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.

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.

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

We compared bacterial cellulose with other materials.

We conducted a cost analysis and risk analysis.

We performed a financial analysis.

We worked on acquiring funding and developing a timeline.

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

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

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

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.

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.

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

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

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

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.

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

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

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

Protocol: YPD Media, BC Production, Downstream Processing BC.

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

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 and 3.

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

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

Results:

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.

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 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 was done for the heterologous gene plasmids to pSEVAb33 

Observations/Results: 

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 with 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.

.

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 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 of hpdR/phpdH inducible promoters to pSEVAb33 

Observations/Results:

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 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: 1^st 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 

2^nd 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 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.

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.

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 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 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 with PCR products, electroporation 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 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 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 .

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.

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.

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.

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.

Observation/Results: The new fragments were successfully amplified.

Week 12 (July 13th-19th)

Aim: To complete reassembly attempts.

Method/Protocol: Performed Gibson assembly conducted porosity assays.

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; 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; 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.

Week 17 (August 17th-23rd)

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

Method/Protocol: Performed PCR; freeze-dried cellulose and measured porosity with the mass-density 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 with new inserts and electroporated them in E. coli.

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

Week 19 (August 31st – September 6th)

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

Method/Protocol: Performed colony PCR and sequencing; grew WT cellulose; attempted electroporation 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; assessed porosity via mass-density method.

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.

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.

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

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

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

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, 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, 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 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

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, K. sucrofermentans Conjugation, K. sucrofermentans Electroporation

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

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

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

Result: No BC was forming.

Week 21 (September 14th-20th)

Aim: BC yield experiment of WT K. Sucrofermentans.

Protocol: BC production

Result: No BC was forming because of contamination.

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.

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.

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.

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.

Week 4 (May 18th-24th)

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

Method/Protocol: Conducted growth experiment 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 for homology arms; ordered degron sequence.

Week 7 (June 8th-14th)

Aim: To improve PCR efficiency while expanding HP work.

Method/Protocol: Optimised PCR 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 for homology arms; performed GGC 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.

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 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; 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 with Re2.8_KlADH4; performed GGC for final pTEV+ expression vectors; designed and cloned sgRNAs for degron fusion; performed PCR 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⁻ 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.

Observation/Results: Degron fusion was successfull.

Week 18 (August 24th-30th)

Aim: To test degron function with expression vectors.

Method/Protocol: Transformed PGK1-degron strain with pTEV+ expression vectors; transformed CEN.PK113-5D WT 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.

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) 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 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 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.

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 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 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 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 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 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.

[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 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 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 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 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 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.

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.

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

Week 6 (June 1st-7th)

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

Protocols: Miniprep, plasmid sequencing

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

Observations/results: Successfully transformed production strain.

Week 9 (June 22nd-28th)

Aim: To produce GFP and CBD:GFP proteins.

Protocols: Protein production & isolation

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, PCR, Gibson Assembly, Heat shock transformation

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

Observations/results: Successfully transformed production strain.

Week 13 (July 20th-26th)

Aim: To produce CBD:Cry3Aa proteins.

Protocols: Protein production & isolation

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

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

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

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.

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.

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.

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.

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.

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.

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). 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.

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%

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.

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.

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.

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.

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:

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:

. 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.

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:

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: