritpal Singh, a scientist with expertise in bacterial cellulose.

**11/04/2025** - Niels interviewed Dr. DW (Desalegn) Etalo from WUR Phytopathology.

**10/05/2025** - Niels reached out to Dr. Ir. Ing Dennis Oonincx and his student Mrtyhe Duijsens for their expertise in animal nutrition for the bioinsecticide protein incorporation against worms.

**01/06/2025** - Iris, Jochem and Camile developed an aim and a plan of action for iHP along with Charlotte and Sophie (supervisors).

**17/06/2025** - Iris interviewed Prof. Dr. Ir. Pascale Daran-Lapujade, from Delft University of Technology, who also provided us the strain *Saccharomyces cerevisiae* IMX1812, deficient in glucose transport.

**22/06/2025** - Camile interviewed Prof. Dr. Ir. Jan van den Ende from Rotterdam School of Management.

**25/06/2025** - Camile and Jochem interviewed Maroushka Blahetek, the CEO and co-founder of Carapace Biopolymers.

**27/06/2025** - Jochem and Camile interviewed Jeroen van Rotterdam, the CEO of Foamlab. Iris interviewed Diego Silva Russi, the CEO \& International Business Consultant Institution NanoCell Farms – Colombia / LATAM.

**18/07/2025** - Jochem and Camile interviewed Jacob P. Rohn, the CEO of Seedforward.

**21/07/2025** - Iris and Jochem interviewed Ece Başak and Şevval Kocaman from SUMATRIX Biotech.

**30/07/2025** - Niels and Yoerik interviewed Zohaib Hussain, a researcher at the Physical Chemistry and Soft Matter group at WUR.

**07/08/2025** - Iris and Camile interviewed Amalia Kafka the Technical Manager of Sustainable Seed Technologies at Euroseeds.

**13/08/2025** - Camile conducted a short interview with a South Holland Beetroot and Wheat Farmer.

**27/08/2025** - Iris contacted and kept in touch with members of the European Commission, via email. After several referrals, the European Commission - DG SANTE - E4 Unit answered some questions for us.

**01/09/2025** - Iris contacted and kept in touch with members of RIVM.

**10/09/2025** - Camile spoke to an ecological farmer.

**16/09/2025** - Camile and Jochem interviewed Bert Smit, who gave us the contact of a farmer.

**17/09/2025** - Jeroen Roelfsema, Samia Ouhajji, and Casper Jongerkrijg (RIVM) were interviewed.

**18/09/2025** - Camile interviewed the farmer through phone.

Events

**29/04/2025** - Anirudhan and Camile had a first meeting with Sowmya Shreedhar to discuss the organisation of the Dutch iGEM meet.

**02/05/2025** - Andra, Niels, Rick and Varsha attended the SynBioNL event held at Utrecht University, where they met Debarun Dhali. He connected them to Robin Reijnen who is involved in the STEP (Student Entrepreneurship Program) offered by StartHub Wageningen.

**11/06/2025** - Iris attended the Bigger Better Biotech Symposium held at Wageningen University. Niels, Rick and Anirudhn had their first STEP meeting with Robin Reijnen.

**11/07/2025** - The BCoated team attended and helped facilitate the Dutch iGEM meet 2025.

**26/07/2025** - Iris, Niels and Anirudhan attended the IGEM Startups Summer School.

**10/08/2025** - Iris visited the SUMATRIX Biotech facility and met the CEO, Semra Ünal Yıldırım.

**05/09/2025** - Niels visited the Stadsbrouwerij Rad van Wageningen to obtain their beer waste stream.

**08/09/2025** - Andra, Anirudhan and Camile gave a guest lecture to WUR students taking a course in ethics.

**21/09/2025** - Anirudhan and Tifara presented our project at a plant synthetic biology workshop hosted by the iGEM UGM Indonesia team.

**25/09/2025** - Anirudhan, Camile, Jochem, Niels, and Tifara visited the Biobased Europe Pilot Plant.

# Functionalisation notebook

---

Tuning the water holding capacity of BC through enzymatic modifications (Varsha)

## Week 1 - Week 2 (May 1st-10th)

**Aim:** To gather knowledge and design
experiments for Bacterial Cellulose (BC) production.

**Method:** Conducted literature review and
drafted experimental plans.

## Week 3 (May 11-17th)

**Aim:** To revive and cultivate
*Komagataeibacter sucrofermentans* from the DSMZ
freeze-dried culture, in liquid and solid growth media.

**Protocol:** [Revival
of Freeze-dried Microbial Cultures from DSMZ](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Observation/Results:** A viable culture was
established as thick BC pellicles were observed at the air-media
interface after 5 days of growth.

## Week 4 (May 18th-24th)

**Aim**: To prepare glycerol stocks and ensure
long-term preservation of *K. sucrofermentans.* .

**Protocol:** [Glycerol
Stocks](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Observation/Results:** Glycerol stocks were
created and stored at 80 °C.

## Week 5 - 6 (May 25th- June 7th)

**Aim**: To determine pH and temperature
conditions for BC production in Hestrin Schramm (HS) Media.

**Protocol:** [HS
Media](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Method:** Non-vented Petri dishes were filled
with 20 mL of HS media adjusted to pH 5, 6, or 7, and 400 μL of active *K.
sucrofermentans* culture. Plates were placed at their
respective temperatures: 25, 30, or 35 °C. Each condition was tested in
triplicate, and controls contained only media. Plates were
incubated for one week, after which BC production was
observed.

**Observation/Results:** At pH 5, BC production
was highest at 25 °C,
forming thick sheets, decreased at 30 °C, and was minimal at 35 °C. At pH 6, substantial BC formed
at 25 °C but with a looser
structure than at pH 5, slightly thinner at 30 °C, and absent at 35 °C. At pH 7, BC was only observed
at 25 °C as a thin, loose
sheet and was absent at 30 °C and 35 °C. All control plates remained
clear under all conditions.

## Week 7 - 8 (June 8th-21st)

**Aim:** To clean and freeze-dry BC
post-production.

**Protocol:** [Downstream
Processing BC.](https://2025.igem.wiki/wageningenur/protocols/index.html#bc-production-and-measurements)

**Observation/Results:** The freeze dried BC were
pale white sheets with a crispy and porous texture.

![](https://static.igem.wiki/teams/5713/notebook/notebookvarsha/varsha-notebook-fig1.webp)

Freeze dried BC

## Week 12 - 13 (July 20th-26th)

**Aim:** To produce BC with Yeast Extract Peptone
Dextrose (YPD) Media.

**Protocol:** [YPD
Media](https://2025.igem.wiki/wageningenur/protocols/#general-lab), [BC
Production, Downstream Processing BC.](https://2025.igem.wiki/wageningenur/protocols/index.html#bc-production-and-measurements)

**Observation/Results:** BC was successfully
produced.

## Week 14 (July 27th-August 2nd)

**Aim:** To assess the water-holding capacity
(WHC) of unmodified BC.

**Protocol:**[Water
Holding Capacity](https://2025.igem.wiki/wageningenur/protocols/index.html#bc-production-and-measurements)

**Results:** The WHC of natural BC was calculated
to be 13 AU amount.

## Week 15 (August 3rd-9th)

**Aim:** To complete Attenuated Total Reflectance
– Fourier Transform Infrared Spectroscopy (ATR-FTIR) training.

**Method:** Training was completed.

**Results:** Gained knowledge on how to operate
the machine ATR-FTIR Spectrometer (Bruker TENSOR II).

## Week 16 - 17 (August 10th – 23rd)

**Aim**: To gather knowledge and design
experiments for the direct functionalisation of BC using enzymatic
treatments, specifically a laccase/TEMPO-mediated system and a
lipase-catalysed system, to introduce new functional groups onto
the BC.

**Method:** Conducted a literature review and
drafted experimental plans.

## Week 18 - 19 (August 24th – August 30th)

**Aim**: To enzymatically modify BC using
laccase/TEMPO- and lipase-mediated catalytic systems to introduce
new functional groups, and characterise these modifications using
ATR-FTIR spectroscopy.

**Method**: The Laccase/TEMPO catalytic system was
used to target and oxidise the -OH groups in BC chains,
introducing aldehyde (-CHO) or carboxyl (-COOH) groups to enhance
BC’s hydrophilicity and, consequently, its WHC. To achieve this,
5m̃L tubes were prepared, each containing 15mg BC. A master mix was
prepared containing the enzyme laccase from *Trametes
versicolor* (0.4mg/mL) and 2,2,6,6-Tetramethylpiperidin-1-oxyl
(TEMPO) (2mg/mL) dissolved in 0.1M̃ sodium acetate buffer (pH 4.5).
3 mL of the master mix was added to the sample tube and the
mixture was incubated at 50 °C for 3 h. After incubation, the
mixture was filtered and the solid was collected. The solid was
washed with ethanol, frozen overnight, freeze-dried, and analysed
by Attenuated Total Reflectance Fourier Transform Infrared
(ATR-FTIR) Spectroscopy (Bruker TENSOR II). Wild-type, untreated
BC was used as a control.

The enzyme lipase, from *Candida rugosa*, was used to
esterify the -OH groups in BC chains with palmitic acid, in an
effort to introduce more hydrophobic functional groups and reduce
the WHC of BC. To achieve this, 1.5m̃L tubes were prepared, each
containing 10mg BC, 50mg palmitic acid, and μL of 1-butanol. 10 mg of lipase
was added, leaving one tube as an enzyme-free control. The
mixtures were incubated at 50 °C for 3 h in a fume hood. After
incubation, the mixtures were filtered, the solids were collected,
washed with ethanol, frozen overnight, freeze-dried, and analysed
by ATR-FTIR spectroscopy.

**Result**: **Table 1.** Expected
FTIR absorption bands of BC samples with corresponding wavenumber
ranges (cm-1) and
characteristic vibrations. The ranges are color-coded to
correspond with the arrow annotations in [2](#fig:FTIRLaccase) and [3](#fig:FTIRLipase).

![](https://static.igem.wiki/teams/5713/notebook/notebookvarsha/varsha-notebook-table1.webp)

![](https://static.igem.wiki/teams/5713/notebook/notebookvarsha/varsha-notebook-fig2.webp)

ATR-FTIR absorbance spectra of BC samples with an
accumulation of 128 scans, 4 cm-1 resolution and range
of 4000-500 cm-1. The axes show wavenumber
(cm-1), and absorbance in arbitrary units (AU). (A) BC
(control); (B) BC incubated laccase/TEMPO reaction mixture for
3 h. Arrows indicate key characteristic vibrations, colour-coded
to correspond with **Table 1**

The Laccase/TEMPO-mediated oxidation of BC was successful as we
see the apperance of the of a C=O band.

![](https://static.igem.wiki/teams/5713/notebook/notebookvarsha/varsha-notebook-fig3.webp)

Figure 4. ATR-FTIR absorbance spectra of BC samples
and palmitic acid with an accumulation of 128 scans,
4 cm-1 resolution and range of
4000-500 cm-1. The axes show wavenumber
(cm-1), and absorbance in arbitrary units (AU). (A) BC
(control); (B) palmitic acid; (C) BC + palmitic acid without
lipase (control); (D) BC incubated in palmitic acid and lipase
reaction mixture for 3 h. Arrows indicate key characteristic
vibrations, colour-coded to correspond with **Table
1** .

The lipase-catalysed esterification of BC was inconclusive.

## Week 19 (August 31st – September 6th)

**Aim**: To calculate and compare the WHC of both
enzymatically treated and untreated BC samples.

**Protocol:** [Water
Holding Capacity](https://2025.igem.wiki/wageningenur/protocols/index.html#culturing-and-transformation)

**Results:**

![](https://static.igem.wiki/teams/5713/notebook/notebookvarsha/varsha-notebook-fig4.webp)

Water holding capacity of BC, expressed in arbitrary
units (AU), before and after incubation in a laccase/TEMPO
reaction mixture. Bars indicate the untreated BC (Control) and BC
incubated for 3 h.

Preliminary data showed that BC samples incubated with the
laccase/TEMPO reaction mixture had higher WHC, around 37 AU, while
the control was 13 AU, meaning there was a 185% increase in WHC
after treatment.

Tuning biodegradability by introducing heterologous genes (Tifara)

## Week 3 (May 11-17th)

**Aim:** To design primers *in silico*.

**Methods/Protocols:** Primers and synthetic gene
were designed *in silico* with Snapgene.

**Observations/Results:** Primers and synthetic
gene were designed and ordered by the end of the week.

## Week 4 (May 18th-24th)

**Aim:** To amplify and purify parts for the
heterologous genes

**Methods/Protocols:**The first PCR was done, and
the parts were amplified and purified. [PCR,
gel electrophoresis, and DNA extraction](https://2025.igem.wiki/wageningenur/protocols/#general-lab) from gel was
done.

**Observations/Results:** The gene was confirmed
by the band size.

## Week 5 (May 25th-31st)

**Aim:** To assemble the heterologous gene
plasmids

**Methods/Protocols:**  First attempt of [golden
gate assembly](https://2025.igem.wiki/wageningenur/protocols/#general-lab) was done for the heterologous gene plasmids to
pSEVAb33

**Observations/Results:**

![](https://static.igem.wiki/teams/5713/notebook/notebooktifara/tifara-notebook-fig1.webp)

*In silico* design of plasmid after Golden Gate
Assembly of bslA gene to pSEVAb33.

## Week 6 (June 1st-7th)

**Aim:** To transform *E. coli* with
heterologous gene construct

**Methods/Protocols:**  First transformation by
heat-shock of *E. coli* DH5a with heterologous gene
construct was done

**Observations/Results:** Few colonies were
observed with bslA plasmid, but no colonies were formed for crdS
plasmid. Colony PCR showed the right band size for bslA
plasmid.

## Week 7 (June 8th-14th)

**Aim:** To amplify synthetic gene (crdS) and
assemble the heterologous gene (crdS) plasmid

**Methods/Protocols:**  Synthetic gene was
amplified due to previous failure in construct and further
assembled with [Golden
Gate Assembly](https://2025.igem.wiki/wageningenur/protocols/#general-lab) with pSEVAb33

![](https://static.igem.wiki/teams/5713/notebook/notebooktifara/tifara-notebook-fig2.webp)

*In silico* design of plasmid after Golden Gate
Assembly of crdS gene to pSEVAb33.

## Week 8 (June 15th-21st)

**Aim:** To design primers *in silico* for
inducible promoters

**Methods/Protocols:** Primers were designed
*in silico* with Snapgene.

**Observations/Results:** Primers were designed
and ordered by the end of the week.

![](https://static.igem.wiki/teams/5713/notebook/notebooktifara/tifara-notebook-fig3.webp)

.

*In silico* design of plasmid of hpdR/phpdH
inducible promoter

## Week 9 (June 22nd-28th)

**Aim:** To amplify and purify parts for inducible
promoters

**Methods/Protocols:** Parts for inducible
promoters were amplified and purified by [PCR,
gel electrophoresis, and DNA extraction](https://2025.igem.wiki/wageningenur/protocols/#general-lab) from gel.

**Observations/Results:** Some parts were
successfully amplified and purified. There were multiple primers
used and multiple PCR to be done, thus continued also next
week.

## Week 10 (June 29th – July 5th)

**Aim:** To amplify and purify parts for inducible
promoters and assemble the inducible promoter plasmids.

**Methods/Protocols:** Several parts for inducible
promoters were amplified and purified and [Golden
Gate Assembly](https://2025.igem.wiki/wageningenur/protocols/#general-lab) of hpdR/phpdH inducible promoters to
pSEVAb33

**Observations/Results:**

![](https://static.igem.wiki/teams/5713/notebook/notebooktifara/tifara-notebook-fig4.webp)

*In silico* design of plasmid after Golden Gate
Assembly of hpdR/phpdH inducible promoter to
pSEVAb33.

## Week 11 (July 6th-12th)

**Aim:** To transform *E. coli* DH5a with
inducible promoter plasmids  and make media for *K.
sucrofermentans* (HS & YPD)

**Methods/Protocols:** [Heat-shock
transformation of *E.coli*](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation) DH5a with inducible promoter
plasmids (hpdR/phpdH) and made media for *K.
sucrofermentans* (HS & YPD)

## Week 12 (July 13th-19th)

Start trying the transformation protocol of *K.
sucrofermentans*

**Aim:** To try the first electroporation
transformation of *K. sucrofermentans.*

**Methods/Protocols:** 1st
Electroporation Protocol:

* Single colonies of *Komagataeibacter* were
  inoculated in 5mL HS broth—0.2% (w/v) glucose and grown at 30ºC
  for 72h
* The cultures were vortexed for 3 min prior to subculturing
  1:20 in 4 tubes of 30 mL fresh HS broth—0.2% (w/v) glucose and
  grown at 30°C for 24 h, with aeration.
* 0.2% (v/v) cellulase (T. reesei ATCC 26921 cellulase,
  Sigma, #C2730) was added into the cultures and continued to grow
  at 30ºC for 4h, with aeration, to breakdown cellulose
  pellicles
* Cultures were vortexed to further dissolve the
  cellulose
* Bacteria were centrifuged at 4100 rpm, 12min, 4ºC
* Supernatant was discarded carefully, and pellets were
  resuspended in 10mL ice- cold 1mM HEPES (pH 7.0)
* All cultures were pooled together in one tube and repeated
  centrifugation as above
* The cultures were washed similarly in 10mL 1mM
  HEPES
* Bacteria were pelleted again at 4100 rpm, 12 min,
  4ºC
* Supernatant was discarded, and pellets were resuspended in
  1.5mL of 15% (v/v)
* 4−7 μL of pure plasmids were added into 100 μL
  electrocompetent cells and transferred into a 0.1 cm Gene Pulser
  Electrocuvette (Bio-Rad).
* *Komagataeibacter* was electroporated at 2.5 kV, 5−6
  ms.
* The cells were recovered by adding 800 μL HS broth−0.2%
  (w/v) glucose supplemented with 0.2% (v/v) cellulase and grown at
  30°C for 16 h, with aeration.
* Following overnight recovery, the cells were spread onto HS
  agar−0.2% (w/v) glucose

**Observations/Results:** Arcing occurred during
electroporation and although the samples were still plated, no
colonies were observed.

## Week 13 (July 20th-26th)

A well-deserved break!

## Week 14 (July 27th-August 2nd)

**Aim:** To redo the transformation of K.
sucrofermentans and cloning of mmsR/pmmsA inducible promoter in
*E.coli* ST18

**Methods/Protocols:** Heat-shock transformation
of *E. coli* ST18

2nd Electroporation Protocol (from previous iGEM
team):

* Inoculate 5mL of HS+0.2 (v/v)cellulase medium with
  Komagataeibacter.
* Incubate at 30°C, 180rpm shaking overnight or until
  cloudy.
* Next day, pour1 0mL of HS+cellulase mediumin to each of 8
  of 50mL tubes.
* To each tube, add seed culture to OD600=0.04 and incubate
  with shaking at 180 rpm, 30 °C overnight until
  OD600=0.4-0.7.
* Before continuing, set up the necessary materials:

  + Pre-cool centrifuge to 4 °C
  + Prepare ice bucket
  + Chill 1mM HEPES bu]er and 15% glycerol bu]er on
    ice
  + Label glycerol stock tubes
* Once the cultures reach desired OD600, take them out of
  incubation and put them on ice for 10 minutes (the tube should
  feel cool).
* From here on, keep cells at 4°C
* After cooling, spin the tubes in a refrigerated (4°C)
  centrifuge for 12 min at 3200g.
* Pour supernatant carefully, taking care not to pour the
  pellet.
* Re-suspend bacteria in 10 mL HEPES: re-suspend first using
  1 mL HEPES and a P1000 pipette, then add 9 mL of HEPES using a
  stripette; it is much easier to re- suspend the pellet fully using
  a P1000.
* At this point can pool 2 samples into one tube to reduce
  handling time.
* Centrifuge again for 14 minutes at 3200 g and 4 °C
  temp.
* Pour supernatant, re-suspend pellet in 10 mL ice-cold HEPES
  on ice as before.
* Centrifuge for 14 minutes at 4100 rpm, 4 °C.
* Pour supernatant and re-suspend pellet in 1 mL ice cold 15%
  glycerol solution. Pool all samples and add glycerol to a total of
  6 mL.
* Pipette 100 μl aliquots into tubes. Store samples on ice
  for immediate use or freeze aliquots in -80 °C. The efficiency of
  electrocompetent cells may reduce after each freezing, so
  immediate use may result in highest efficiencies.
* Set up the electroporator with settings at 2.5 kV, 5-8 ms,
  400 Ohm resistance, 25 μF capacitance.
* Prepare 15 mL culture tubes containing 800 μL HS+cellulase
  media.
* Prepare ice bucket, place plasmid DNA and electrocuvettes
  on ice and thaw electrocompetent cells on ice. NB! Make sure DNA
  is desalinated before use – ionic solutions can cause
  arcing.
* Add 2 μL (20-200 ng/μL) of plasmid DNA to 100 μL of
  concentrated cells in a cold microcentrifuge or PCR tube and mix
  well by pipetting. Don’t add plasmid DNA to one aliquot of
  electrocompetent cells for negative control.
* Transfer the cell/DNA mixture to a cold electroporation
  cuvette. Dry any water condensate outside of the cuvette, place
  the cuvette into the electroporator, and apply the pulse.
* Transfer the pulsed cells into 800 μL of HS+cellulase
  medium in a culture tube.
* Incubate the culture tubes with shaking (170 rpm) at 30 °C
  for 4 hours – 16 hours (large incubation time does not result in
  reduced efficiencies).
* When culturing for 4 hours, spin the culture at max
  available RPM for 10 minutes, resuspend in 200 μl and plate on an
  HS-agar plate with an appropriate antibiotic
  concentration.
* Grow plates at 30 °C inverted, colonies will appear in
  24-72 hours

**Observations/Results:** Three colonies were
observed from 4 different plates and was tested with colony PCR,
however, there were no bands detected which means that
the transformation failed

## Week 15 - Week 16 (August 3rd-16th)

**Aim:**To try different electroporation settings
on *K. sucrofermentans* electroporation protocol

**Methods/Protocols:** The 2nd electroporation
protocol was done with different electroporation settings (mixture
of 2.5kV, 1.8kV, 400 ohm, 200 ohm, and usage of 1mm or 2mm
cuvette).

**Observations/Results:** More colonies were
observed but colony PCR did not confirmed the presence of the
plasmid.

## Week 17 (August 17th-23rd)

**Aim:**To try a new protocol on *K.
sucrofermentans* and send plasmids for sequencing.

**Methods/Protocols:** The new electorporation
protocol on *K. sucrofermentans* were tried out. Plasmids
were sent for sequencing and primers were ordered (16S primers and
internal primers for bcsA synthase gene).

**Observations/Results:** Transformation succeeded
with empty plasmid with very high transformation efficiency. More
can be read in the Wet Lab results page. Internal primers also
detected that previous colonies formed with previous
transformation protocol was not *K. sucrofermentans* and
were contaminants.

## Week 18 (August 24th-30th)

**Aim:** To transform *K. sucrofermentans*
with plasmids that carry heterologous genes and inducible
promoters and construct the negative control (phpdH) for inducible
system.

**Methods/Protocols:** The new [electroporation
protocol](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation) was used.

**Observations/Results:** bslA was successfully
transformed in *K. sucrofermentans*, but other plasmids
were unsuccessfully transformed.

## Week 19 (August 31st – September 6th)

**Aim:** To transform *K. sucrofermentans*
with crdS, phpdH (negative control) and hpdR/phpdH plasmid.

**Methods/Protocols:** crdS, phpdH (negative
control) and hpdR/phpdH plasmid were transformed into *K.
sucrofermentans*.

**Observations/Results:** phpdH (negative control)
and hpdR/phpdH was successfully transformed into *K.
sucrofermentans*, but crdS did not come out with any
result.

## Week 20 (September 7th-13th)

**Aim:** To prepare and run plate reader assay for
hpdR/phpdH inducible system and to grow *K.
sucrofermentans* with *bslA* plasmid.

**Methods/Protocols:** Plate reader assay was done
for hpdR/phpdH indudcible system and *K. sucrofermentans*
with *bslA* plasmid were grown

**Observations/Results:** Plate reader assay
result was not good and cells did not really grow. Repetition
should be done.

## Week 21 (September 14th-20th)

**Aim:**To prepare and run second plate reader
assay for hpdR/phpdH incudible system and downstream process
*K. sucrofermentans* with *bslA* plasmid

**Methods/Protocols:** Second plate reader assay
was prepared and done with single measurements at time 0h, 2h, 4h,
and 6h.

**Observations/Results:** Plate reader assay data
was collected, analysed, and made into plots. *K.
sucrofermentans* with *bslA* plasmid could not be
downstream processed because the BC was still very thin and could
not even be taken out from the flask, thus it is not possible
either to test the biodegradability of the resulted BC with wild
type.

Porosity measurements (Andra)

## Week 2 (May 4th-10th)

**Aim:** To design a plasmid construct for GFP
reporter expression under pLux promoter control.

**Method/Protocol:** Used *in silico*
cloning tools to plan Gibson assembly in pSEVA331 backbone, using
a pSEVA64-pLux-GFPuv plasmid to obtain the desired fragment.

**Observation/Results:** A complete design map for
the pSEVA331-pLux-GFPuv construct was generated.

![](https://static.igem.wiki/teams/5713/notebook/notebookandra/andra-notebook-fig1.webp)

Final *in silico* assembly of
pSEVA331-pLux-GFPuv.

## Week 3 (May 11-17th)

**Aim:** To extend the plasmid design with
motility and sugar metabolism genes, and to complete essential lab
training.

**Method/Protocol:** Performed additional *in
silico* fragment design for Gibson assembly; attended lab
safety training and tour.

**Observation/Results:** Designed primers and
*in silico* assembly of porosity-related genes in
pSEVA331-pLux-GFPuv plasmid. Acquired safety clearance for lab
work.

## Week 4 (May 18th-24th)

**Aim:** To generate DNA fragments for Gibson
assembly.

**Method/Protocol:** Ordered required primers;
performed [PCR](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation)
amplification of target fragments.

**Observation/Results:** Successful PCR products
obtained for downstream cloning.

## Week 5 (May 25th-31st)

**Aim:** To amplify and purify fragments for
Gibson assembly.

**Method/Protocol:** Performed [Q5
PCR and gel electrophoresis](https://2025.igem.wiki/wageningenur/protocols/#general-lab) to obtain fragments for Gibson
assembly. Isolated the fragments from the gel.

**Observation/Results:** Fragments obtained to
continue Gibson assembly next week.

## Week 6 (June 1st-7th)

**Aim:** To assemble the first version of the GFP
reporter plasmid and learn electronic lab notebook usage.

**Method/Protocol:** Performed [Gibson
assembly](https://2025.igem.wiki/wageningenur/protocols/#general-lab) with PCR products, [electroporation](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation)
in *E. coli*; followed ElabJournal training.

**Observation/Results:** After 24 hours a large
amount of colonies grew in the selective plate, leading to the
need to perform a re-streak.

## Week 7 (June 8th-14th)

**Aim:** To verify successful Gibson assembly
clones.

**Method/Protocol:** Performed [OneTaq
colony PCR](https://2025.igem.wiki/wageningenur/protocols/#general-lab) screening and submitted samples for Plasmidsaurus
sequencing.

**Observation/Results:** All the samples sent for
sequencing showed large DNA fragments missing.

## Week 8 (June 15th-21st)

**Aim:** To validate plasmid backbones and
establish a baseline for cellulose production.

**Method/Protocol:** Sequenced plasmids;
inoculated WT *Komagataeibacter* in YPD medium and grew in
agar plates. Collected the bacterial cellulose (BC) and [downstream
processed](https://2025.igem.wiki/wageningenur/protocols/#bc-production-and-measurements) it.

**Observation/Results:** The pSEVA331 plasmid
backbone showed no mutations. The pSEVA64-pLux-GFPuv plasmid
showed multiple fragments missing, leading to the necessity to
order an RBS-LuxR fragment; After two days a BC sheet could be
collected and [downstream
processed](https://2025.igem.wiki/wageningenur/protocols/#bc-production-and-measurements) .

![](https://static.igem.wiki/teams/5713/notebook/notebookandra/andra-notebook-fig2.webp)

Bacterial cellulose sheet after downstream
processing.

## Week 9 (June 22nd-28th)

**Aim:** To assess cellulose porosity and
communicate results visually.

**Method/Protocol:** Produced cellulose sheets;
tested Water Flux Assay for small scale; tested [mass-density
method](https://2025.igem.wiki/wageningenur/protocols/#bc-production-and-measurements).

**Observation/Results:** The water flux assay
showed to be hard to adapt. The mass-density method resulted in a
very high porosity measurement, probably caused by a not proper
removal of the surface water on the cellulose sheet.

![](https://static.igem.wiki/teams/5713/notebook/notebookandra/andra-notebook-fig3.webp)

Attempt to create a structure for water flux
measurement.

## Week 10 (June 29th – July 5th)

**Aim:** To improve construct design and refine
cellulose porosity testing.

**Method/Protocol:** Redesigned DNA fragments and
ordered a pLux-RBS-LuxR synthetic fragment; repeated porosity
assays.

**Observation/Results:** The water flux assay was
again attempted by using a Büchner filter slightly smaller that
the cellulose pellicle. This approach seemed to be the most
successful method to allow standardisation.

![](https://static.igem.wiki/teams/5713/notebook/notebookandra/andra-notebook-fig4.webp)

Ordered fragment.

![](https://static.igem.wiki/teams/5713/notebook/notebookandra/andra-notebook-fig5.webp)

New *in silico* assembly with the ordered
fragment.

![](https://static.igem.wiki/teams/5713/notebook/notebookandra/andra-notebook-fig6.webp)

Attempt of measuring the water flux with a Büchner
filter.

## Week 11 (July 6th-12th)

**Aim:** To generate DNA for reassembly and
improve project communication.

**Method/Protocol:** Performed new [PCRs,
gel elecrophoresis and isolation of the PCR fragments from
gel](https://2025.igem.wiki/wageningenur/protocols/#general-lab).

**Observation/Results:** The new fragments were
successfully amplified.

## Week 12 (July 13th-19th)

**Aim:** To complete reassembly attempts.

**Method/Protocol:** Performed [Gibson
assembly](https://2025.igem.wiki/wageningenur/protocols/#general-lab) conducted [porosity
assays](https://2025.igem.wiki/wageningenur/protocols/#bc-production-and-measurements).

**Observation/Results:** The water flux assay was
attempted again but, after a comparison with data from literature,
it was clear that the method was not reliable. This brought to the
decision to rely, for porosity measurement, only on the water flux
assay and potentially to SEM microscopy.

## Week 13 (July 20th-26th)

Holiday! Ciao!

## Week 14 (July 27th-August 2nd)

Holiday! Ciao!

## Week 15 (August 3rd-9th)

**Aim:** To assemble and test constructs, isolate
genomic DNA and standardise cellulose production for porosity
measurement.

**Method/Protocol:** Performed [Gibson
assembly](https://2025.igem.wiki/wageningenur/protocols/#general-lab); electroporated *E. coli*; isolated
*Komagataeibacter* genome to amplify genes related to
porosity; inoculated *Komagataeibacter* in 1000 mL
erlenmeyer flasks at OD 0.3.

**Observation/Results:** 24 hours after
electroporation of *E.Coli* with the Gibson-assembled
pSEVA331-pLux-GFPuv, a large amount of colonies grew. The plate
was re-streaked on another selective plate;
*Komagataeibacter* genome was successfully isolated.

## Week 16 (August 10th-16th)

**Aim:** To verify assemblies, isolate functional
genes, and begin downstream BC processing and documentation.

**Method/Protocol:** Performed sequencing, [Q5
PCR, gel electrophoresis, and gel isolation](https://2025.igem.wiki/wageningenur/protocols/#general-lab); recorded pH
changes during BC processing; reinoculated cultures.

**Observation/Results:** The sequencing results
showed that the pSEVA331-pLux-GFPuv assembly was successful. The
desired genes, related to porosity, were amplified from
*Komagataeibacter* and isolated. After three days a thick
BC mat was obtained. The culture’s pH dropped from 6.5 to 3, while
the final OD600 was 0.43.

![](https://static.igem.wiki/teams/5713/notebook/notebookandra/andra-notebook-fig7.webp)

Erlenmeyer flask with grown bacterial cellulose after
three days.

## Week 17 (August 17th-23rd)

**Aim:** To prepare fragments for Gibson assembly
and test porosity via dried samples.

**Method/Protocol:** Performed [PCR](https://2025.igem.wiki/wageningenur/protocols/#general-lab);
freeze-dried cellulose and measured porosity with the [mass-density](https://2025.igem.wiki/wageningenur/protocols/#bc-production-and-measurements)
method.

**Observation/Results:** The pSEVA331-pLux
fragment was successful amplified to be assembled with the desired
genes; A high porosity was recorded.

## Week 18 (August 24th-30th)

**Aim:** To clone additional functional genes.

**Method/Protocol:** Performed [Gibson
assemblies](https://2025.igem.wiki/wageningenur/protocols/#general-lab) with new inserts and [electroporated](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation)
them in *E. coli*.

**Observation/Results:** After electroporation,
colonies grew on the selective plate.

![](https://static.igem.wiki/teams/5713/notebook/notebookandra/andra-notebook-fig8.webp)

*E. coli* grown on selective LB plates after
electroporation.

## Week 19 (August 31st – September 6th)

**Aim:** To verify assemblies, perform cellulose
production to get more data.

**Method/Protocol:** Performed colony [PCR](https://2025.igem.wiki/wageningenur/protocols/#general-lab)
and sequencing; grew WT cellulose; attempted [electroporation](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation)
in *Komagataeibacter* (unsuccessful).

**Observation/Results:** Colony PCR confirmed the
constructs assembled the previous week; electroporation into
*Komagataeibacter* was unsuccessful; cellulose sheets were
produced and downstream processed.

## Week 20 (September 7th-13th)

**Aim:** To gather more porosity data.

**Method/Protocol:** Produced [cellulose](https://2025.igem.wiki/wageningenur/protocols/#bc-production-and-measurements);
assessed porosity via [mass-density
method](https://2025.igem.wiki/wageningenur/protocols/#bc-production-and-measurements).

**Observation/Results:** A high porosity was
obtained, in line with previous results. Data were analysed and
average porosity in WT cellulose was determined.

## Week 21 (September 14th-20th)

**Aim:** To analyse cellulose structure
microscopically.

**Method/Protocol:** Performed SEM imaging of
cellulose at the Wageningen Electron Microscopy Center; started
analysis of SEM images using ImageJ software.

**Observation/Results:** The SEM images showed
visible pores in the cross-section, while the surface was
uniform.

![](https://static.igem.wiki/teams/5713/notebook/notebookandra/andra-notebook-fig9.webp)

SEM image of BC at 30 μm scale.

![](https://static.igem.wiki/teams/5713/notebook/notebookandra/andra-notebook-fig10.webp)

SEM image of BC at 5 μm scale.

![](https://static.igem.wiki/teams/5713/notebook/notebookandra/andra-notebook-fig11.webp)

SEM image of BC at 1 μm scale.

## Week 22 (September 21-27th)

**Aim:** To complete the data analysis of SEM
images through ImageJ software.

**Method/Protocol:** Used ImageJ to determine
pores’ areas.

**Observation/Results:** The majority of pores had
an area below 0.1 μ2. Each image showed also the presence
of few very large pores.

Conjugation of *K. sucrofermentans* (Daan)

## Week 8 (June 15th-21st)

**Aim**: To complete *in silico* design of
primers and synthetic fragments required for amplification of glf,
glk, pgm, galU, ndk, secB and knockout fragments for egl, zwf, and
tal.

**Result**: Ordered primers and fragments

## Week 9 (June 22nd-28th)

**Aim**: To conduct PCR amplification of gene
fragments.

**Protocol**: [PCR](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Result**: Most of the genes were correctly
amplified, some required retaking and PCR optimisation.

## Week 10 (June 29th – July 5th)

**Aim**: To conduct PCR amplification of gene
fragments.

**Protocol**: [PCR](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Result**: More genes that were correctly
amplified.

## Week 11 (July 6th-12th)

**Aim**: To conduct PCR amplification of gene
fragments and perform golden gate cloning into storage vectors for
modular cloning (pSB1C3).

**Protocol**: [Golden
Gate and PCR](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Result**: Achieved first batch of modular
cloning plasmids with genes.

## Week 12 (July 13th-19th)

**Aim**: To conduct PCR amplification of gene
fragments, Golden Gate cloning into pSB1C3, Gibson assembly of
BcsABCD into pMETA23 overexpression vector, transformation of
successful vectors into *E. coli* DH5a cells.

**Protocol**: [PCR,
Golden Gate, Gibson Assembly](https://2025.igem.wiki/wageningenur/protocols/#general-lab), [transformation](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation)
*E. coli*

**Result**: Established some modular cloning
plasmids in *E. coli* and created more plasmids with genes
from *K. sucrofermentans.*

## Week 13 (July 20th-26th)

**Aim**: To conduct PCR amplification of gene
fragments, golden gate cloning into pSB1C3, Gibson assembly of
BcsABCD into pMETA23 overexpression vector, transformation of
successful vectors into *E. coli* DH5a cells.

**Protocol**: [PCR,
Golden Gate, Gibson Assembly](https://2025.igem.wiki/wageningenur/protocols/#general-lab), [transformation](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation)
*E. coli*

**Result**: Established some modular cloning
plasmids in *E. coli* and created more plasmids with genes
from *K. sucrofermentans.*

## Week 14 (July 27th-August 2nd)

**Aim**: To transform successful pSB1C3 vectors in
*E. coli* DH5a.

**Protocol**: [transformation](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation)
*E. coli*

**Result**: All modular cloning plasmids done

## Week 15 (August 3rd-9th)

Holiday

## Week 16 (August 10th-16th)

Holiday

## Week 17 (August 17th-23rd)

**Aim**: To do modular golden gate cloning of
pSB1C3 vectors to implement genes into overexpression vectors
(pMETA23).

**Protocol**: [Golden
Gate](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Result**: A couple of overexpression plasmids
finished, but the majority failed.

## Week 18 (August 24th-30th)

**Aim**: To do modular golden gate cloning of
pSB1C3 vectors to implement genes into overexpression vectors
(pMETA23). Conjugation of pSB1C3 plasmids into *K.
sucrofermentans.* Electroporation of knock-out plasmid in
*K. Sucrofermentans*.

**Protocol**: [Golden
Gate](https://2025.igem.wiki/wageningenur/protocols/#general-lab), [*K.
sucrofermentans* Conjugation, *K. sucrofermentans*
Electroporation](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation)

**Result**: Successful transformation of test
plasmid in *K. sucrofermentans*.

![](https://static.igem.wiki/teams/5713/notebook/notebookdaan/daan-notebook-fig1.webp)

Colony PCR proof of working conjugation. The first 12
bands show correct height (300 bp) for the test plasmid
(pSEVAb33). The next 12 bands confirm all colonies are *K.
sucrofermentans* by amplification of the BscA gene. The last
four bands are WT *K. sucrofermentans*, starting with 2
confirmations the plasmid was not in there orginally and ending
with confirmation of the correct height for the BscA gene.

## Week 19 (August 31st – September 6th)

**Aim**: Electroporation of succesful
overexpression vectors to *K. sucrofermentans.* BC yield
experiment of *WT K. Sucrofermentans.* Coding for the
wiki.

**Protocol**: *K. sucrofermentans* [electroporation](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation)

**Result**: Electroporation seemed successful but
was later found out to be contaminated.

## Week 20 (September 7th-13th)

**Aim**: BC yield experiment of overexpressed secB
in *K. Sucrofermentans*. Confirmation of successful
conjugation.

**Protocol**: [BC
production](https://2025.igem.wiki/wageningenur/protocols/#bc-production-and-measurements)

**Result**: No BC was forming.

## Week 21 (September 14th-20th)

**Aim**: BC yield experiment of WT *K.
Sucrofermentans*.

**Protocol**: [BC
production](https://2025.igem.wiki/wageningenur/protocols/#bc-production-and-measurements)

**Result**: No BC was forming because of
contamination.

Protein functionalisation through secretion by *S. cerevisiae* (Jochem)

## Week 1 (May 1st-3rd)

**Aim:** To gather knowledge and design detailed
experiments for bacterial cellulose (BC) production. Investigate
cultivation conditions and possibilities.

## Week 2 (May 4th-10th)

**Aim:** Work on protein design. Select which
elements the protein should contain.

## Week 3 (May 11-17th)

**Aim:** To start in the lab, prepare media and
start a *K. sucrofermentans* culture for the first time.
Learn about the yeast toolkits which are compatible with Golden
Gate assembly.

## Week 4 (May 18th-24th)

**Aim:** To check cultures from last week for
first cellulose production. To make glycerol stocks from our
strain and store at -80. To Search for DSP protocols. To start in
BC production in an erlenmeyer.

## Week 5 (May 25th-31st)

**Aim:** To design protein part of the
project.

## Week 6 (June 1st-7th)

**Aim:** To conduct a shaking experiment and test
different pH.

## Week 7 (June 8th-14th)

**Aim:** To Test DSP and learn freezedrying.

## Week 8 (June 15th-21st)

**Aim:** To conduct a platereader experiment to
measure the effect of cellulase and general growth. To do an
agitation experiment.

**Observation/Results:** Cellulase does not seem
to work.

## Week 9 (June 22nd-28th)

**Aim:** To check the cellulase enzymes. To design
plasmids

## Week 10 (June 29th – July 5th)

**Aim:** To design plasmids.

## Week 11 (July 6th-12th)

**Aim:** To design plasmids and order fragments
and primers. To learn more about MoClo cloning and available
toolkits.

## Week 12 (July 13th-19th)

**Aim:** To Prepare a new -80 order SP toolkit
from Addgene. To produce and miniprep of golden gate fragments. To
process IDT order.

## Week 13 (July 20th-26th)

**Aim:** To get an introduction to bioreactor and
perform a temperature experiment.

**Protocol**: Golden gate (GG) 1, Transformation,
PCR

**Observation/Results:** No colonies were
observed.

## Week 14 (July 27th-August 2nd)

**Aim:** To do Sanger sequencing of GG 1 PCR
products. To Learn and setup how the DASGIP bioreactor works and
operates. To run the bioreactor run. To do an *In situ*
coating experiment.

**Observation/Results:** No growth in the
bioreactor. Sanger sequencing samples were not concentrated
enough. *in situ* doest not work well yet.

## Week 15 (August 3rd-9th)

**Aim:** To do Golden Gate 2 with only mUkG1
samples. Transformation of GG Colony PCR

## Week 16 - Week 17 (August 10th-23rd)

Summer holiday

## Week 18 (August 24th-30th)

**Aim:** To do electroporation with T7 production
strain Goldengate 3 with all samples

## Week 19 (August 31st – September 6th)

**Aim:** To start an extra agitation experiment
Final bioreactor run Colony PCRs for *E. coli* Measure BC sheets for
WHC and yield. Plasmidsaurus + minipreps Transform yeast with 11
different plasmids.

![](https://static.igem.wiki/teams/5713/notebook/notebookjochem/jochem-notebook-fig1.webp)

Comparison of the bioreactor by eye, part of the
harvested and freeze dried sheet/pellicle and SEM of the shown
sheet.

## Week 20 (September 7th-13th)

**Aim:** To measure BC sheets for WHC and yield.
Check yeast after transformation. Measuring protein production in
yeast.

**Method/Protocol:** Platereader experiment for
OD600, OD588, OD512 and fluorescence.

**Observation/Results:** Successful transformation
in 10/11 plates.

![](https://static.igem.wiki/teams/5713/notebook/notebookjochem/jochem-notebook-fig2.webp)

Growth characteristics of transformed yeast. (A) OD600
measurement of 6 different plasmid strains in non-induced and
induced conditions over a 160 hour time period. (B) Comparison of
the final OD600 in non-induced and induced samples.

![](https://static.igem.wiki/teams/5713/notebook/notebookjochem/jochem-notebook-fig3.webp)

Fluorescent intensity for protein detection. (A)
Fluorescent at 510 nm normalised per cell over a 160 hour time
period. (B) Comparison of the final fluorescent intensity at 510nm
between non-induced and induced conditions. Comparison is made
between the samples that show a noticeable increase in
fluorescent.

# Production platform notebook

---

Ethanol homeostasis circuit (Camile)

## Week 1 (May 1st-3rd)

**Aim:** To generate a *rho*⁻ yeast strain
for mitochondrial function studies.

**Method/Protocol:** Attempted to make a [*rho*⁻
mutation in *S. cerevisiae* CEN.PK113-7D.](https://2025.igem.wiki/wageningenur/protocols/#co-culture)

**Observation/Results:** Wrong strain was
used.

## Week 2 (May 4th-10th)

**Aim:** To create the correct *rho*⁻
strain.

**Method/Protocol:** Generated *rho*⁻
mutation in CEN.PK113-5D.

## Week 3 (May 11-17th)

**Aim:** To verify the *rho*⁻ mutation.

**Method/Protocol:** Confirmed *rho*⁻
mutation via phenotypic checks.

**Observation/Results:** The *rho*-
mutation was successfully induced. This is confirmed with replica
plating and an overnight growth experiment.

![](https://static.igem.wiki/teams/5713/notebook/notebookcamile/camile-notebook-fig1.webp)

Results of replica plating after two growth cycles on
plates containing EtBr. The YPGlycerol p[late contains four
distinct colonies. The YPD plate, containing glucose, contains
many yeast colonies.

## Week 4 (May 18th-24th)

**Aim:** To validate null-model predictions and
advance cloning.

**Method/Protocol:** Conducted [growth
experiment](https://2025.igem.wiki/wageningenur/protocols/#co-culture) and HPLC measurements to assess yeast growth and
ethanol production under different glucose concentrations

**Observation/Results:** Growth experiment results
are shared with Rick to validate the model.

## Week 5 (May 25th-31st)

**Aim:** To prepare expression constructs and
design a Golden Gate Cloning (GGC) strategy for building a
promoter reporter vector.

**Method/Protocol:** Performed in silico design of
pTEV expression vectors; performed PCR for Re2.8 promoter
reporter.

**Observation/Results:** A complete GGC design for
the promoter reporter vector was generated; the PCR needs
optimisation since no amplification occurred.

## Week 6 (June 1st-7th)

**Aim:** To prepare for degron fusion
strategy.

**Method/Protocol**: Designed homology template
for degron fusion; isolated *S. cerevisiae* genomic DNA and
performed [PCR](https://2025.igem.wiki/wageningenur/protocols/#general-lab)
for homology arms; ordered degron sequence.

## Week 7 (June 8th-14th)

**Aim:** To improve PCR efficiency while expanding
HP work.

**Method/Protocol:** Optimised [PCR](https://2025.igem.wiki/wageningenur/protocols/#general-lab)
for Re2.8 promoter reporter by using a range of annealing
tempeatures and adding high GC-content enhancer.

**Observation/Results:** PCR for Re2.8 vector is
still not successfull, a new polymerase that is suitable for long
DNA fragments is needed. PCR for homology arms also has to be
optimised with a range of Tms and high GC content enhancer.

## Week 8 (June 15th-21st)

**Aim:** To advance cloning.

**Method/Protocol:** Performed final [PCR](https://2025.igem.wiki/wageningenur/protocols/#general-lab)
for homology arms; performed [GGC](https://2025.igem.wiki/wageningenur/protocols/#general-lab)
for degron fusion template and pTEV+ expression vector.

**Observation/Results:** Colonies with the correct
insertion can be screened because they lose their red colour. In
addition, less colonies are expected for the negative control
where one fragment is not added to the reaction mix. For some
plasmids this is not the case. These need optimisation. For other
GGC reactions a few colonies are inocculated, miniprepped and
digested. Some plasmids show the correct digestion pattern, they
can be sequenced.

![](https://static.igem.wiki/teams/5713/notebook/notebookcamile/camile-notebook-fig2.webp)

Results of replica plating after two growth cycles on
plates containing EtBr. The YPGlycerol p[late contains four
distinct colonies. The YPD plate, containing glucose, contains
many yeast colonies.

## Week 9 (June 22nd-28th)

**Aim:** To improve cloning efficiency.

**Method/Protocol:** Optimised GGC conditions
adjusting the temperature for the restriction enzymes, the number
of cycles and the final step from ligation to digestion; optimised
[PCR](https://2025.igem.wiki/wageningenur/protocols/#general-lab)
for promoter reporter vector with primestar GXL polymerase.

**Observation/Results:** Correct vectors have been
obtained; new polymerase enabed correct PCR amplification of
Re2.8.

## Week 10 (June 29th – July 5th)

**Aim:** To refine construct design.

**Method/Protocol:** Modified pTEV+ expression
vector design and ordered primers to make this design.

## Week 11 (July 6th-12th)

**Aim:** To expand the genetic toolkit for
promoter analysis.

**Method/Protocol:** Assembled Re2.8\_KlADH4
construct; designed promoter library.

## Week 12 (July 13th-19th)

**Aim:** To confirm construct correctness.

**Method/Protocol:** [Verified
plasmid digests; sequenced plasmids](https://2025.igem.wiki/wageningenur/protocols/#general-lab); ordered primers and
sequences for promoter library.

**Observation/Results:** Sequencing confirmed
correct constructs.

## Week 13 - Week 14 (July 20th- August 2nd)

Holiday

## Week 15 (August 3rd-9th)

**Aim:** To establish transformations and expand
cloning.

**Method/Protocol:** [Transformed
CEN.PK113-5D WT](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation) with Re2.8\_KlADH4; performed [GGC](https://2025.igem.wiki/wageningenur/protocols/#general-lab)
for final pTEV+ expression vectors; designed and cloned sgRNAs for
degron fusion; performed [PCR](https://2025.igem.wiki/wageningenur/protocols/#general-lab)
for promoter library.

## Week 16 (August 10th-16th)

**Aim:** To validate expression constructs and
gather field-level input.

**Method/Protocol:** Completed all pTEV+
expression vectors; [transformed
CEN.PK113-5D *rho*⁻](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation) with CRISPR toolkit; performed
plate reader assays with Re2.8\_KlADH4 to assess promoter dynamic
range; interviewed a farmer.

**Observation/Results:** Expression vectors were
validated and promoter activity was quantified.

## Week 17 (August 17th-23rd)

**Aim:** To establish degron strains and expand
promoter library assembly.

**Method/Protocol:** Repeated CRISPR toolkit
transformation of CEN.PK113-5D rho⁻; confirmed degron fusion with
[colony
PCR.](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Observation/Results:** Degron fusion was
successfull.

![](https://static.igem.wiki/teams/5713/notebook/notebookcamile/camile-notebook-fig3.webp)

Results of replica plating after two growth cycles on
plates containing EtBr. The YPGlycerol p[late contains four
distinct colonies. The YPD plate, containing glucose, contains
many yeast colonies.

## Week 18 (August 24th-30th)

**Aim:** To test degron function with expression
vectors.

**Method/Protocol:** [Transformed
PGK1-degron strain](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation) with pTEV+ expression vectors; [transformed
CEN.PK113-5D WT](https://2025.igem.wiki/wageningenur/protocols/#culturing-and-transformation) with promoter library vectors; wrote wiki main
page content.

**Observation/Results:** Yeast colonies grew on
selective medium, so the transformation has succeeded.

## Week 19 (August 31st – September 6th)

**Aim:** To characterise promoter strength and
update project communication.

**Method/Protocol:** Performed promoter dynamic
range assays in plate reader.

## Week 20 (September 7th-13th)

**Aim:** To validate degron activity in the
circuit, analyse data and finalise documentation.

**Method/Protocol:** Performed additional promoter
dynamic range assays; conducted growth experiment with plate
reader to confirm degron activity; performed plate reader data
analysis in python.

**Observation/Results:** All plate reader assays
need optimisation, since the fluorescence windows for excitation
and emission show overlap. The data is therefore not useful.

Establishing the cross-feeding and obtaining a basis for the co-culture (Iris)

## Week 3 (May 11-17th)

**Aim:** - To prepare media to use in the
following weeks; YPD, YPM, YPG. To have autoclaved agar ready when
plates are needed to be poured. - A *S. cerevisiae* strain
that would not consume glucose was researched.

**Methods/Protocols:** - 3x yeast extract and
peptone was prepared ([YEP
protocol](https://2025.igem.wiki/wageningenur/protocols/#general-lab)) and autoclaved as a 1L liquid broth. 500 mL 10x
dextrose, maltose and glycerol was prepared and autoclaved as
described in the protocol. These were mixed and used as needed. -
Industrial Biotechnology department of Delft University was
contacted for the strain *S. cerevisiae* deficient of
hexose transporters.

**Observations/Results:** - Sterile media was
prepared and ready for use. - Paperwork for the transport of the
strain *S. cerevisiae* IMX1812 was prepared.

## Week 4 (May 18th-24th)

**Aim:** - To have liquid inoculations of *K.
sucrofermentans* and *S.cerevisiae* prepared in [YPD](https://2025.igem.wiki/wageningenur/protocols/#general-lab)
in 30°C incubator shaking at 250 rpm. - Designing 96 well-plate
growth kinetics experiments for both strains by researching
relevant concentrations from literature.

**Methods/Protocols:** - *K.
sucrofermentans* and *S.cerevisiae* was inoculated from
plates according to their respective protocols. - The [96
well-plate growth kinetics experiment protocols](https://2025.igem.wiki/wageningenur/protocols/#co-culture) were prepared
according to the minimum and inhibitory concentrations found in
literature, and step-sizes for concentration series were
determined to facilitate pipetting.

**Observations/Results:** - The liquid inoculums
for both strains show growth. - The protocol for the 96-well plate
experiments were ready.

![](https://static.igem.wiki/teams/5713/notebook/notebookiris/iris-notebook-fig1.webp)

Example of Calculations to Prepare the Protocol for
the 96-Well Plate Growth Kinetics Experiment

![](https://static.igem.wiki/teams/5713/notebook/notebookiris/iris-notebook-fig2.webp)

Example of 96 well plate layout, *K.
sucrofermentans*

## Week 5 (May 25th-31st)

**Aim:** - To have sterile 914 mM potassium
acetate, 481 mM sodium gluconate, and 2055 mM ethanol stock
solutions in 2% YPD prepared for use in the 96-well plate
experiments. - To start preparing YPM + ethidium bromide (EtBr)
agar plates for mitochondrial knock-out experiments upon arrival
of *S. cerevisiae* IMX1812.

**Methods/Protocols:**  - The weight of potassium
acetate and sodium gluconate needed was calculated using their
molar weights (98.14 g/mol and 218.14 g/mol respectively) , and
the volume of ethanol needed was calculated using the molar weight
and density of pure ethanol (46.07g/mol, 0.789 g/mL). -These were
mixed with YEP, glucose and water and sterilised by passing
through a 2 μm membrane filter. - 1 mg/mL sterile EtBr was
prepared by passing through a 2 μm membrane filter. - 2x [YPM
liquid media](https://2025.igem.wiki/wageningenur/protocols/#general-lab) was prepared according to protocol.

**Observations/Results:** - The stocks were ready
to use for the 96 well plate experiments. - The stocks were ready
to prepare the ethidium bromide plates. The arrival of
*S.cerevisiae* IMX1812 was delayed due to the paperwork
process. The 96-well plate kinetics experiment was decided to be
carried out with *rho*- and WT *S. cerevisiae*
CEN.PK113-5D, and the changes in their growth parameters were
assumed to be analogous to rho- and WT *S. cerevisiae*
IMX1812.

## Week 6 (June 1st-7th)

**Aim:** To carry out the 96 well plate kinetics
experiment with *rho-* and WT *S. cerevisiae*
CEN.PK113-5D.

**Methods/Protocols:**  The [96
well-plate](https://2025.igem.wiki/wageningenur/protocols/#co-culture) was prepared and inserted into the plate reader as
described in the protocol.

**Observations/Results:** See results in the Wet
Lab - Production Platform page.

## Week 7 (June 8th-14th)

**Aim:** To carry out the 96-well plate kinetics
experiment with *K. sucrofermentans*.

**Methods/Protocols:**  The [96
well-plate](https://2025.igem.wiki/wageningenur/protocols/#co-culture) was prepared and inserted into the plate reader as
described in the protocol.

**Observations/Results:** Contamination was
observed in the wells of this plate, this experiment needed to be
repeated.

## Week 8 (June 15th-21st)

**Aim:** - To knock out mitochondrial function
from *S. cerevisiae* IMX1812 by plating on ethidium bromide
YPM plates for three rounds and selecting successful colonies.
through replica plating. - Repeating the 96-well plate experiment
for *K. sucrofermentans.*

**Methods/Protocols:** -For each round of ethidium
bromide plating, two or more days were necessary, because of the
stress inflicted. From each round, the colony that is smaller and
rounder is selected since it is an indication of loss of activity
of mitochondria.

- The [96
well-plate](https://2025.igem.wiki/wageningenur/protocols/#co-culture) was prepared and inserted into the plate reader as
described in the protocol.

**Observations/Results:** - The colonies from the
second round of plating were inoculated in liquid YPM to alleviate
stress and support growth before the third round of plating. - The
data was analysed and fit to the Richard’s growth function.

## Week 9 (June 22nd-28th)

**Aim:** - To complete the third round of ethidium
bromide plating for replica plating. - To analyse the results of
the growth kinetics experiments and implementing this data into
the model to help design the co-culture protocol.

**Methods/Protocols:** - The liquid inoculum from
the second round of ethidium bromide plating was plated for a
third round, and growth was expected within 3 days. - See dry lab
- consortium section to see the simulations done using the data
from the growth kinetic experiments. The results from these were
used to inform the duration to grow the pre-cultures and the
ratios for diluting both *K.sucrofermentans* and *S.
cerevisiae*.

**Observations/Results:** - Growth was not
observed on the third round of plates, the liquid inoculum from
the second round of plating was plated again. - From the model
simulations, it was decided that the *K.sucrofermentans*
would be grown for two days as a preculture, while the rho- *S.
cerevisiae* IMX1812 would be grown for around 30 hours. The
aim for the optical density’s before diluting for the co-culture
were 0.01 for *S. cerevisiae* and 2.5 for
*K.sucrofermentans*. The dilutions for the first test
coculturing were decided to be 1/50 for the bacterium and 1/100
for the yeast.

## Week 10 (June 29th – July 5th)

**Aim:** -To obtain the rho- mutated *S.
cerevisiae* IMX1812 from replica plating. - To prepare all the
media for the samples and controls needed for the co-culture and
inoculating the precultures of both strains.

**Methods/Protocols:** - The second trial for the
third round of ethidium bromide plating showed growth. The replica
plating was carried out according to protocol with duplicates on
YPM and YPG plates. - The [media
needed for the co-culture experiment](https://2025.igem.wiki/wageningenur/protocols/#general-lab) was prepared. The
pre-cultures were not inoculated since the rho- mutated *S.
cerevisiae* IMX1812 was not ready in time.

**Observations/Results:** - It was observed that
only a few colonies were still able to grow on the YPG plates,
which is an indication of respiration. The YPM1 and YPG1 plates
were aligned to select small and round colonies furthest from the
colonies showing growth from the YPG plates.

![](https://static.igem.wiki/teams/5713/notebook/notebookiris/iris-notebook-fig3.webp)

A. Results of Three Rounds of EtBr + Maltose Plating
of *S. cerevisiae* IMX1812 B. Results of Replica Plating on
Maltose and Glycerol to Select Succesful Colonies

[H]

## Week 11 (July 6th-12th)

**Aim:** -To inoculate the precultures for the
co-culture experiment and set-up the first co-culture experiment.
-To design guide RNAs and homology arms to engineer acetate
utilisation to the rho-mutated *S.cerevisiae*

**Methods/Protocols:** - The [pre-culture](https://2025.igem.wiki/wageningenur/protocols/#co-culture)
was prepared as described in the protocol, however cellulase was
not added as indicated. - The guide RNA and homology arms were
designed according to the non-commercial Ellis Lab CRISPR/Cas9
guide for yeast written by Will Shaw. (These were not amplified
and transformed into the yeast at the end because of the time
restrictions)

**Observations/Results:** - The preculture for
*K.sucrofermentans* did not reach the OD as expected, so
the protocol was updated to include cellulase in the preculture to
help release and recover the cells from the cellulose matrix, for
a more concentrated inoculum for the subsequent co-culture.

## Week 12 (July 13th-19th)

**Aim:** To inoculate *K.sucrofermentans*
and *S.cerevisiae* for the precultures and beginning the
co-culture by the end of the week.

**Methods/Protocols:** The pre-cultures were
grown, and the [co-cultures](https://2025.igem.wiki/wageningenur/protocols/#co-culture)
were set up according to the final protocol. Time-point samples
for the first day were taken, and centrifuged according to the
protocol to obtain the supernatant and pellets separately to
freeze and use in later HPLC and genomic analysis.

**Observations/Results:** The co-culture was
allowed to grow over the weekend.

## Week 13 (July 20th-26th)

**Aim:** To analyse the results from the
co-culture.

**Methods/Protocols:** The cellulose produced in
the monocultures and co-cultures were observed. The endpoint
sample for HPLC was taken and the supernatant was freezed for
later analysis.

**Observations/Results:** It was observed that the
needle included in the Erlenmayers for time point sampling had
interfered with the cellulose formation as a sheet. As a result,
the cellulose yield could not be assesed. The samples for HPLC and
assesing survival were frozen to analyse after holiday.

## Week 14 - Week 15 (July 27th-August 9th)

A well-deserved break!

## Week 16 (August 10th-16th)

**Aim:**To analyse the HPLC samples from the
previous co-culture and setting up the new co-culture to ensure
that the needle does not interfere with the cellulose sheet
formation.

**Methods/Protocols:** The [HPLC
samples were analysed](https://2025.igem.wiki/wageningenur/protocols/#co-culture) according to the protocol and the needle
was stabilised in the midpoint of the stopper so that it does not
come in contact with the media, and the samples are taken by
tilting the Erlenmeyer.

**Observations/Results:** Formate formation
instead of acetate was observed in the *K.sucrofermentans*
monocultures. Most peaks were inconclusive. Since this co-culture
was not succesful in terms of the cellulose, the results were not
further analysed for presentation or graphing. The new set-up for
sampling with the needle had to be tested with a new co-culture
trial.

## Week 17 (August 17th-23rd)

**Aim:**To carry out a new co-culture experiment
and test the new sampling method and seeing if it prevents
interfering with pellet formation.

**Methods/Protocols:** The [co-culture
experiment](https://2025.igem.wiki/wageningenur/protocols/#co-culture) was set-up according to the protocol once
again.

**Observations/Results:** The cellulose sheets
formed as expected, showing that the new-set up is succesful. The
HPLC samples were frozen for analysis in the coming weeks, and
samples from the co-culture duplicates were frozen to plate on
YPD+YPM plates to check if both strains survived.

## Week 18 (August 24th-30th)

**Aim:**To compare the outcomes of cellulose
production from the monoculture to the co-culture.

**Methods/Protocols:** The [washing/downstream
processing protocol](https://2025.igem.wiki/wageningenur/protocols/index.html#bc-production-and-measurements) was followed to sterilise the cellulose
sheets. The sheets were freeze-dried according to the protocol to
weigh and calculate the yield, titer, and productivity.

**Observations/Results:** There was not a
remarkable higher production of bacterial cellulose in the
co-culture, which was hypothesised to be due to the lack of an
acetate sink from the model, which supports the initial design
that yeast consuming acetate from the medium and fermenting it to
ethanol will boost yield.

## Week 19 (August 31st – September 6th)

**Aim:**To asses the dynamics of metabolite
consumption and production in the co-culture experiment with HPLC,
and checking if both strains have survived by plating on YPM+YPD
plates.

**Methods/Protocols:** The areas obtained were
converted to concentrations using the calibration curves made
according to the standard samples with known concentrations. The
plates assessing if both strains survived were compared to known
monoculture plates to compare phenotypes.

![](https://static.igem.wiki/teams/5713/notebook/notebookiris/iris-notebook-fig4.webp)

Calibration curves obtained for each metabolite from
the standards with known concentrations

**Observations/Results:** See results in the wet
lab page. Overall, the co-survival and ethanol transfer from the
yeast to the bacterium was deemed successful.

## Week 20 - Week 21 (September 7th-20th)

**Aim:**To gather all results and preparing
graphs and figures to fit the format agreed by the team.

**Methods/Protocols:** A python script prepared by
Rick was used to follow the standards for the figures.

**Observations/Results:** All of the figures were
prepared.

# Seed coating & application notebook

---

Use case 1: Controlled delivery of pesticides (Niels)

## Week 2 (May 4th-10th)

**Aim:** Following lab introduction tour.

**Results:** learned how to work in the lab
safely.

## Week 3 - Week 4 (May 11-24th)

**Aim:** To design GFP (fusion) proteins and
primers *in silico*.

**Methods:** SnapGene, AlphaFold.

**Results:** Ordered DNA fragments and primers

## Week 5 (May 25th-31st)

**Aim:** To begin with plasmid assembly for GFP
and CBD:GFP production.

**Protocols:** [PCR,
Gibson Assembly, Heat shock transformation](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

## Week 6 (June 1st-7th)

**Aim:** To finish with plasmid assembly for GFP
and CBD:GFP production.

**Protocols:** [Miniprep,
plasmid sequencing](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Observations/results:**
Fully assembled plasmids for the production of GFP and
CBD:GFP.

## Week 8 (June 15th-21st)

**Aim:** To transform the assembled plasmid into
the production strain.

**Protocols:** [Electroporation](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Observations/results:** Successfully transformed
production strain.

## Week 9 (June 22nd-28th)

**Aim:** To produce GFP and CBD:GFP proteins.

**Protocols:** [Protein
production & isolation](https://2025.igem.wiki/wageningenur/protocols/#proteins)

**Observations/results:** GFP and CBD:GFP protein
solutions.

## Week 10 (June 29th – July 5th)

**Aim:** To design Cry3Aa fusion protein and
primers *in silico*.

**Protocols:** SnapGene, AlphaFold

**Observations/results:** Parts were ordered.

## Week 11 (July 6th-12th)

**Aim:** Validation of the binding of the CBD to
BC, CBD:Cry3Aa plasmid assembly.

**Protocols:** [CBD
Binding Validation, Fluorescence measurement](https://2025.igem.wiki/wageningenur/protocols/#proteins), [PCR,
Gibson Assembly, Heat shock transformation](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Observations/results:** Concluded CBD can bind
to BC, assembled CBD:Cry3Aa plasmid.

## Week 12 (July 13th-19th)

**Aim:** To transform the CBD:Cry3Aa plasmid into
the production strain.

**Protocols:** [Electroporation](https://2025.igem.wiki/wageningenur/protocols/#general-lab)

**Observations/results:** Successfully transformed
production strain.

## Week 13 (July 20th-26th)

**Aim:** To produce CBD:Cry3Aa proteins.

**Protocols:** [Protein
production & isolation](https://2025.igem.wiki/wageningenur/protocols/#proteins)

**Observations/results:** CBD:Cry3Aa protein
solution.

## Week 14 (July 27th-August 2nd)

**Aim:** To validate whether CBD is also able to
bind to wheat bran.

**Protocols:** [CBD
binding validation, Fluorescence measurment](https://2025.igem.wiki/wageningenur/protocols/#proteins)

**Observations/results:** Concluded that CBD can
bind to wheat bran.

## Week 15 (August 3rd-9th)

**Aim:** To follow mealworm training at the
department of Animal Nutrition.

**Results:** Learned how to properly rear and
handle insects.

## Week 16 - Week 17 (August 10th-23rd)

**Aim:** Vacation

**Methods/protocols:** Drinking beer in
Germany

## Week 18 (August 24th-30th)

**Aim:** To investigate the effects of CBD:Cry3Aa
on mealworms.

**Protocols:** [Pesticide
assay](https://2025.igem.wiki/wageningenur/protocols/#use-cases)

**Observations/results:** Weight of mealworms
recorded prior to exposure.

## Week 19 (August 31st – September 6th)

**Aim:** To investigate the effects of CBD:Cry3Aa
on mealworms.

**Protocols:** [Pesticide
assay](https://2025.igem.wiki/wageningenur/protocols/#use-cases)

**Observations/results:** Weight and mortality
rates of mealworms recorded following exposure.

## Week 20 (September 7th-13th)

**Aim:** To process data.

**Methods:** RStudio, statistics

**Observations/results:** Processed data for the
wiki.

Use case 2: Parasitic weed suppression (Yoerik)

## Week 12 (July 13th-19th)

**Aim:** To start up in the lab.

**Method:** Conducted literature review and
drafted experimental plans.

**Observation/Results:** Initial steps were taken
towards developing a coating protocol.

## Week 13 (July 20th-26th)

**Aim:** To start devising a method to sterilise
seeds for plans down the line.

**Method/Protocol:** Started working with rice
(*Oryza sativa*) and developing a sterilisation protocol
for it by using different bleach concentrations and durations of
this step.

**Observations/Results:** Sterilisation did not
work initially due to endophytes, beneficial bacteria that are
naturally present, within the seed.

![](https://static.igem.wiki/teams/5713/notebook/notebookyoerik/yoerik-notebook-fig1.webp)

Rice covered in endophytes unsuccessfully removed with
the present sterilisation protocol.

## Week 14 (July 27th-August 2nd)

**Aim:** To continue devising a sterilisation
protocol.

**Method/Protocol:**Decided to switch crops to
sorghum (*Sorghum bicolor*) var. Teshdale, a cultivar
susceptible to *Striga hermonthica*.

**Observations/Results:**Surface sterilisation of
this crop was easier and resulted in minimal complications.

![](https://static.igem.wiki/teams/5713/notebook/notebookyoerik/yoerik-notebook-fig2.webp)

Array of rice, barley and sorghum with different
bleach intensities and durations.

## Week 15 (August 3rd-9th)

**Aim:** To devise a protocol for coating the
seeds.

**Method/Protocol:** Started with attempting to
develop seed coatings initially through a ziploc bag method.
Interviewed Zohaib Hussain about alternatives as this had a low
throughput and started working on those.

**Observations/Results:** Bag method had a low
throughput for creating a coating - so other methods were
considered instead.

![](https://static.igem.wiki/teams/5713/notebook/notebookyoerik/yoerik-notebook-fig3.webp)

Rice covered by the ziploc bag method, n indicates
amount of coating rounds each taking 1.5 hours.

## Week 16 (August 10th-16th)

**Aim:** To start working on an *ex situ*
coating method that requires the material to be dissolved. Also
continued working on the *In situ)* method as recommended
by Zohaib.

**Method/Protocol:** Tried out different solvents
and temperatures to solubilise bacterial cellulose, including
N–methylmorpholine-N-Oxide. *In situ* coating was initially
performed in erlenmeyer flasks.

![](https://static.igem.wiki/teams/5713/notebook/notebookyoerik/yoerik-notebook-fig4.webp)

Bacterial cellulose (BC) broken by rigorous shaking in
an erlenmeyer.

**Observations/Results:** Solubilising the BC was
unsuccesful. We instead continued developing the *in situ*
method, although initial erlenmeyer attempts were
unsuccessful.

## Week 17 (August 17th-23rd)

**Aim:** To continue developing an *In
situ* coating method.

**Method/Protocol:** Coating method development
was continued to be done in well plates, where we initially
compared the different sizes and their effect on seed coating, as
well as the effect of shaking or static cultures.

![](https://static.igem.wiki/teams/5713/notebook/notebookyoerik/yoerik-notebook-fig5.webp)

Bacterial cellulose (BC) grown in a 96 well plate with
a seed, coating it.

**Observation/Results:** The initial switch to
well plates appeared promising and resulted in higher success
rates, although not high enough yet for use in greenhouse assays
**(Figure [6](#fig:treatments))**. the agitated
cultures resulted in higher coating success rates for specific
treatments in a larger variety of culturing conditions,
specifically well sizes. This may indicate that the PDA+S
treatment causes bacteria to more effectively deposit BC on site,
whereas without PDA+S, bacteria are washed off during agitation,
leading to detached BC pellicles.

![](https://static.igem.wiki/teams/5713/notebook/notebookyoerik/yoerik-notebook-fig6.webp)

Effects of seed coating treatment, well-size, and
culturing conditions on coating success rate. (A) Effect of static
versus continuous shaking culture (n=32). (B) Within continuous agitation
or (C) static cultures, non-treated seeds, scarified (S),
polydopamine (PDA)-coated, or PDA+S treated seeds were coated
*in situ* (n=32).
Identical letters signify no statistical differences as
investigated using logistic regression followed by Tukey
HSD.

## Week 18 (August 24th-30th)

**Aim:** To continue developing an *In
situ* coating method by adding in optimisations.

**Method/Protocol:** Mechanical scarification and
polydopamine-HCl steps were added to enhance the success rate of
the coating procedure.

**Observations/Results:** Adding these two steps
increased the success rate of coatings to 80%

![](https://static.igem.wiki/teams/5713/notebook/notebookyoerik/yoerik-notebook-fig7.webp)

Effects of seed coating treatment on coating success
rate with the optimised protocol. Non-treated seeds, scarified
(S), PDA treated, or PDA+S treated seeds were coated *in
situ* (n=32). Identical
letters signify no statistical differences as investigated using
logistic regression followed by Tukey HSD.

## Week 19 (August 31st – September 6th)

**Aim:** To take the next steps towards developing
the protocol in washing and drying seeds.

**Method/Protocol:** Seeds were washed with
sterile MiliQ and dried.

**Observations/Results:** Washed seeds were
successfully cleansed of medium and dried, enabling them to be
used in greenhouse experiments with minimal contamination
risks.

![image](https://static.igem.wiki/teams/5713/notebook/notebookyoerik/yoerik-notebook-fig8.webp) Coated seeds
are drying.

## Week 20 (September 7th-13th)

**Aim:** To assess the effect of the coating
procedure on the germination rate.

**Method/Protocol:** Seeds were placed on filter
paper and treated with demi water to identify germination
rates.

**Results:** Inconclusive, experiments need to be
repeated.

# Dry lab notebook

---

Consortium model (Anirudhan)

## Week 1 - Week 2 (May 1st-10th)

**Aim:** To conduct background research on ODE
based models.

**Methods:** Reviewed literature articles.

## Week 3 - Week 5 (May 11-31st)

**Aim:** To conduct background research on ODE
based models, and gain familiarity with coding.

**Methods:** Reviewed literature articles, online
tutorials on python.

## Week 6 (June 1st-7th)

**Aim:** To map the metabolic pathway involved in
the monocultures of *S. cerevisiae* and *K.
sucrofermentans*.

**Methods:** Understand metabolic pathway related
to consumption and utilisation of carbon source, relating to the
production of biomass and cellulose.

## Week 7 (June 8th-14th)

**Aim:** To map the metabolic pathway involved in
the monocultures of *S. cerevisiae* and *K.
sucrofermentans*.

**Methods:** Understand metabolic pathway related
to consumption and utilisation of carbon source, relating to the
production of biomass and cellulose.

**Observation/Results:** Metabolic map indicating
the consumption and utilisation of growth media components was
established.

## Week 8 (June 15th-21st)

**Aim:** Establish cross-feeding mechanism for the
co-culture system.

**Methods/Protocol:** Establish metabolic pathway
accounting for the production of ethanol by *S. cerevisiae*
and consumption of ethanol by *K. sucrofermentans*. Also
establish metabolic pathway accounting for the production of
acetate by *K. sucrofermentans* and consumption by *S.
cerevisiae*.

**Observation/Results:** Metabolic pathway
accounting for the cross-feeding mechanism for the co-culture was
established.

![](https://static.igem.wiki/teams/5713/drylabconsortium/drylabconsortium-fig1.webp)

Metabolic pathway of consortium.

## Week 9 - Week 11 (June 22nd- July 12th)

**Aim:** To establish the computational system for
simulation.

**Method/Protocol:** Write the metabolic pathways
as a system of ODEs.

**Observation/Results:** The ODEs accounting for
the rate of change of metabolic compounds, over time was
mathematically expressed.

## Week 12 - 13 (July 13th-26th)

**Aim:** To establish the computational
system.

**Methods/Protocol:** Python was chosen as the
system to build model on, the ODEs were written down and each
parameter, and variable was initalised on the programme code.

**Observation/Results:** The computational system
was established.

## Week 14 -16 (July 27th- August 16th)

**Aim:** To identify values of various parameters
for initial simulations.

**Method/Protocol:** Conduct literature review for
parameter values.

**Observation/Results :** Parameter sets were
identified.

## Week 17 (August 17th-23rd)

**Aim:** To perform initial monoculture
simulations, and fit the data to laboratory experiments by using a
scoring function.

**Method/Protocol:**
(1) Run the simulations based on initial values, and adjust
parameters until the biomass simulation qualitatively matches
laboratory data.
(2) Establish upper and lower bounds for the various parameters
based on biologically feasible ranges.
(3) Establish a scoring function to identify parameter sets that
best match the laboratory data.
(4) Run the scoring function for 10,000 iterations, with each
iteration using a Latin Hypercube Sampling to generate random
parameter values within the bounds. This performs global
optimisation.

**Observation/Results:** Optimal parameter sets
were identified.

## Week 18 (August 24th-30th)

**Aim:** To fine tune the model.

**Method/Protocol:** To the optimal parameter
values identified, establish an upper and lower bound on a sum and
difference of 5% respectively, and run the scoring function for
10,000 iterations, with each iteration generating random parameter
values within the bound. This performs local optimisation.

## Week 19 (August 31st – September 6th)

**Aim:** To perform co-culture simulations.

**Method/Protocol:** Perform simulations,
accounting for the cross feeding mechanisms.

**Observation/Results:** Co-culture simulations
were performed, differences were observed between simulation
results and laboratory experiments.
An overproduction of acetate was also observed, leading to
unrealistic biomass simulations.

## Week 20 (September 7th-13th)

**Aim:** To fine tune the model and perform
predictions.

**Method/Protocol:** Acetate sink was incorporated
to the cross feeding mechanism, to account for the consumption of
acetate.

**Observation/Results:** Model qualitatively
matches the laboratory data.

## Week 21 (September 14th-20th)

**Aim:** To perform Data analysis and
documentation.

**Method/Protocol:** Analyse data based on the
change in environmental concentrations of compounds at start and
end of simulation. Additionally, draw conclusions based on
comparison with laboratory data.

**Observation/Results:** Acetate sink was
hypothesised to represent an unaccounted parameter.
Parameter values were noted down, and documentation was
performed.

Ethanol homeostasis circuit (Rick)

## Week 1 - Week 3 (May 1st-17th)

**Aim:** To develop a dynamic model that describes
the genetic circuit implemented in *S. cerevisiae*.

**Method:** Formulate differential equations that
represent key biological processes of the genetic circuit.

## Week 4 - Week 5 (May 18th-31st)

**Aim:** To script the dynamical model in python
code.

**Method:** Implement the dynamic model in Google
Colaboratory based on guidance from supervisor.

## Week 6 (June 1st-7th)

**Aim:** To fit the dynamic model to experimental
ethanol, glucose, and biomass data from *S.
cerevisiae*.

**Method:** Manually adjust model parameters to
improve the fit to the experimental data.

## Week 7 (June 8th-14th)

**Aim:** To evaluate the goodness-of-fit of the
model to the experimental data.

**Method/Protocol:** Generate plots to compare the
model versus the experimental data.

**Observations:**

![](https://static.igem.wiki/teams/5713/drylabcircuit/drylab-circuit-fig3.webp)

Comparison between the sped up null model simulation
and the *S. cerevisiae* experimental data collected in the
lab for initial glucose concentrations of 2%, 5%, and 10% (w/v).
The top panels show the model simulation. The bottom panels show
the experimental data. Error bars represent standard deviation. 2%
(w/v) condition is indicated in brown. 5% (w/v) condition is
indicated in blue. 10% (w/v) condition is indicated in yellow. The
ODEs and parameter values as described in the model section were
used, except V\_{\text{max,Eth,int}} was adjusted
from 5300 to 3500. To speed up the system, all ODEs were
multiplied by 7.

## Week 8 (June 15th-21st)

**Aim:** To determine if our PGK1 and GMP1 version
of the genetic circuit reduce ethanol production in *S.
cerevisiae*.

**Method:** Perform optimisations towards the
desired ethanol concentration for both the PGK1 and GPM1 version
of genetic circuit given predefined bounds.

**Observations/Results:**

![](https://static.igem.wiki/teams/5713/drylabcircuit/drylab-circuit-fig4.webp)

. The null model is indicated in brown. The PGK1 model is
indicated in blue. The GPM1 model is indicated in yellow. An
initial condition of 5% (w/v) glucose was used.

Model simulations of the null model, the PGK1 model,
and the GPM1 model. For the PGK1 and GPM1 model, the simulation
with the lowest score was visualised. PGK1 and GPM1 models were
optimised towards the desired external ethanol behaviour (dashed
black line, 1% (v/v)/171 mmol L^{-1}

## Week 9 (June 22nd-28th)

**Aim:** To identify important parameters within
the genetic circuit and to visualise the effect of one of these
parameters.

**Method:** Assess distribution of optimised
parameters and manually adjust the dissociation constant of the
promoter.

**Observations/Results:**

![](https://static.igem.wiki/teams/5713/drylabcircuit/drylab-circuit-fig5.webp)

Distribution of 1% best optimised parameter sets of
PGK1 model. Parameters that did not have a range were omitted. A
uniform distribution indicates little effect of the parameter on
the desired behaviour. A non-uniform distribution indicates an
effect of the parameter on the desired behaviour. X-axis
corresponds to the pre-defined bounds of the parameter +/- 10%.
The median is indicated by the dashed red line. An initial
condition of 5% (w/v) glucose was used.

![](https://static.igem.wiki/teams/5713/drylabcircuit/drylab-circuit-fig6.webp)

Qualitative effect of dissociation constant (K\_d) of the ethanol-inducible promoter
on external ethanol concentration in PGK1 model. Simulations were
performed with the best optimised PGK1 model parameter set, except
for K\_d. K\_d was varied per simulation (see
legend). The desired external ethanol concentration is indicated
by the dashed black line (171 mmol L^{-1}). An initial condition of 5%
(w/v) glucose was used.

## Week 10 (June 29th – July 5th)

**Aim:** 1) To explore the impact of ethanol
consumption by *K. sucrofermentans* on the behaviour of the
genetic circuit. 2) To explore the effect of population volume on
the reaction rates.

**Method:** 1) Manually adjust the parameter in
the dynamic model that represent ethanol consumption by *K.
sucrofermentans*. 2) Manually adjust the initial *S.
cerevisiae* population volume.

**Observations/Results:**

![](https://static.igem.wiki/teams/5713/drylabcircuit/drylab-circuit-fig7.webp)

Qualitative effect of K\_{uptake} on external ethanol
concentration in PGK1 model. Simulations were performed with the
best optimised PGK1 model parameter set, except for K\_{uptake}. K\_{uptake} was varied per simulation
(see legend). The desired external ethanol concentration is
indicated by the dashed black line (171 mmol L^{-1}). An initial condition of 5%
(w/v) glucose was used.

![](https://static.igem.wiki/teams/5713/drylabcircuit/drylab-circuit-fig8.webp)

Effect of initial *S. cerevisiae* population
volume on external glucose and external ethanol concentrations in
PGK1 model. Simulations were performed with the best optimised
PGK1 model parameter set. The initial *S. cerevisiae*
population volume (V\_{int}) was
varied per simulation (see legend). The desired external ethanol
concentration is indicated by the dashed blackline (171 mmol
L^{-1}). An initial condition of
5% (w/v) glucose was used.

{% endblock %}

{% extends "layout.html" %}
{% block header\_bg %}
background-image: url('https://static.igem.wiki/teams/5713/v3/engineering-v3-v3.webp');
padding-top: 500px;
background-position: 50% 80%
{% endblock %}
{% block title %}
BCoated Engineering
{% endblock %}
{% block page\_content %}

# ENGINEERING

---

Contents

+ [**Introduction**](#introduction)
+ [Consortium
  engineering](#consortium-engineering)
  - [**Cycle
    1: characterising the monocultures and parameterising the
    model**](#cycle-1-characterising-the-monocultures-and-parameterising-the-model)
  - [**Cycle 2: constructing
    the co-culture**](#cycle-2-constructing-the-co-culture)

- [**Ethanol
  homeostasis circuit**](#ethanol-homeostasis-circuit)
  * [**Cycle 1: model design
    and validation**](#cycle-1-model-design-and-validation)
  * [**Cycle
    2: build a genetic circuit based on model predictions**](#cycle-2-build-a-genetic-circuit-based-on-model-predictions)

# **Introduction**

To achieve stability in our engineered biological systems, we
relied on Design–Build–Test–Learn (DBTL) cycles as the foundation
of our workflow. By using this iterative approach, we gradually
redefined our ideas and moved towards effective solutions. Dry lab
and wet lab efforts complement each other, creating strong DBTL
cycles that combine modelling-based predictions with experimental
validation. This approach ensured that our workflows were
efficient, data-driven, integrated, and grounded in rational
approaches. Although our project spans several modules for the
development of our process and product, here we highlight two of
the particularly strong DBTL cycles used for the production
platform:

1. **Consortium engineering –** We aimed to investigate and
   implement the initial and operational conditions to support a
   robust co-culture of *Komagataeibacter sucrofermentans* and
   *Saccharomyces cerevisiae*. This subproject went through
   two DBTL iterations, combining modelling and experimental efforts
   to understand and optimise the balance between the two
   species.
2. **Ethanol homeostasis circuit –** We aimed to design a
   synthetic genetic circuit that stabilises the ethanol
   concentration in the culture medium to 1% (v/v), thereby
   maintaining optimal conditions for bacterial cellulose (BC)
   production in *K. sucrofermentans[1](#ref-Montenegro-Silva2024)*. This subproject
   progressed through two iterations of the DBTL cycle, each bringing
   us closer to a functional circuit.

# Consortium engineering

Our project followed a structured DBTL approach to construct a
stable yeast–bacteria co-culture for enhanced BC production,
inducibility of the system, and functionalisation of the material.
In two DBTL cycles, we simultaneously developed the model and
experimental co-culture.

1. **Characterising the monocultures and parameterising the
   model:**

   Our first iteration focused on establishing the baseline
   characteristics of potential toxicities, metabolic preferences,
   and kinetic parameters. These were also simulated by feeding the
   data acquired in the lab into a mathematical model for both
   strains individually, before introducing the added complexity of
   the co-culture interactions.
2. **Constructing the co-culture:**

Using insights from cycle 1, we extended the model to predict
co-culture dynamics and guided the design of experimental
inoculation strategies. We then compared the results observed in
the lab to model simulations to test our hypotheses.

[Learn
more about our wet lab](https://2025.igem.wiki/wageningenur/production-platform/#establishing-the-cross-feeding-and-obtaining-a-basis-for-the-co-culture)

[Learn
more about our dry lab](https://2025.igem.wiki/wageningenur/consortium-model/)

### **Cycle 1: Characterising the monocultures and parameterising the model**

Design

We built an ordinary differential equations-based model
(ODE-based model) inspired by a model for the growth of
*Escherichia coli* by Millard et al.[2](#ref-Millard2021),
to simulate the metabolic pathways of *S. cerevisiae* and
*K. sucrofermentans* within a consortium. The Millard
models were modified by updating the parameter values within a
biologically relevant range.

The choice to design a consortium was inspired by Gilbert
et al., 2021[3](#ref-Gilbert2021), who established a
synthetic community (Syncom) of *S. cerevisiae* and *K.
rhaeticus* as a way of incorporating programmable
functionalities to BC using the toolbox of yeast. From our human
practices [(Dr. Amritpal Singh)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#stakeholder-amritpalsingh), we
determined that we could achieve higher BC yields in a consortium.
*K. sucrofermentans* was shown to achieve higher yields of
BC when 1% (v/v) ethanol is provided, which can be produced by
*S. cerevisiae*[1](#ref-Montenegro-Silva2024). Gilbert et
al., 2021[3](#ref-Gilbert2021) used a method of codependence to design a stable consortium. This
method was not applicable to our co-culture, so we designed a
cross-feeding mechanism to impose codependence. The overview of
the cross-feeding mechanism is described in [(Figure 1)](#fig:P). For the ethanol not to be
self-consumed, a mitochondrial knockout was performed in the
yeast.

![](https://static.igem.wiki/teams/5713/engineering2/engineering-2-fig1.webp)

Overview of the design of the production platform.
*K. sucrofermentans* consumes glucose to polymerise
cellulose. The intermediate, Glucose-6-P, is the branching point
to cellulose production, glycolysis, and pentose phosphate pathway
(PPP). As a byproduct of cellulose production,
*Komagataeibacter* sp. produces gluconate and acetate. This
acidifies the medium, and eventually becomes toxic and reduces
yield. An engineered *S. cerevisiae* can be introduced into
the medium to consume the excess acetate to alleviate this
drawback, which also prevents glyceraldehyde-3-phosphate flux to
glycerol. To avoid competition for glucose in the medium, *S.
cerevisiae* without hexose transporters can be utilised. The
strain *S. cerevisiae* IMX1812 grows primarily on maltose.
When the mitochondria is knocked out, the maltose will only be
fermented to ethanol instead of respired. When a responsive
ethanol circuit is introduced, ethanol can be maintained at a
concentration that boosts cellulose yield by providing excess ATP
and reducing the flux to gluconate. Introducing *S.
cerevisiae* also has additional benefits such as expanding the
range of substrates that *K. sucrofermentans* could utilise
due to the enzyme invertase (not shown in figure).

Build

We prepared the respiration deficient *S. cerevisiae*
IMX1812 (*rho-* *S. cerevisiae* IMX1812) through
three rounds of ethidium bromide (EtBr) plating [(Figure 2)](#fig:EtBr Rounds).

![](https://static.igem.wiki/teams/5713/engineering2/engineering-2-fig2.webp)

Three rounds of plating *S. cerevisiae* on
ethidium bromide and maltose plates to induce the loss of
mitochondrial DNA.

We set up the monoculture growth kinetics experiments for both
strains. *K. sucrofermentans* and *rho- S.
cerevisiae* were grown in 96-well plates with three replicates
in concentration series of ethanol, gluconate, acetate, and a
combination of gluconate and acetate. These experiments were used
to find the growth parameter values that are used in the model
under different circumstances. In addition, the change in the
growth of the respiration deficient strain compared to the
wild-type (WT) was observed.

Test

The colonies from the third round of EtBr plating were tested
[through
replica plating](https://2025.igem.wiki/wageningenur/protocols/#co-culture) on maltose and glycerol with two replicates [(Figure 3)](#fig:rcoculture rep).

![](https://static.igem.wiki/teams/5713/engineering2/engineering-2-fig3.webp)

Replica plating the third round of the ethidium
bromide plates on maltose and glycerol. Glycerol can not be
consumed if respiration is not present. The YPG plates only have a
few colonies, showing that most of the colonies lost their
mitochondrial function. The successful colonies are also smaller
since they can generate less energy.

Learn

We deemed the mitochondrial knockout experiment to be
successful, as little to no colonies grew on glycerol.

We fit the OD vs. time data obtained from the growth
experiments to a Richards growth function using a Python script.
We integrated Michaelis-Menten parameters (\mu, K{\_m}, V{\_{max}}) into the model, and simulated
the behaviour of each strain.

We fit the model to the monoculture growth curve data [(Figure 4)](#fig:New_img). After a successful
fit, we identified the optimal values of the various metabolic
parameters. Based on monoculture experiments on varying media, we
also obtained parameters involved in the cross-feeding
mechanism.

![](https://static.igem.wiki/teams/5713/engineering2/engineering-2-fig4.webp)

Comparison of model simulation with experimental
growth data. (A) Visualisation of data fit for *K.
sucrofermentans*. (B) Visualisation of data fit for *S.
cerevisiae*.

### **Cycle 2: Constructing the co-culture**

Design

Based on the simulations carried out for the co-culture, we
built the model for the consortium. This model incorporated the
cross-feeding mechanism, and additional fine-tuning was performed
to account for an acetate sink. We did this because
unrealistically high acetate production was observed within the
simulation. Simultaneously, we designed a model that does not
include this acetate sink, with the aim of comparing simulation
results to experimental data.

Using the predictions made by the co-culture model, we aimed to
investigate the effect of the initial balance of the microbes
(inoculation concentrations) on the final cellulose yield of the
co-culture.

Build

We inoculated pre-cultures of *K. sucrofermentans* and
*rho- S. cerevisiae* 48 hours and around 30 hours prior to
the pre-cultures, and until the OD reached 2.5 and 0.01
respectively. The media required for the monoculture controls and
co-cultures, namely, YPD (glucose), YPM (maltose), and YPD+M
(glucose and maltose) were prepared. The bacterium was diluted
1/50 while the yeast was diluted 1/100 in the media.

Test

After four days, we analysed co-culture results. Sugar
consumption and the concentrations of cross-feeding metabolites
were measured with HPLC. To do this, duplicate samples were
centrifuged at 6000 g for 12 minutes. The supernatant was frozen
and later used for HPLC. In addition, a small amount of the
co-culture was plated on YPD+M to assess if both strains had
survived.

Learn

We confirmed that both strains survived by comparing the sample
plated from the co-culture to the monoculture plates of the
strains [(Figure 5)](#fig:irisplates). The
phenotypically distinct characteristics in the (potential)
co-culture plate matched the observed characteristics from the
monoculture plates, so the organisms both survived.

![](https://static.igem.wiki/teams/5713/engineering2/engineering-2-fig5.webp)

(A) The (duplicate) co-culture samples was plated on a
YPD+M plate to assess if both strains survived after four days.
(B) Visualisation of *K. sucrofermentans* monoculture to
compare. (C) Visualisation of *S. cerevisiae* monoculture
to compare.

We observed differing results between the model simulation and
the laboratory experiments, mainly in the levels of acetate
production. The model simulates an overproduction of acetate,
which was not observed in the lab, thus to modify the model we
incorporated an acetate sink. The model’s simulation with the
acetate sink qualitatively matched the results seen in the
laboratory.

From the HPLC results of the *K. sucrofermentans*
monoculture, we noticed that there was a carry-over of maltose
(0.5 g/L, around 5.5% of the amount in the co-culture) [(Figure 6A)](#fig:Iris_img_1). No maltose was
added to the original monoculture sample, so was thought to be
left from the previous injection. Moreover, low production levels
of ethanol and acetate, with a relatively higher proportion of
ethanol, were observed. We brainstormed two potential hypotheses
to explain the results:

(1) There could have been yeast contamination at a very low
concentration within the HPLC samples, leading to the production
of ethanol.
(2) The *Komagataeibacter* produced ethanol, which is not
described for its metabolism.

Of the two, we consider (1) to be more likely, since our
model’s prediction was able to show a production of ethanol [(Figure 7B)](#fig:fig_compiled) at a higher
concentration than that of acetate [(Figure 7B)](#fig:fig_compiled), when accounting for *S. cerevisiae* as
a contaminant [(Figure 7B&D)](#fig:fig_compiled), and there
was around 0.5 g/L maltose in the medium. This qualitatively
matched the HPLC results [(Figure 6A)](#fig:Iris_img_1), suggesting
that we likely did have a contamination.

![](https://static.igem.wiki/teams/5713/engineering2/engineering-2-fig6.webp)

HPLC results showing concentrations (g/L) for glucose, maltose,
acetate and ethanol for the start and end of (A) *K.
sucrofermentans* monoculture and (B) co-culture. We obtained
the concentrations by constructing calibration curves for each
metabolite from peak areas of known standard solutions (7.5, 2.5,
2, 1.5, 1, 0.5, 0.25, 0.1). Linear regression equations from these
calibration curves were then applied to convert sample peak areas
into concentrations (g/L).

![](https://static.igem.wiki/teams/5713/engineering2/engineering-2-fig7.webp)

Model predictions (A) Concentration of environmental
ethanol over time in the consortium. (B) Concentration of
environmental acetate and environmental ethanol at the end point
(70 hours - 72 hours) in the consortium. (C) Comparison of
external acetate and ethanol concentration over time in the
consortium. (D) Growth of *K. sucrofermentans* and *S.
cerevisiae* (contaminant).

# **Ethanol homeostasis circuit**

A central challenge in developing a stable yeast–bacteria
consortium for BC production is controlling the ethanol
concentration. *S. cerevisiae* naturally produces ethanol
at levels exceeding the desired threshold of 1% (v/v), which risks
destabilising the system and compromising BC production by *K.
sucrofermentans*[1](#ref-Montenegro-Silva2024). To overcome this, we
applied a structured DBTL approach to iteratively design, model,
and test an ethanol homeostasis circuit. Across two DBTL cycles,
we progressively advanced both our wet lab and dry lab work:

1. Model design and validation: We began by designing an ODE
   model for *S. cerevisiae* metabolism and ethanol production
   and validating whether the model described *S. cerevisiae*
   under normal circumstances. When this was confirmed, the model was
   extended to describe the ethanol inducible circuit.
2. Build genetic circuit based on model predictions: After
   validation of the model, it was used to guide design choices for
   the wet-lab circuit design and help with the experimental
   implementation of the genetic circuit.

[Learn
more about our wet lab](https://2025.igem.wiki/wageningenur/production-platform/#s.-cerevisiae-ethanol-homeostasis-circuit)

[Learn
more about our dry lab](https://2025.igem.wiki/wageningenur/ethanol-homeostasis-circuit-model/)

### **Cycle 1: Model design and validation**

Design

The synthetic circuit was conceptually
developed to allow for the inhibition of ethanol production when
threshold concentrations are reached. The synthetic circuit
consists of a promoter that senses ethanol concentrations. This
promoter regulates the expression of *pTEV+*. pTEV+ is a protease that cleaves a specific amino acid sequence. We used this
for the targeted degradation of PGK1 or GPM1, key enzymes in
ethanol production [(Figure 8)](#fig:ethanol circuit). When *pTEV+* is expressed, this now
causes degradation of PGK1 and therefore a decrease in ethanol
production. When the ethanol concentration drops as a result of
this, the signal transduction also ceases, and the inhibition of
ethanol production stops.

![](https://static.igem.wiki/teams/5713/engineering2/engineering-2-fig8.webp)

Overview of ethanol homeostasis circuit. From the
medium, glucose is taken up by *S. cerevisiae* and
fermented into ethanol. PGK1 and GPM1 are two enzymes involved in
fermentation. Ethanol diffuses into the environment across the
membrane of *S. cerevisiae*. Ethanol in the external
environment may be taken up by *K. sucrofermentans* for
biomass production. The ethanol inside *S. cerevisiae*
induces transcription of *pTEV+*. pTEV+ degrades either PGK1 or
GPM1. We hypothesised that this will reduce the ethanol production
rate in *S. cerevisiae*. DPG:
3-phospho-D-glyceroyl-phosphate. G3P: 3-phospho-D-glycerate. 2-PG:
2-phospho-D-glycerate. Dotted arrows indicate summarised metabolic
reactions.

The circuit was modelled with ordinary differential equations
(ODEs). The model was based on Michaelis-Menten equations and the
fermentation pathway in *S. cerevisiae*. Literature values
for growth rates, promoter activity, and degradation rates were
used as initial parameter estimates. The null-model behaved
plausibly *in silico*, but the model predicted slower
*S. cerevisiae* growth than expected. This suggested that
either the parameter estimates were inaccurate or that the model
was oversimplifying growth behaviour. Experimental data were
needed to confirm which explanation was correct.

Build

Before ethanol homeostasis circuit
simulations were run to test the functioning of the synthetic
circuit, parameter estimates first had to be validated. To this
end, experiments were performed to assess *S. cerevisiae*
growth, glucose consumption, and ethanol production without the
synthetic circuit. To obtain these data, a *rho-* strain was developed. This
strain lacked respiratory capacity and had energy and biomass
production coupled to the production of ethanol, which matched the
model.

Test

The *rho-* strain was grown for 24
hours in glucose concentrations of 2% (w/v), 5% (w/v) and 10%
(w/v), all in triplicate. Every hour for the initial 8 hours, and
then after 22 hours, a measurement was taken to determine OD600,
the glucose percentage (w/v), and the ethanol percentage (v/v).
OD600 was measured with a spectrophotometer, and ethanol and
glucose concentrations were determined with HPLC.

Learn

We used the experimental *S.
cerevisiae* data to fine-tune and validate the model, such
that it could be used to make predictions to help with the
experimental implementation of the genetic circuit. Initially, the
experimental data showed faster ethanol accumulation than the
model. The model parameters were successfully optimised so that
the null-model matched the experimental data [(Figure 9)](#fig:1). We used the
validated model to make predictions about whether 1) the genetic
circuit indeed reduces the *S. cerevisiae* ethanol
production rate, 2) which version (PGK1 or GMP1) of the genetic
circuit to implement in the lab, and 3) which parameters to
engineer in the lab.

![](https://static.igem.wiki/teams/5713/engineering2/engineering-2-fig9.webp)

Comparison between the rescaled null model simulation
and the *S. cerevisiae* experimental data collected in the
lab for initial glucose concentrations of 2%, 5%, and 10% (w/v).
The top panels show the model simulation. The bottom panels show
the experimental data. Error bars represent standard deviation. 2%
(w/v) condition is indicated in brown. 5% (w/v) condition is
indicated in blue. 10% (w/v) condition is indicated in yellow. The
ODEs and parameter values as described in the model section were
used, except V\_{\text{max,Eth,int}} was adjusted
from 5,300 to 3,500. To rescale the system, all ODEs were
multiplied by seven.

### **Cycle 2: Build a genetic circuit based on model predictions**

Design

From the model, we learned that the
synthetic circuit can indeed be used to reduce the *S.
cerevisiae* ethanol production rate. According to the model, it
did not matter whether PGK1 or GPM1 is targeted for degradation in
the actuator domain of the synthetic circuit. For the wet-lab we
therefore decided to focus solely on the development of the
circuit with PGK1. Finally, the model helped identify parameters
that were important for the functioning of the synthetic circuit [(Figure 10)](#fig:2). All parameters
that were related to promoter activity showed non-uniform
distributions, which meant that their activity is important.
Therefore, different promoters with a range of dynamic ranges were
designed.

![](https://static.igem.wiki/teams/5713/engineering2/engineering-2-fig10.webp)

Distribution of 1% best optimised parameter sets of
PGK1 model. Parameters that did not have a range were omitted. A
uniform distribution indicates little effect of the parameter on
the desired behaviour. A non-uniform distribution indicates an
effect of the parameter on the desired behaviour. X-axis
corresponds to the pre-defined bounds of the parameter +/- 10%.
The median is indicated by the dashed red line. An initial
condition of 5% (w/v) glucose was used. The four parameters on the
top row relate to the promoter. \alpha\_0 is the leaky transcription
rate of *pTEV+* (mmol L^{-1} h^{-1}), \alpha\_1 is the transcription rate of
*pTEV+* when ethanol is maximised (mmol L^{-1} h^{-1}), n is the cooperativity of the
promoter and K\_{d} is the
dissociation constant of the promoter (mmol L^{-1}). The parameters on the bottom
row relate to pTEV+. K\_{d,mRNA1}
is the degradation rate of pTEV+ mRNA (h^{-1}), \beta\_1 is the translation efficiency
of *pTEV+* (h^{-1}), K\_{d,TEVd1} is the N-degron degradation
rate (h^{-1}) and K\_{TEV} is the saturation constant of
pTEV+ degradation (mmol L^{-1}).

Build

The build of the synthetic circuit in
the wet-lab was split up into two parts: the signal transducer and
actuator domain were designed separately from the promoter. For
the actuator, genome engineering with CRISPR/Cas9 was used to fuse
an inactive bidirectional degron to *PGK1*. This inactive
degron is activated by the signal transducer pTEV+. Plasmids were
designed to express *pTEV+* under the Z\_{3}EVpr, which is \beta-estradiol inducible. With the
signal transducer and actuator domain, the effect of the circuit
can be tested upon activation by a promoter with a known dynamic
range. For the ethanol-inducible promoter, we developed a modular
cloning strategy that allowed ethanol-responsive Upstream
Activation Sequence (UASe) repeats, isolation sequences, and core
promoter elements to be easily combined in a single Golden Gate
cloning step. Using this approach, we constructed 12 promoter
variants by pairing four different core elements with one, two, or
three UASe repeats.

Test

Due to time constraints, this testing
step has not yet been completed, but we aim to perform it shortly.
By splitting up the circuit into a sensor domain and a domain
containing the signal transducer and actuator, we are able to
explore the effect of different levels of circuit activity on the
production of ethanol and cell growth. Varying amounts of \beta-estradiol will be added to the
growth medium. During a 24 hour growth experiment, samples will be
collected to measure OD600, glucose concentration, and ethanol
concentration. Similarly, ethanol promoter activity can be tested
in a range of ethanol concentrations to find the promoter dynamic
ranges. These measurements are taken continuously in a plate
reader experiment, since promoter activity can be measured with
fluorescence.

Learn

Once results about the circuit activity
have been obtained, they can be coupled back to the model. The
results of the signal transduce and effector domain can be used to
further validate the model. When varying the \beta-estradiol concentrations,
resulting in different levels of Z\_3EV promoter activity, this changes
the extent to which the ethanol homeostasis circuit is active. We
expect this to result in different *S. cerevisiae*
behaviour. Information about the cell growth and ethanol
production for different activity levels of the ethanol
homeostasis circuit can be compared to the model prediction. If
the model predicts similar behaviour as the experimental results,
we can further specify model parameters. This can then be used to
guide improvements for future designs of the experimental genetic
circuit. In addition, the experimental promoter dynamic range
assays help determine the true parameter values of the
ethanol-inducible promoter available in the lab. Simulating the
model with the experimentally identified promoter-related
parameters will help identifying promoters that help stabilise the
ethanol concentration at 1% (v/v). This will provide concrete
suggestions for the final version of the ethanol homeostasis
circuit in which the actuator domain is combined with an
ethanol-inducible promoter.

(1)

Montenegro-Silva, P.; Ellis, T.;
Dourado, F.; Gama, M.; Domingues, L. Enhanced Bacterial
Cellulose Production in *Komagataeibacter
Sucrofermentans*: Impact of Different PQQ-Dependent
Dehydrogenase Knockouts and Ethanol Supplementation.
*Biotechnology for Biofuels and Bioproducts*
**2024**, *17*. <https://doi.org/10.1186/s13068-024-02482-9>.

(2)

Millard, P.; Enjalbert, B.;
Uttenweiler-Joseph, S.; Portais, J.; Létisse, F. Control and
Regulation of Acetate Overflow in Escherichia Coli.
*eLife* **2021**, *10*, e63661. <https://doi.org/10.7554/eLife.63661>.

(3)

Gilbert, C.; Tang, T.-C.; Ott, W.;
Dorr, B. A.; Shaw, W. M.; Sun, G. L.; Lu, T. K.; Ellis, T.
Living Materials with Programmable Functionalities Grown from
Engineered Microbial Co-Cultures. *Nature Materials*
**2021**, *20* (5), 691–700. <https://doi.org/10.1038/s41563-020-00857-5>.

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{% block header\_title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
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## What Should this Page Contain?

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Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

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