Team:Amsterdam/Notebook

Forbidden FRUITS

February

In February the iGEM fun started by trying to compose a team by Robin and Dennis.

March

In the beginning of the month, the final team was formed and discussions about the individual scientific tasks were held. Besided, outreach material, such as a group photo, a logo and an outreach strategy was made. Unfortunately, on March 13th the intelligent lockdown in the Netherlands started. As a consequence no real life meetings could be held and all team members had to work from at home. By the end of the month, we lost a team member and half of the remaining team decided not to start iGEM related scientific work until June 1st. But the remaining team members started to do literature research to prepare the lab work.
Highlights:

  • 13th: start corona measures in the Netherlands - intelligent lockdown (shift to online meetings and working from at home)
  • 27th: zoom meeting with European teams and iGEM HQ about Corona - Dennis, Hugo and Filipe

April

Due to the Corona virus restrictions people still needed to work from home, but this did not stop us from reaching out and trying to attract sponsors! Also, the foundation for the dry- and wet lab projects was laid during individual meetings with supervisors and some courses.
Highlights:

  • 1th: receive letter of recommendation from Stanley Brul (UvA, SILS)
  • 14th: receive letter of recommendation from Bas Teusink (VU, AIMMS)
  • Robin, Vicky and Samira joined the NL iGEM grousapp
  • 24th: SILS (UvA) is will be sponsoring the team
  • 24th: Coffee hour with iGEM ambassador about the Giant Jamboree not taking place in Boston - Dennis

Wetlab

  • Producing salicylic acid with Synechocystis (Samira)
    • Literature research to find the function of the one gene (pyk2) which needs to be knocked out in order to growth couple salicylic acid production.

Drylab

  • 8th: intro course FF - Joeri & Max
  • 14th: programming in Python - Joeri
  • 15th: Object oriented programming and UML -Joeri
  • 17th: FF API - Joeri
  • 22nd: extended OOP -Joeri
  • 23rd: Linear Programming - Max
  • 24th: MILP and Duality - Max
  • LP recap -Max, Dennis, Samira

May

Because of the continuing Coronavirus restrictions, we were waiting for the labs to open. But in the meanwhile, we used our time well: we started drylab projects, prepared our experiments, designed primers and did literature research. Besides, there was time to work on the social media campaign.
Highlights:

  • 11th: meeting with VU demonstrator lab about possible sponsorship/collaboration. (Joris, Samira, Filipe, people from VU)
  • 13th: brainstorm with iGEM ambassador about Benelux meetup - Dennis and Samira
  • 13th: Contact with Jeroen Hugenholtz of WUR about possible applications of Forbidden FRUITS in the current biotechnology industry - Samira and Kelly
  • 15th-17th: iGEM opening weekend
  • 20th: Discussion about the business aspect of Forbidden FRUITS and on how to perform market research with Eugene from iGEM Leiden -Joris, Samira, Robin

May 4rd - 10th

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Literature research to find methods to knockout or knockdown essential genes in cyanobacteria.
  • Lactic acid bacterial cell factory (Robin): doing literature research to using lactic acid bacteria as cell factories. A possible strain could be Lactococcus Lactis.
  • Multiwell cultivator (Vicky): discuss research plan with Joeri. Determine the aim of the study: calibration of optic sensors

Drylab

  • Merging Databases (Dennis): start working on a code to parse information from the databases
  • Global Plumber (Samira): Familiarise with Forbidden FRUITS API and try to design toy models to test future linear programming.
  • Cheap lunches (Vicky): start to get familiar with Linear Programming

May 11th - 17th

Human Practices

  • Survey: brainstorm about ways to approach people about a survey concerning the technologies we are developing. This appeared to be quite challenging due to the complexity of the project.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Decide on strategies to knockout pyk2 and design primers for those strategies
  • Lactic acid bacterial cell factory (Robin): doing literature research to using lactic acid bacteria as cell factories.

Drylab

  • Merging Databases (Dennis): The first version of the MetaNetX parser is finished, although there are still some abnormalities. Working on improving the parser
  • Global Plumber (Samira): Familiarise with Forbidden FRUITS API and try to design toy models to test the future linear program.
  • Cheap lunches (Vicky): Design programming strategy

May 18th - 24th

Human Practices

  • Survey: Set up a first version of the survey in the format which was decided on next week.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Primer design. There might be a possibility to work in the lab of the demonstrator lab (which is officially sponsoring us), but this is difficult to realize.
  • Lactic acid bacterial cell factory (Robin): doing literature research to using lactic acid bacteria as cell factories.

Drylab

  • Merging Databases (Dennis): The first version of the MetaNetX parser is finished, although there are still some abnormalities. Working on improving the parser
  • Global Plumber (Samira): Familiarise with Forbidden FRUITS API and try to design toy models to test the future linear program.

May 25th - 31st

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Primer design. There might be a possibility to work in the lab of the demonstrator lab (which is officially sponsoring us), but this is difficult to realize.
  • Lactic acid bacterial cell factory (Robin): doing literature research to using lactic acid bacteria as cell factories.

Drylab

  • Merging Databases (Dennis): The first version of the MetaNetX parser is finished, although there are still some abnormalities. Working on improving the parser
  • Global Plumber (Samira): Familiarise with Forbidden FRUITS API and try to design toy models to test the future linear program.

June

In June we have our first real life meeting and the restrictions to allow wetlab work are discussed. By the end of the month, the first work in the lab is started! Besides, since the university courses are over, our team is finally on full force. During this month we are continuing the design for the survey, start programming on the wiki and discuss the safety aspects of our research.
Highlights:

  • 2nd: first real life meeting with the team (excluding supervisors) at Science Park
  • 3rd: Discussion of computational side of the iGEM Leiden project with Marijn, Sebastian and Eugene from the Leiden team - Samira, Dennis
  • 12th: First real life meeting with the team and supervisors at Science Park
  • 13th: Help from Zakaria Laaraj (fellow member of the demonstrator lab) with the crowdfunding campaign - Vicky, Joris
  • 24th: first day of lab work!

June 1st - 7th

Human Practices

  • Survey: Review survey content, approaching possible distributors

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Literature research to find methods to knockout essential genes in cyanobacteria.
  • Lactic acid bacterial cell factory (Robin): doing literature research to using lactic acid bacteria as cell factories.

Drylab

  • Merging Databases (Dennis): Working on improving the parser
  • Global Plumber (Samira): A final test database was composed, which contained all possible modifications and could yield several strategies with a different yield. The next step is to built a database to verify the possible output the future plumber gives.
  • Cheap lunches (Vicky): Familiarising with programming in Forbidden FRUITS
  • GPRA network (Mingdong): study the concept of gene protein reaction associations
  • Optimizing the modification strategy (Kelly): Familiarising with programming in Forbidden FRUITS
  • Sequence optimization (Joris): Familiarising with programming in Forbidden FRUITS

June 8th - 14th

Human Practices

  • Survey: This week, we sent the survey to an expert in the field (a professor at the VU) for feedback. Unfortunately, we never got a response.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): A decision was made on the modification strategy. Corresponding primers were designed.
  • Lactic acid bacterial cell factory (Robin): Digging into research on R05537 reaction in L. Lactis which is proving harder than expected.

Drylab

  • Merging Databases (Dennis): Working on improving the parser
  • Global Plumber (Samira): Building a simulation plumber. In order to be able to verify the solutions the future plumber will produce and to further explore the problem, we wanted to build a test database using a crude force simulation.
  • Cheap lunches (Vicky): Familiarising with programming in Forbidden FRUITS
  • GPRA network (Mingdong): study the concept of gene protein reaction associations
  • Optimizing the modification strategy (Kelly): Familiarising with linear programming in Forbidden FRUITS
  • Sequence optimization(Joris): Familiarising with programming in Forbidden FRUITS

June 15th - 21th

Human Practices

  • Survey: Continuing improving survey content.
  • Online workshops about genetic modification (Samira and Vicky): a first set up was made to educate people about genetic modification and biotechnology. This could help us to explore the way people think about genetic modification.
  • Market Research (Joris and Robin): brainstorm about possible strategies to get familiar with stakeholders in the industry and about the motivation of MMP members to develop Forbidden FRUITS

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Primers were ordered.
  • Producing Mannitol with Synechocystis (Dennis): Strategy is discussed and primers were designed and ordered. The protocols and sequences are being revised while waiting for the lab to open up for students.
  • Producing salicylic acid with E.coli (Joris): Determine an experimental strategy using an JW6 (Wang, et al. 2019, University of Georgia) E.coli strain.
  • Lactic acid bacterial cell factory (Robin): organizing details of the modification strategy L. lactis with the supervisor on a minimal medium. In order to do so, we needed to re-run Forbidden FRUITS with adjustments.

Drylab

  • Merging Databases (Dennis): Working on improving the parser.
  • Global Plumber (Samira): Continue working on building a simulation plumber. The simulation was performed on a test database.
  • Cheap lunches (Vicky): Familiarising with Object Oriented Programming and try to get familiar with the problem of cheap lunches.
  • GPRA network (Mingdong): Building a toy model with gene protein reaction association in Forbidden FRUITS. To understand the problem, this was done manually first. Later, the building of a toy model was automated.
  • Optimizing the modification strategy (Kelly): Familiarising with Linear Programming
  • Sequence optimization (Joris): Literature research on sequence optimization

June 22nd - 28th

Human Practices

  • Survey: Sent survey out to safety office of the VU
  • Online workshops about genetic modification (Samira and Vicky): Do literature research on opinions about genetic modification.
  • Market Research (Joris and Robin): meeting with head of MMP to discuss the entrepreneurial aspect and funding of Forbidden FRUITS
  • Benelux meetup: start to prepare the team presentation

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Experiments are prepared, protocols in benchling are set up.
  • Producing Mannitol with Synechocystis (Dennis): First week of lab work. Started to knockin the susA gene by transfecting synechocystis with a plasmid containing the susA gene and a spectinomycin resistance gene. Transfected cells were selected by growing them on a plate with 10µg/mL of spectinomycin.
  • Producing lactate with Synechocystis (Kelly): Experiments were prepared and primers were ordered.
  • Producing salicylic acid with E.coli (Joris): Determine an experimental strategy using an JW6 (Wang, et al. 2019, University of Georgia) E.coli strain. Get in touch with the Demonstrator lab at the VU about the possibility of using their lab.
  • Lactic acid bacterial cell factory (Robin): Organizing supervision of this project (at the VU)

Drylab

  • Merging Databases (Dennis): Tried to get an overview based on what similarities the merger merges reactions.
  • Global Plumber (Samira): Try to implement a possible strategy in the Global Plumber, but this proves to be difficult. In the end, this method is not even feasible at all.
  • Cheap lunches (Vicky): Implement pathfinding methods on a toy model. Getting familiar with the Forbidden FRUITS API.
  • GPRA network (Mingdong): Getting familiar with linear programming and learning ElementTree API and exploring processing of xml format files in order to relate these two in a logical manner.
  • Optimizing the modification strategy (Kelly): Familiarising with Linear Programming, focus on duality.
  • Sequence optimization (Joris): Literature research on sequence optimization and work on a strategy to implement.

July

In July, the wet lab projects were gradually started up. But our projects using E.coli and L.lactis ran into problems with obtaining the correct strain for the designed strategy, so these needed to be postponed. Also, some team members joined the RIVM safe by design webinar to work on the safety aspect of our work. Besides wet lab, we started to incorporate human practices into our project: by holding a workshop about genetic modification we learned about ways to communicate about our application. Besides, we were approached by NEMOkennislink to write a blog for their biotechnology website and we reached out to Micropia (a museum about microbes) about a possible collaboration. And last but not least, we met with other iGEM teams from BeNeLux and presented our project and gave a webinar about linear programming.
Highlights:

  • 4th: first FB workshop (Genetic modification: the how to’s and why’s) - Vicky and Samira
  • 7th: RIVM safe by design webinar by Petra Hoogerhuis - Vicky, Joris, Mingdong, Kelly
  • 21st: discuss blog ideas for NEMO kennislink - Robin & Kelly
  • 22nd-25th: BeNeLux meetup with other iGEM teams from the BeNeLux
    • 23th: team presentation - Joris and Vicky
    • 24th: webinar about linear programming - Kelly and Samira
  • 30th: meeting with Ruben Janssen and Jasper from Micropia about possible collaboration - Kelly and Joris

June 29th - July 5th

Human Practices

  • Survey: Waiting for reply from safety office of the VU
  • Online workshops about genetic modification (Samira and Vicky): First online workshop/discussion about genetic modification was held. Unfortunately there was only 1 attendant. For the next webinar, we will prepare a more detailed promotion plan.
  • Benelux meetup: start to prepare the team presentation
  • Market Research (Joris and Robin): process meeting about entrepreneurship

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Experiments are prepared, protocols in benchling are set up.
  • Producing Mannitol with Synechocystis (Dennis): Cells are growing and hopefully the transfection has succeeded
  • Producing lactate with Synechocystis (Kelly): Uploading all protocols to Benchling. Start lab work next week.
  • Producing salicylic acid with E.coli (Joris): Determine an experimental strategy using an JW6 (Wang, et al. 2019, University of Georgia) E.coli strain.
  • Lactic acid bacterial cell factory (Robin): There are issues concerning gene identification necessary for the salicylate strategy in L.Lactis. The enzymes involved in reaction R05537 are undefined. Discussing on how to proceed.

Drylab

  • Merging Databases (Dennis): Halted due to holiday
  • Global Plumber (Samira): Working on 2 strategies in designing Plumber: one approach is based on elementary flux modes and duality while the other is only focused on the difference between the dual and primal.
  • Cheap lunches (Vicky): Working on path finding for cheap lunch strategies while getting to know the API. Gradually moving to more complicated programming tasks.
  • GPRA network (Mingdong): Parsing of gene protein reaction association from a pseudo model is now possible. This approach will be tested on different scenarios. Besides, a larger pseudo model with gene protein reaction associations will be created to validate this approach
  • Optimizing the modification strategy (Kelly): Familiarising with Linear Programming, focus on duality.
  • Sequence optimization (Joris): Literature research on sequence optimization and work on a strategy to implement.

July 6th - 12th

Human Practices

  • Survey: Waiting for reply from safety office of the VU
  • Online workshops about genetic modification (Samira and Vicky): Work out a more detailed promotion plan for the next workshop.
  • Market Research (Joris and Robin): process meeting about entrepreneurship
  • Benelux meetup: start to prepare the team presentation
  • Safe by design: attending and reviewing the webinar held by the RIVM

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Start of labwork postponed due to illness
  • Producing Mannitol with Synechocystis (Robin): To determine if the transfection has succeeded, 10 colonies were chosen to grow on a new plate.
  • Producing lactate with Synechocystis (Kelly): Postponed due to possible Corona contamination (quarantine).
  • Producing salicylic acid with E.coli (Joris): Waiting for a reply from the university of Georgia concerning obtaining the JW6 (Wang, et al. 2019, University of Georgia) E.coli strain.
  • Lactic acid bacterial cell factory (Robin): There are issues concerning gene identification necessary for the salicylate related to the strategy infor L.Lactis. These enzymes involved in reaction R05537 are undefined. Discussing on how to proceed.

Drylab

  • Merging Databases (Dennis): Halted due to holiday
  • Global Plumber (Samira): Incorporating faucets (input reactions) and sinks (output reaction) in the plumber
  • Cheap lunches (Vicky): Working on path finding for cheap lunch strategies while getting to know the API. Gradually moving to more complicated programming tasks
  • GPRA network (Mingdong):The Gene-Protein-Reaction-Association processing script is finished and validated. The next step is to put all gene protein associations of the toy model and the reversibility of each reaction in a sbml-format file. This file can be parsed and components of a problem for Forbidden FRUITS can be generated.
  • Optimizing the modification strategy (Kelly): Literature research to possible strategies
  • Sequence optimization (Joris): Literature research on sequence optimization and work on a strategy to implement.

July 13th - 19th

Human Practices

  • Survey: Waiting for reply from safety office of the VU
  • Online workshops about genetic modification (Samira and Vicky): Start doing research on the content for the second workshop.
  • Market Research (Joris and Robin): process meeting about entrepreneurship
  • Benelux meetup: Finalize the team presentation (Vicky and Joris) and compose a webinar about linear programming in biology (Samira and Kelly)
  • Safe by design: reviewing the webinar

Wetlab

  • Producing salicylic acid with Synechocystis (Joris): fragments to replace pyk1 with the irp9 gene under regulation of the strong J23119 promoter were created using fusion PCR.
  • Producing Mannitol with Synechocystis (Robin): The colonies were grown. PCR was used to check if the 6kb fragment was inserted in the genome of the cells. Unfortunately, this was not the case, possibly due to the large size of the DNA fragment. Single colonies were replated on fresh plated with a higher concentration of spectinomycin (50 µL)
  • Producing lactate with Synechocystis (Kelly): Postponed due to possible Corona contamination (quarantine).
  • Producing salicylic acid with E.coli (Joris): Waiting for a reply from the university of Georgia concerning obtaining the JW6 (Wang, et al. 2019, University of Georgia) E.coli strain. Contact with another supervisor at the VU about the possibility of working in their lab.
  • Lactic acid bacterial cell factory (Robin): The strategy for L.Lactis appeared to be wrong. A new reaction was found. Literature research was performed. We will try to borrow genes from Arthrobacter that would not only allow for growth-coupled production of salicylate but also involve the degradation of PAH (which is a carcinogenic compound). Although the approach sounds promising, we were still not able to find all the answers necessary and therefore, we contacted the authors of the paper.

Drylab

  • Merging Databases (Dennis): Halted due to holiday
  • Global Plumber (Samira): Incorporating faucets (input reactions) and sinks (output reaction) in the plumber
  • Cheap lunches (Vicky): To become more familiar with the pathfinding methods in Forbidden FRUITS, try to implement pathfinding for “branched” pathways.
  • GPRA network (Mingdong): Expand the Gene Protein Reaction processing script into a script which can parse the components of a problem for Forbidden FRUITS.
  • Optimizing the modification strategy (Kelly): Literature research to possible strategies
  • Sequence optimization (Joris): Literature research on sequence optimization and work on a strategy to implement.

July 20th - 27th

Human Practices

  • Survey: The safety board of the VU wants to discuss the survey with the Ethics Committee of the VU. We have to wait for their approval until August 10th.
  • Online workshops about genetic modification (Samira and Vicky): Prepare and practice the presentation for the second workshop. Promote on social media and within personal networks. The recordings of the first webinar were posted.
  • Market Research (Joris and Robin): process meeting about entrepreneurship
  • Benelux meetup: Finalize the team presentation (Vicky and Joris) and webinar about linear programming in biology (Samira and Kelly). 22-25th the meetup was held. We processed the feedback of the other teams and the judges to improve our projects.
  • Safe by design: reviewing the webinar
  • NEMO kennislink blog: invited to send a test blog for the Dutch public about our project.
  • Micropia workshops: first contact with Ruben from Micropia about possible collaboration. We would like to use their museum to involve people in the cell factory research.

Wetlab

  • Producing salicylic acid with Synechocystis (Joris): fragments to replace pyk1 with the irp9 gene under regulation of the strong J23119 promoter were transformed into Synechocystis. Meanwhile, the construction of the DNA fragments to replace pyk2, without disturbing the transcriptional unit was started. This was unsuccessful, probably due to a wrong annealing temperature. To find the optimal annealing temperatures for the fragments, temperature gradient PCR will be performed.
  • Producing Mannitol with Synechocystis (Dennis): 6 colonies were grown. PCR was used to check if the 6kb fragment was inserted in the genome of the cells. 3 out of 6 colonies had the susA gene in their genome.
  • Producing lactate with Synechocystis (Kelly): Prepare BG11 medium stock.
  • Producing salicylic acid with E.coli (Joris): Waiting for a reply from the university of Georgia concerning obtaining the JW6 (Wang, et al. 2019, University of Georgia) E.coli strain. Contact with another supervisor at the VU about the possibility of working in their lab.
  • Lactic acid bacterial cell factory (Robin): An enzyme in a reaction in Arthrobacter sp. F101 is identified, so that we can ultimately move forward with the larger lactis plan. Further study of this reaction will be fruitful, since it involves the degradation of the carcinogen PAH.

Drylab

  • Merging Databases (Dennis): Halted due to holiday
  • Global Plumber (Samira): Incorporating faucets (input reactions) and sinks (output reaction) in the plumber
  • Cheap lunches (Vicky): Creating a toy database with reactions that make cheap lunch strategies. Thinking of different types of cheap lunch strategies and how they might make the reaction coupling more complicated. The cheap lunch strategies are implemented using the Forbidden FRUITS API.
  • GPRA network (Mingdong): Familiarize with the tools needed to further develop the gene protein reaction association: Unittest in python, HTML, CSS, bootstrap and Database API.
  • Optimizing the modification strategy (Kelly): designing of a new function to mark downstream metabolites
  • Sequence optimization (Joris): A MinMax tool was created by setting up a diagram. Besides the start was made in creating a simple DNA transcriber, the next step is to extend it to the codon table.

July 28th - August 2nd

Human Practices

  • Survey: Wait for approval from the VU ethics committee until August 10th.
  • Online workshops about genetic modification (Samira and Vicky): Give the second workshop about genetic modification in biotechnology. Unfortunately our promotion strategy was not very successful: we only had 1 participant.
  • Market Research (Joris and Robin): process meeting about entrepreneurship
  • Safe by design: Trying to find safe by design coach (holiday delays)
  • NEMO kennislink blog (Kelly): start writing a test blog.
  • Micropia workshops (Kelly and Joris): first skype meeting with Ruben from Micropia about possible collaboration

Wetlab

  • Producing salicylic acid with Synechocystis (Joris): The construction of the DNA fragments to replace pyk2, without disturbing the transcriptional unit was still not successful for some fragments. To find the optimal annealing temperatures for the fragments, temperature gradient PCR was performed, new template DNA was extracted from Synechocystis WT and new primer solutions were prepared
  • Producing Mannitol with Synechocystis (Dennis): The quality of the inserts were studied. By amplification of the full sequence of the insert, it was checked if the gene was incorporated by single or double crossover. Besides, it was tested if susA was inserted into the genome by amplification of the last part of the vector. Results were looking promising.
  • Producing lactate with Synechocystis (Kelly): New primers were designed. In order to overexpress pyk2, this gene was amplified from WT Synechocystis. Pyk2 was inserted into a vector using enzymatic digestion and transformed into E.coli.
  • Producing salicylic acid with E.coli (Joris): Waiting for a reply from the university of Georgia concerning obtaining the JW6 (Wang, et al. 2019, University of Georgia) E.coli strain. Contact with another supervisor at the VU about the possibility of working in their lab.
  • Lactic acid bacterial cell factory (Robin): The ordering of the Arthrobacter strain is difficult and therefore a backup plan using Pseudomonas sp JM2 was designed.
  • Multiwell cultivator (Vicky): Getting familiar with the 24-well cultivator by reading documentation.

Drylab

  • Merging Databases (Dennis): Halted due to holiday
  • Global Plumber (Samira): Incorporating faucets (input reactions) and sinks (output reaction) in the plumber
  • heap lunches (Vicky): Creating classes to implement the cheap lunch strategies into the Forbidden FRUITS API, which is complicated due to the highly integrated structure of the Forbidden FRUITS programme.
  • GPRA network (Mingdong): Rewriting the GPRA parsing method with the libsbml library. Adding general features (like printing functions) to the current GPRA object.
  • Optimizing the modification strategy (Kelly): Creating a toy database
  • Sequence optimization (Joris): A MinMax tool was created by setting up a diagram. Besides the start was made in creating a simple DNA transcriber, the next step is to extend it to the codon table.

August

Deadlines are approaching and we all feel the effect of working in lockdown. Although there are slightly more opportunities to meet each other, we realize that we need to discuss some aspects of the programming and story telling in real life. By the end of the month, we were able to organize some meetings at Science Park to start integrating our drylab work. On the Human Practices side, we made plans to give workshops in micropia and we got the opportunity to write a blog for the Dutch public, which gave us new energy and hope to continue!
Highlights:

  • 3th: Second facebook workshop (Genetically modified organisms today?!) - Vicky & Samira
  • 6th: meeting with micropia considering experiments for workshops - Ruben Janssen, Jasper - Kelly & Joris
  • 19th: Thank you video is published
  • 25th (Vicky): Meeting with Uchicago team about possible collaboration
  • 31st: Lecture about LPs in FF by Samira to team

August 3rd - 9th

Human Practices

  • Survey: Wait for approval from the VU ethics committee until August 10th.
  • Online workshops about genetic modification (Samira and Vicky): Review the promotion strategy for the workshops. Start collecting information and outlining the third workshop.
  • Market Research (Joris and Robin): brainstorming about potential stakeholders. Set up an interview about the design process of MMP
  • Safe by design:Trying to find safe by design coach (holiday delays). Formulating questions.
  • NEMO kennislink blog (Kelly): We are selected for writing a blog. Kelly has been working on the content of the first blog
  • Micropia workshops: brainstorm about experiments.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Halted due to holiday
  • Producing Mannitol with Synechocystis (Dennis): The glgc gene was introduced into the genome. PCR amplification confirmed the introduction of susA and glgc in the synechocystis culture. We continued to achieve a full segregation of the susA gene in Synechocystis.
  • Producing lactate with Synechocystis (Kelly): A plasmid (pHKH_PcpcBA_pyk2) which had the pyk2 was created. The quality of the plasmid was verified using PCR amplification of the PcpcBA_pyk2 fragment on this plasmid showed that the concentration was too but low (below 10 ng/ul) for ligation. Therefore, the plasmid was remade. The concentration of this plasmid in the ligation mixture was high enough for ligation in E.coli.
  • Producing salicylic acid with E.coli (Joris): There has been contact with the university of Georgia concerning obtaining the JW6 (Wang, et al. 2019, University of Georgia) E.coli strain. We need to wait for a Material Transfer Agreement (MTA) to be signed.
  • Lactic acid bacterial cell factory (Robin): Research has been done to find a new strain and it seems that the salicylate strategy in L.lactis can go forward. We have been in touch with a lab in Houston who confirmed the enzymes which we need are not yet identified. The new strain will be: Pseudomonas nitroreducens. Also, we are in touch with the VU to get the strains ordered.
  • Multiwell cultivator (Vicky): Getting familiar with the 24-well cultivator by reading documentation.

Drylab

  • Merging Databases (Dennis): Halted due to holiday
  • Global Plumber (Samira): The current Plumber does not correctly predict modifications for all databases, therefore the desired/undesired constraints in the linear programme are tweaked to make it fit all types of databases.
  • Cheap lunches (Vicky): Trying to implement pathfinding for cheap lunch strategies. The idea is to use “normal” pathfinding once a metabolite that can be directly growth coupled is found when tracing back in the additional chain of reactions that produce the target metabolite. Because it is important to take advantage of what is already programmed, understanding what the different methods in the different classes involved do is an important part of the process.
  • GPRA network (Mingdong): New examples have been made and a script to parse sbml files has been added. Exploring tools which already exist within Forbidden FRUITS.
  • Optimizing the modification strategy (Kelly): Exploring the OOP in Forbidden FRUITS
  • Sequence optimization (Joris): Exploring the Forbidden FRUITS API

August 10th - 16th

Human Practices

  • Survey: Finally received a reply from the VU ethics committee. They suggested that we should write a data management plan, so Kelly and Samira worked on that. For help, we reached out to the safety officer at the science faculty of the VU.
  • Online workshops about genetic modification (Samira and Vicky): Preparing the presentation for the third workshop.
  • Market Research (Joris and Robin): brainstorming about potential stakeholders. Set up an interview about the design process of MMP. In touch with the VU about following a market research course in the autumn.
  • Safe by design:Trying to find safe by design coach (holiday delays). Joris has set up a framework for the safe by design infographic.
  • NEMO kennislink blog (Kelly): Writing the first blog and discussing this blog with NEMOkennislink
  • Micropia workshops (Mingdong and Samira): set up a first version of an experimental plan for the small workshops in Micropia.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Halted due to holiday
  • Producing Mannitol with Synechocystis (Dennis): The introduction of susA in the genome was confirmed. Sequencing showed no mutations in the gene. We continued to achieve a full segregation of the susA gene in Synechocystis.
  • Producing lactate with Synechocystis (Kelly): The replicative plasmid pHKH_PcpcBA_pyk2containing the pyk2 was transformed into E.coli WT. To check if there are mutations in the plasmid, the plasmid was isolated from E.coli and sent for sequencing. The initial plan was to replace the spectinomycin resistance gene in the plasmid with a chloramphenicol resistance gene. However, study has shown that this is not feasible. A different replicative plasmid already containing the chloramphenicol resistance cassette was obtained
  • Producing salicylic acid with E.coli (Joris): The MTA for the strain has been sent to the head of MMP. When the MTA is signed by both parties, the strain willl be shipped to Amsterdam.When the strain arrives, we plan to implement the irp9 gene from Yersinia enterocolitica in this strain to realize the production of salicylic acid.
  • Lactic acid bacterial cell factory (Robin): Finalizing strain order. We have been in touch with Vera, DSMZ, and researchers to decide on the strain. Because reaction R05537 is not well studied, it’s difficult to decide which strain of Pseudomonas nitroreducens to order.
  • Multiwell cultivator (Vicky): Studied data reports from 24-well-cultivator and the circuit diagram to try to understand possible causes for the behavior of some plots. Some results and saturation of the sensors with certain wavelengths might be due to the circuit design, this might not be easy to address. Designing better ways to approach the calibration issues

Drylab

  • Merging Databases (Dennis): The MetaNetX database was updated. This meant that the files were changed, so the current parsing method was adjusted. The updated database contains the double amount of information, which means that there is more information to find reactions and compounds in Forbidden FRUITS!
  • Global Plumber (Samira): We are trying a different approach on the Plumber: only irreversible reactions and adding faucets are considered. The sinks were added later. The main problem lies in the prediction of knockouts, since the linear program doesn’t strictly need to knock a gene out in order to redirect the flux.
  • Cheap lunches (Vicky):.Creating new methods and attributes in the cheap lunch class to make the strategy search work for all types of strategies. This is achieved by following the algorithm step by step to see where it goes and where new/overriding methods might be useful.
  • Optimizing the modification strategy (Kelly): Writing an approach to find flux consuming essential reactions upstream of the production reaction and relocate them to a position downstream of the production reaction. To achieve this, we need to find reactions with the same essential compound in a database. These can be placed downstream of the production metabolite if feasible.
  • Sequence optimization (Joris): Exploring the Forbidden FRUITS API

August 17th - 23rd

Human Practices

  • Survey: Kelly and Samira worked on a data management plan. For help, we reached out to the safety officer at the science faculty of the VU.
  • Online workshops about genetic modification (Samira and Vicky): Preparing the presentation for the third workshop.
  • Market Research (Joris and Robin): brainstorming about potential stakeholders. Set up an interview about the design process of MMP. In touch with the VU about following a market research course in the autumn.
  • Safe by design:Trying to find safe by design coach (holiday delays). Joris has set up a framework for the safe by design infographic.
  • NEMO kennislink blog (Kelly): Writing the first blog and discussing this blog with NEMOkennislink.
  • Micropia workshops (Mingdong and Samira): set up a first version of an experimental plan for the small workshops in Micropia.
  • CRISPRCon 2020 (Robin): Because of a participation in CRISPRCon of previous years, Robin was approached by the CRISPRCon organisation to host a discussion. She is in touch with the organisation to explore this possibility to hold a discussion related to cell factory engineering.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Colony PCR to amplify the irp9 gene from the colonies which were transfected showed that only one out of 20 colonies had the irp9 gene incorporated. A colony PCR was performed to check if the pyk1 gene was absent in this colony. Besides, we tried to obtain fragments by PCR to add a promoter in front of the sll1276 and after pyk2 to make sure this gene does not get disturbed by knocking out pyk2. However, the constructs containing pyk2 were difficult to obtain. To find the optimal annealing temperature a temperature gradient PCR was performed and new gDNA was extracted from a fresher Synechocystis culture.
  • Producing Mannitol with Synechocystis (Dennis): We continued to increase the antibiotic spectinomcin and chlorampenicol concentration to achieve a full segregation of the susA gene in Synechocystis. Besides, the effect of using sucrose to push the use of this gene is studied.
  • Producing lactate with Synechocystis (Kelly): Transformation of the pHKH_PcpcBA_pyk2 plasmid into E.coli was confirmed by colony PCR. Another plasmid (MDH::PcpcBA_mlth) was extracted from E.coli. This plasmid will be incorporated in the WT Synechocystis strain to create SMG001. SMG001 is Synechocystis strain, where we want to introduce the product reaction mlth with a strong promoter PcpcBA and remove the mdh gene to prevent the from Oxaloacetate to malate reversed reaction. Besides a liquid culture of synechocystis was made and BG-11 and LB plates with kanR were prepared.
  • Producing salicylic acid with E.coli (Joris): The MTA for the strain has been sent to the head of MMP. When the MTA is signed by both parties, the strain willl be shipped to Amsterdam.When the strain arrives, we plan to implement the irp9 gene from Yersinia enterocolitica in this strain to realize the production of salicylic acid.
  • Lactic acid bacterial cell factory (Robin): Finalizing strain order. We have been in touch with Vera, DSMZ, and researchers to decide on the strain. Because reaction R05537 is not well studied, it’s difficult to decide which strain of Pseudomonas nitroreducens to order.
  • Multiwell cultivator (Vicky): Data reports from 24-well-cultivator were used to analyse the correlation between the measurements of different wavelengths at different intensities after calibration with red light.

Drylab

  • Merging Databases (Dennis): The parsing script of MetaNetX database is adjusted such that it correctly processes the data after the MetaNetX update. This was easier than expected. The next step is to improve the merger (what does it do?). We started by designing two dummy databases to test the current merger and to see which parts are not working correctly.
  • Global Plumber (Samira): Knockouts are forced by a constraint which forces flux through reactions that are not knocked out. Since this approach is not generic, we have been trying to change the undesired constraints such that the same is achieved.
  • Cheap lunches (Vicky): Implementing finder methods from Forbidden FRUITS in the cheap lunch strategy finding.
  • GPRA network (Mingdong): A class to make gene protein associations from sbml files was made. In order to incorporate this into the Forbidden FRUITS structure, the database structure and several classes were studied, as well as object oriented programming in general.
  • Optimizing the modification strategy (Kelly): We have been writing a code which can find an upstream, essential flux consuming reaction. So far the code will find all the upstream reactions of the production reaction. Starting at the product forming reaction, it traces back the reactions producing the product all the way back to the substrate consuming reaction.
  • Sequence optimization (Joris): Exploring the Forbidden FRUITS API

August 24th - 30th

Human Practices

  • Survey: Kelly and Samira worked on the data management plan
  • Online workshops about genetic modification (Samira and Vicky): Preparing the presentation for the third workshop.
  • Market Research (Joris and Robin): brainstorming about potential stakeholders. Set up an interview about the design process of MMP. In touch with the VU about following a market research course in the autumn.
  • Safe by design:Trying to find safe by design coach (holiday delays). Joris has set up a framework for the safe by design infographic.
  • NEMO kennislink blog (Kelly): Discussed the second blog with NEMOkennislink.
  • Micropia workshops (Mingdong and Samira): set up a first version of an experimental plan for the small workshops in Micropia.
  • CRISPRCon 2020 (Robin): A discussion session hosted by Robin is confirmed by the CRISPRCon 2020 organization.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): We have sequenced irp9 from the colony which was transformed with the irp9 construct, unfortunately there were 2 mutations. We are going to redo the transformation. Also we found out that one of the primers (to make a construct with pyk2) to make the construct to introduce a promoter between sll1276 and pyk2 was incorrect, a correct primer was ordered. Also, to a plasmid containing elements for CRISPR-Cpf1 modification, gRNA to find pyk2 was added. This plasmid was transformed into E.coli to later knockout pyk2 in Synechocystis.
  • Producing Mannitol with Synechocystis (Dennis): We continued to increase the antibiotic (which one?) concentration to achieve a full segregation of the susA gene in Synechocystis. Besides, the effect of using sucrose to push the use of this gene is studied.
  • Producing lactate with Synechocystis (Kelly): Waiting for segregation of the smg001 strain.
  • Producing salicylic acid with E.coli (Joris): The MTA for the strain has been sent to the UvA. When the MTA is signed by both parties, the strain will be shipped to Amsterdam.We are designing primers and an experimental strategy to be able to start at the VU as soon as the strain arrives.
  • Lactic acid bacterial cell factory (Robin): We are waiting for the strains to arrive from Germany.
  • Multiwell cultivator (Vicky): We are trying to find ways to plot the 24-well cultivator data to see the relation between wavelength and intensity.

Drylab

  • Merging Databases (Dennis): Finally 100.1% merging on compounds and 70% on reactions was achieved. Although this is a big improvement, a lot of errors still need to be fixed and the reactions merging could still be improved. The next step is to merge all databases instead of two at a time.
  • Global Plumber (Samira): We have connected the addition of faucets and sinks to knockouts, so no excessive faucets and sinks are added.We were still trying to find a way to force flux through only those reactions which don’t have knockouts upstream.
  • Cheap lunches (Vicky): The current cheap lunch strategy finder is working with a toy database. The next step is to validate it on an actual database.
  • GPRA network (Mingdong): The Gene Protein Reaction Association parsing method was incorporated in the database module, although this still requires further improvement. We are planning on incorporating the model transformation in the database module as well. To test the current method a paper (specify) which uses pFBA to verify their results on the iAF1260 model was studied.
  • Optimizing the modification strategy (Kelly): At the moment, the algorithm is able to find possible flux consuming essential reactions upstream of the production reaction. The next step is to confirm that these reactions consume flux.
  • Sequence optimization (Joris): We are struggling with how to incorporate the sequence optimizer for heterologous expression in Forbidden Fruits. We are discussing how and where to implement this tool and how to build it based on the %MinMax algorithm.

September

After the holiday season, things started to get rolling in September. With people coming back from their holidays, we were finally able to handle some wet lab related issues and the other Human Practice projects are now really getting started. Because we can meet more often in person, we were able to connect the dots between our individual projects and to create a story. Unfortunately, we have bad luck with ordering the strains for the E.coli and L.lactis strategy. But we came across a perhaps more promising approach using a fast growing cyanobacterial strain!
Highlights:

  • 4th: first blog posted on biotechnologie.nl
  • 4th: ‘Thank you’ video is sent out to our crowdfunding sponsors
  • 5th: Third facebook workshop (Exploring the potential of (genetically modified) microbes) - Samira & Vicky
  • 8th: trip to micropia to discuss possible workshop
  • 8th: publishing of JOGL interview
  • 11th: discuss FF pipeline - Kelly, Joris, Vicky, Samira
  • 214th: post second blog on biotechnology.nl
  • 28th: CRISPR-Con “ideas Marketplace” discussion: “The Ethical Implications of Creating Anything in Anything”- Robin
  • 28th: survey is published

August 31st - September 6th

Human Practices

  • Survey: Kelly discussed the data management plan with the data management office of the VU beta faculty. While we are making this plan, we are reaching out to the Ethics Committee to get the official approval and be able to start sending out our survey.
  • Online workshops about genetic modification (Samira and Vicky): Presentation of the third facebook workshop (Exploring the potential of (genetically modified) microbes) was a success, although we only had one attendant. This attendant had a more critical opinion about genetic modification, which helped us in understanding the ‘other side’ of the genetic modification story. In this webinar we gave a dummy explanation about our projects and metabolic networks, so we hope we can reach out to more people with the recordings.
  • Market Research (Joris and Robin): brainstorming about potential stakeholders. Set up an interview about the design process of MMP.
  • Safe by design:Trying to find safe by design coach (holiday delays). Joris has set up a framework for the safe by design infographic.
  • NEMO kennislink blog (Kelly): The first blog is posted on biotechnologyie.nl. Start on writing the second blog.
  • Micropia workshop: Revising the plan for the workshops in Micropia. We are collecting all materials and chemicals we need to test the experiments we have designed in the lab.
  • CRISPRCon 2020 (Robin):Preparing the discussion session.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): The amplification of fragments to add the irp9 under regulation of the J23119 promoter while knocking out pyk1 were recreated using phusion PCR. Sequencing results showed no mutations in the fragment. To introduce a promoter between sll1276 and pyk2, we tried to make DNA fragments using PCR amplification. This proved to be more difficult to be expected, so the protocol was optimized once more using temperature gradient PCR. Also, the plasmid containing CRISPR-Cpf1 elements and pyk2 gRNA was sequenced. No mutations were present in the plasmid.
  • Producing Mannitol with Synechocystis (Dennis): Adding sucrose to the media didn’t give the expected result yet. The Synechocystis cells are growing culture with and one without sucrose. Hopefully they will show more segregation over time. Other methods to force the cells to incorporate the susA gene in their genomes, for example adding salt to the media, are explored.
  • Producing lactate with Synechocystis (Kelly): We are still trying to construct the ppc::PcpcBA_NAPD_ME2_Kan_Cm plasmid to knockout the ppc gene. After adding the chloramphenicol resistance cassette in E.coli using restriction enzymes, digestion and ligation to this plasmid, the results were confirmed using colony PCR. The plasmids were sent for sequencing. Next, we will conjugate the pHKH_PcpcBA_pyk2 plasmid from E.coli to Synechocystis.
  • Producing salicylic acid with E.coli (Joris): There is still no information about the status of the MTA at the UvA. The details about the experiments are discussed. The plasmid containing the irp9 gene is ready, so the strain can be transformed as soon as it arrives.
  • Lactic acid bacterial cell factory (Robin): The strains have arrived. Experiments are prepared and the necessities for the culture medium are studied.
  • Multiwell cultivator (Vicky): We are analyzing plots of the 24-well cultivator data to see the relation between wavelength and intensity. Besides, the first steps to performing experiments with the cultivator were made.

Drylab

  • Merging Databases (Dennis): The merging method is tested. There are still a lot of mistakes that keep coming up when trying to find all exceptions. The merger is planned to be finished next week, so we can precede with merging multiple databases.
  • Global Plumber (Samira): The Plumber seems to correctly predict the combination knockins and knockouts to add the production reaction in all the test databases. Next step is to see if the program is working on a full Synechocystis model (iSynCJ816). Besides, we are considering to add gene-usage relations to make the Plumber minimize the number of genes which need to be knocked in or out. We are also studying the solver to see if we can get a solution pool with other (sub)optimal strategies.
  • Cheap lunches (Vicky): Studied the structure of the database files to see if they are useful in the cheap lunch finder class. At the moment, as well the database as the model are read correctly. We are still looking for good compounds which can be used for testing.
  • GPRA network (Mingdong): We succeeded in transforming the reactions-metabolites network of the E.coli iAF1260 model into a gene-based network with gene-protein-reaction information. We validated and reproduced the results on the transformed network and original network with pFBA. The validation will be continued and a first attempt to integrate this method in the Forbidden FRUITS pipeline will be made in the coming weeks.
  • Optimizing the modification strategy (Kelly):Change from defining upstream metabolites to downstream metabolites. Rewrite the function for finding essential metabolites
  • Sequence optimization (Joris): The plan is to first build a sequence optimizer for codon usage, then build an extra element to input the desired reaction. This element will link the reaction to a protein. Codon optimization can be performed by reverse translation of the protein.

September 7th - 13th

Human Practices

  • Survey: While waiting for the reply of the ethics committee, we have been in touch with the Dutch tv show ‘Keuringsdienst van Waarde’ about them spreading out our survey in their networks. This tv show attracts people with a critical mind about current industrial practices and consumers behaviour.
  • Market Research (Joris and Robin): An interview about the design process of MMP was held with some core members of the research group. We are working on a plan to interview potential stakeholders on their view on the project.
  • Safe by design:Trying to find safe by design coach (holiday delays).
  • NEMO kennislink blog (Kelly): Writing the second blog and making a short vlog about working on programming and working in the lab. Besides, a clear infographic was made by Joris
  • Micropia workshop (Joris, Vicky and Mingdong): Testing the experiments in the lab to see if it is workable. We had to adjust our plan slightly for safety reasons, but most experiments were successful. We are still searching for other fun experiments to explain about cell factories.
  • CRISPRCon 2020 (Robin):Preparing the discussion session.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Finally the constructs to introduce a promoter between sll1276 and pyk2 are correctly made using phusion PCR. It appeared that the phusion polymerase (with proofreading capacity) was not able to amplify certain gene fragments, while the Taq polymerase (without proofreading capacity) was.
  • Producing Mannitol with Synechocystis (Dennis): The medium with sucrose showed minor improvements to the segregation of the susA gene. In order to increase our chances we designed new experiments to see if the segregation could be improved. As susA is a key part of making mannitol and mannitol can be used to combat osmotic stress, adding salt to the media could select the microbes with high production of mannitol in a Darwinistic way, making susA essential. Besides, the gene to be replaced by susA (glgc) is essential for making glycogen during daytime. The glgc gene will not be essential if the light intensity is limited. Without light, the cells will not be able to do photosynthesis and thus will not have energy. Therefore, we added glucose to the media to keep the cells happy.
  • Producing lactate with Synechocystis (Kelly): Sequencing showed that there were two non-synonymous mutations in the ppc::PcpcBA_NAPD_ME2_Kan_Cm plasmid to knockout the ppc gene. This will be repaired by fusion PCR. Therefore primers are ordered. We continued to push segregation in SMG001 to incorporate the mlth, production reaction and knock-out the mdh gene.
  • Producing salicylic acid with E.coli (Joris): There is still no information about the status of the MTA at the UvA. The details about the experiments are discussed. The plasmid containing the irp9 gene is ready, so the strain can be transformed as soon as it arrives.
  • Lactic acid bacterial cell factory (Robin): The strains were collected from the VU and transported to Science Park. There are many uncertainties concerning the supervision of this project. The experimental strategy is revised and information about gene candidates for the enzyme of study is collected.
  • Multiwell cultivator (Vicky): The system of the 24-well cultivator was explored while taking some measurements. Future growth experiments were prepared.

Drylab

  • Merging Databases (Dennis): The merging method is almost finished. Working on debugging.
  • Global Plumber (Samira): The iSynCJ816 model was loaded into the Plumber. To validate the approach we are planning to run a brute force simulation (to calculate all possible combinations of modifications) on the iSynCJ816 model to see if the strategy the plumber produces is feasible. To achieve this, the model first has to be transformed to a model with only irreversible reactions. Code from the gene-protein-relation transformation will be adjusted for this purpose.
  • Cheap lunches (Vicky): Trying to figure out the way of validating the cheap lunch strategy finder using a real database instead of a toy database.
  • GPRA network (Mingdong): We are looking for studies which performed a gene-protein-reaction transformation and validated their approach using pFBA to verify our approach with a few more established cases. Besides, the integration of this method in the Forbidden FRUITS pipeline was discussed.
  • Optimizing the modification strategy (Kelly): Working on a method to efficiently find reactions downstream of the production reaction.
  • Sequence optimization (Joris): Organized the sequence optimizer in smaller modules: going from KEGG reaction to gene to DNA/protein sequence, optimize the sequence using codon frequencies and optimize the GC-content.

September 14th - 20th

Human Practices

  • Waiting for the reply of the ethics committee. Waiting for a reply from the Dutch tv show ‘Keuringsdienst van Waarde’ about them spreading out our survey in their networks.
  • Market Research (Joris and Vicky): The interview about the design process of MMP was held with some core members of the research group was processed. This gave us insight into the story of MMP in general, stable production within Synechocystis by trying different how’s. From growth-coupling (FRUITS) to fitness coupling (faster growing under specific conditions) and now the narrative we are trying to tell in Forbidden FRUITS.
  • Safe by design:Trying to find safe by design coach (holiday delays).
  • NEMO kennislink blog (Kelly): Post second blog on biotechnology.nl
  • Micropia workshop (Joris): We are still searching for other fun experiments to explain about cell factories. Finalizing the experimental design and making information sheets.
  • CRISPRCon 2020 (Robin):Preparing the discussion session.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): We conjugated the pWD199 plasmid containing CRISPR-Cpf1 elements and pyk2 gRNA together with the pWD133 plasmid to facilitate the conjugation to Synechocystis in order to modify the pyk2 gene. To the mixture containing E.coli with the plasmids and Synechocystis we also added the gene constructs to place the J23119 (stronger) or J23104 (weaker) promoter between pyk2 and sll1276. We hope to knockout pyk2 after adding the promoter in front of the neighbouring gene using CRISPR-Cpf1.
  • Producing Mannitol with Synechocystis (Dennis): Experiments are postponed due to illness.
  • Producing lactate with Synechocystis (Kelly): Primers were ordered to repair the non-synonymous mutations in the ppc::PcpcBA_NAPD_ME2_Kan_Cm plasmid by phusion PCR. We continued to push segregation in SMG001. Next, we will conjugate the pHKH_PcpcBA_pyk2 plasmid from E.coli to Synechocystis.
  • Producing salicylic acid with E.coli (Joris): The MTA to obtain the strain is still not signed by the UvA. Thus we decided to order the commercial strain ATCC 31884 (good reference) to be able to finally start our experiments.
  • Lactic acid bacterial cell factory (Robin): Still trying to find a feasible experimental strategy given the expertise of the supervisors and the amount of time left.
  • Multiwell cultivator (Vicky): Growth experiments in the 24-well cultivator were started. Some intensity measurements were taken to validate the calibration of the diodes. A R report is made to analyse the calibration data.
  • Producing lactate with Synechococcus UTEX 2973 (Robin and Joris): Because of the continuous problems with obtaining the E.coli strain and with the strategy design for L.Lactis, we started on a new, promising strain. The fast growing cyanobacterium Synechococcus UTEX 2973 should be relatively easy to manipulate. This week we started by constructing the replicative plasmid (pSEVA-451) with PpsbA2-mlth and PcpcBA-mlth by digestion and ligation. The resulting pSEVA-451-PpsbA2-mlth and -PcpcBA-mlth was transformed into E.coli.

Drylab

  • Merging Databases (Dennis): All merger options are checked and work as expected. We are moving on to multiple database merging. The reaction merging still has some small problems, but we will try to resolve this along the way.
  • Global Plumber (Samira): The iSynCJ816 model can now be transformed into a network with only irreversible reactions. This method will be validated using FBA. When this method is valid, we will run a brute force simulation (to calculate all possible combinations of modifications) on the iSynCJ816 model to see if the strategy the plumber produces is feasible. We also have been designing a strategy to incorporate gene-protein-relations in the Plumber.
  • Cheap lunches (Vicky): Trying to figure out the way of validating the cheap lunch strategy finder using a real database instead of a toy database.
  • GPRA network (Mingdong): The network transformation methods were moved from the examples to the database and combined in a simple pipeline. This was confirmed to be workable. Besides the design of a gene-protein-reaction association (GPRA) entry was changed by means of how it connects reaction and gene products, how a GPRA is presented and how it deals with the database.
  • Optimizing the modification strategy (Kelly): Designing a strategy using object oriented programming, modify the current function to this style.
  • Sequence optimization (Joris): Organized the sequence optimizer in smaller modules: going from KEGG reaction to gene to DNA/protein sequence, optimize the sequence using codon frequencies and optimize the GC-content.

September 21st - 27th

Human Practices

  • Survey: Received an official approval from the ethics committee. Waiting for a reply from the Dutch tv show ‘Keuringsdienst van Waarde’ about them spreading out our survey in their networks. In the meanwhile we are setting up a method to spread our survey among university students in Amsterdam.
  • Market Research (Joris and Robin): We have established contact with Photanol, Corbion and DSM in order to book a short interview. Thinking about interview questions for this interview. Besides, Joris and Robin have followed an entrepreneurship course from ACE/IXA, which gave them inspiration and tools for performing the market research.
  • Safe by design: Stanley Brul has agreed to be our safe-by-design coach. We are working on finishing the safe-by-design infographic.
  • NEMO kennislink blog (Kelly): Brainstorming about the content of the third blog.
  • Micropia workshop (Joris, Kelly and Samira): This week, we discussed our experimental set-up with Micropia. We are setting up a timetable for the team members to be present in the museum during the week of sustainability (October 10th-17th). Besides, we have been finalizing the information sheets.
  • CRISPRCon 2020 (Robin):Preparing the questions for the discussion session

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Because there was no difference in growth between the positive (pWD199 plasmid without gRNA, no gene constructs) and negative (pWD199 plasmid with gRNA, no gene constructs) control, we decided to redo the conjugation of positive and negative control strain to see if this was due to the experimental procedure or if the gRNA is not correctly folded.
  • Producing Mannitol with Synechocystis (Dennis): We are growing new lines of cells in the dark to make sure glgc is not essential. However, because the cells can not perform photosynthesis this also resulted in extreme slow growth. The added osmotic stress could have helped the segregation, but it is too early to see. Hopefully they will grow more over the weekend, we hope to see more next week.
  • Producing lactate with Synechocystis (Kelly): The SMG001 strain was still not segregated. Besides, primers to repair the non-synonymous mutations in the PcpcBA_NAPD_ME2_Kan_Cm plasmid did not arrive yet. However, we conjugated the pHKH_PcpcBA_pyk2 plasmid to Synechocystis using E.coli in the SMG002, SMG003 and SMG004 strains in order to introduce pyk2 in these strains. In these strains the mlth gene, responsible for the production of lactate, is present under the control of promoters of different strengths.
  • Producing salicylic acid with E.coli (Joris): Waiting for the commercial strain ATCC 31884 (good reference) to arrive.
  • Multiwell cultivator (Vicky): A R report was made for last week's growth experiments. Early conclusions about the 24-well-plate calibrations were drawn based on intensity measurements at different wavelengths. So far it seems that the model created with the red LED does not give the expected predicted intensity when applied to other wavelengths.
  • Producing lactate with Synechococcus UTEX 2973 (Robin and Joris): We constructed the pSEVA451_mlth with the two different promoters by digestion and ligation. Sequencing confirmed that there were no mutations. The resulting pSEVA-451-PpsbA2-mlth and -PcpcBA-mlth was transformed into E.coli.

Drylab

  • Merging Databases (Dennis): The merger is now able to merge all databases. The last step to complete it is to merge it with the sbml files which contain the metabolic networks.
  • Global Plumber (Samira): The iSynCJ816 model transformation to irreversible reactions was validated using FBA. The faucet on the coupling metabolite was replaced by a production pathway, which was tested using a test database. Over the weekend a brute force simulation (to calculate all possible combinations of modifications) on the iSynCJ816 model was run for pyruvate as coupling metabolite to see if the strategy the plumber produces is feasible. We also have working on a strategy to incorporate gene-protein-relations in the Plumber
  • Cheap lunches (Vicky): Creating a script to validate the cheap lunch strategy finder with the merged iSynCJ816 model and BIGG database.
  • GPRA network (Mingdong): We redesigned the Gene Product and GPRA entries using referencing. Currently, when creating a GPRA, all Gene Products in this GPRA will register this GPRA itself, so we can enter the GPRA through a Gene Product entry. The SBMLGPRAFactory was also redesigned to be in line with the methods of all other factories. Besides, we are trying to change the methods of adding a GPRA entry into the database to prevent double entries.
  • Optimizing the modification strategy (Kelly): Designing a strategy using object oriented programming and the already existing pathfinding methods to make a pathway from the essential metabolite to one of the downstream metabolites.
  • Sequence optimization (Joris): Setting up the modular structure for the objects in the sequence optimizer.

October

October is a month filled with iGEM. We are all working hard to finalize our projects, although for the wetlab projects this is not possible due to a late start. Besides the science, the human practices branch of the projects is also on full force: the survey is being filled in by many students, our workshops in Micropia are a success and we get involved in a tool to incorporate ethics in the design of biotech processes. A lot of work, but also a lot of fun!
Highlights:

  • 11th-25th: Daily workshops in Micropia
  • 13th: Recording a short video for the Demonstrator Lab open day - Samira
  • 19th: Closing of the survey about genetic modification in biotechnology
  • 20th: Discussion with Athena institute (VU) about incorporating ethics in the design of biotechnological processes
  • 21th: Third blog was posted on biotechnologie.nl
  • 22th: presentation about our project to the RIVM

September 28th - October 24h

Human Practices

  • Survey: We started spreading our survey on social media and the UvA buildings. Because of time limitations, we limit our target audience to university students in Amsterdam.
  • Market Research (Joris and Robin): We are carefully setting up questions for the interviews. Besides, Joris and Robin are following an entrepreneurship course from ACE/IXA, which gave them inspiration and tools for performing the market research.
  • Safe by design: We are working on finishing the safe-by-design infographic.
  • Micropia workshop (Joris): We used feedback from Micropia to further shape the workshop. Together with the promotion team of Micropia we recorded a small promotional video and decided on a promotion strategy. We also discussed the effect of the new Dutch COVID-19 measures on the workshops.
  • NEMO kennislink blog (Kelly): Working on the content of the third blog.
  • CRISPRCon 2020 (Robin): Robin hosted a discussion session on CRISPRcon “ideas Marketplace” on the ethics of what Forbidden FRUITS can allow us to do with genetic engineering. During this session she spoke with someone from ELSI (Ethical, Legal and Social Implications research) and someone from Keystone Policy center working at various points of supply chain for GMOs in agriculture.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): Again there was no difference in growth between the positive (pWD199 plasmid without gRNA, no gene constructs) and negative (pWD199 plasmid with gRNA, no gene constructs) control. This suggests that the gRNA might not be correctly processed. To prove this hypothesis we redid the conjugation experiment with a negative control using a plasmid which contains a gRNA which is known to work (pWD193).
  • Producing Mannitol with Synechocystis (Dennis): Because cells are grown in the dark to make sure glgc is not essential, they grow extremely slow. We have tried colony PCR to see if segregation is progressing but failed due to low concentrations of cells. We decided on a new protocol for colony PCR.
  • Producing lactate with Synechocystis (Kelly): The SMG001 strain was still not segregated. The non-synonymous mutations in the PcpcBA_NAPD_ME2_Kan_Cm plasmid were repaired using phusion PCR. The conjugation of the pHKH_PcpcBA_pyk2 plasmid to Synechocystis using worked for the SMG003 and SMG004 strains but not for the SMG002 Strain. A fresh culture of SMG002 was prepared to redo the conjugation.
  • Producing salicylic acid with E.coli (Joris):The E. coli strain JW6 (Wang, et al. 2019, University of Georgia) has arrived.
  • Multiwell cultivator (Vicky): A second growth experiment was set up. We are analysing the results from the first growth experiment .It seems that the growth in the plates is not exponential and it does not match the growth curves from the preculture, but no conclusions can be drawn yet.
  • Producing lactate with Synechococcus UTEX 2973 (Robin and Joris): We constructed DNA fragments to knockout the ppc gene and to insert a kanamycin resistance cassette. Colony PCR showed that the transformation of the pSEVA-451-PpsbA2-mlth plasmid was less successful (some false positives) than the transformation of the pSEVA-451-PcpcBA-mlth (few false positives). The transformed plasmids were purified and sent for sequencing.

Drylab

  • Merging Databases (Dennis): The merger was finalized and will be implemented in the working version of Forbidden FRUITS soon.
  • Global Plumber (Samira): Unfortunately the simulation of the iSynCj816 model was too computationally intensive and thus aborted. A new validation approach using FBA and FVA was set up. Besides the desired and undesired constraints were improved to make the program more generic. This set of rules was feasible for multiple test databases. Because we discovered that the current BIGG version of the iSynCJ816 was incomplete, we are adjusting the algorithm to use the older synechocystis model for validation.
  • Cheap lunches (Vicky): Testing the validation method for the cheap lunch finder class.
  • GPRA network (Mingdong): Made NetworkBuilder(s) classes, with BaseNetworkBuilder, GPRANetworkBuilder and NetworkBuilder. The network transformation functions are now settled into those classes. Also made corresponding changes in the pFBA example when making networks.
  • Network Builder (Mingong):Because of the increasing complexity of building a network from a cell model and the database, we decided to make an additional class specified in network building.
  • Optimizing the modification strategy (Kelly): A class was made to find essential and downstream metabolites. Currently, we are adapting existing functions to make it compatible for making downstream paths.
  • Sequence optimization (Joris): Setting up the modular structure for the objects in the sequence optimizer.
  • Forbidden FRUITS pipeline (Samira, Mingdong): Together with our supervisors (Max Guillaume and Joeri Jongbloets), we discussed the concepts and definitions of the component of the Forbidden FRUITS pipeline. The implementation of the gene protein reaction association and plumber were discussed in detail.

October 5th - 11th

Human Practices

  • Survey: Our survey is up and running. We are promoting it as much as possible.
  • Market Research (Joris and Robin): Joris and Robin are still following an entrepreneurship course from ACE/IXA, which gave them inspiration and tools for performing the market research. Some interviews with different companies were arranged
  • Safe by design: The safe-by-design infographic is nearly finished. We are in contact with Cecile from RIVM the risk assessment of Forbidden FRUITS. We are planning a short discussion in the coming week.
  • NEMO kennislink blog (Kelly): Working on the content of the third blog.
  • Micropia workshop (Joris): We started the first workshop in Mircropia this week. We were able to speak to many people and educate many children. As well the visitors, as the staff in Micropia, as us were enthusiastic about the result.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): The conjugation to study if the current pyk2 gRNA is functional was unsuccessful. Therefore, we redid the experiment. In the meanwhile, new gRNA was designed. We ligated the gRNA in the pWD199 plasmid and transformed E.coli. Besides, a start was made to synthesize the DNA template fragments for the CRISPR-Cpf1 experiment. Next week, we will continue preparing an E.coli strain for CRISPR-Cpf1 with the new gRNA.
  • Producing Mannitol with Synechocystis (Dennis): Cells are not segregated yet. We will continue the growth experiment under various conditions.
  • Producing lactate with Synechocystis (Kelly): The SMG001 strain was still not segregated. The reparation of the non-synonymous mutations in the PcpcBA_NAPD_ME2_Kan_Cm plasmid was continued. To check if it worked out, the repaired plasmid will be sequenced.. The strains SMG003 and SMG004 are almost ready to knock out the ppc gene.
  • Producing salicylic acid with E.coli (Joris): We prepared a suitable medium for the pyruvate dependent E. coli strain JW6 (Wang, et al. 2019, University of Georgia) has arrived. We transformed this strain with the pFL-irp9 plasmid to insert the irp9 gene. We cultivated this strain in a medium without pyruvate, using the pyruvate dependent strain and wildtype as negative and positive control.
  • Multiwell cultivator (Vicky): The third growth experiment was set up. We are studying the growth under different light conditions with added bicarbonate. Meanwhile, we are analysing the results from the first growth experiments.
  • Producing lactate with Synechococcus UTEX 2973 (Robin): Colony PCR showed that the transformation of the pSEVA-451-PpsbA2-mlth plasmid was successful.

Drylab

  • Merging Databases (Dennis): A minor bug needs to be resolved before the merger can be implemented in the working version of Forbidden FRUITS. Documentation was added to the readme file.
  • Global Plumber (Samira):Validation of the Plumber using the older synechocystis model was continued. The program is slow and there are still some things going on which need further investigation.
  • Cheap lunches (Vicky): Before we can validate the cheap lunch strategy, the code needs to be debugged and wait for the final adjustments in the database file.
  • GPRA network (Mingdong): We documented and improved readability of the code.
  • Network Builder (Mingong): The network builders were redesigned.
  • Optimizing the modification strategy (Kelly): The class finds essential and downstream metabolites was tested on test databases. However, some adjustments are still necessary to be able to separate between native upstream and native downstream metabolites.

October 12th - 18th

Human Practices

  • Survey: Our survey is up and running. Already 280 people have filled it in.
  • Market Research (Joris and Robin): Employees from different companies in the biotechnology industry (amongst which DSM, Corbion and Photanol) were interviewed about their vision of the use of our computational tools in the design of biotechnological processes in their organization and to discuss their industrial process.
  • Safe by design: The safe-by-design infographic is finished and sent to the safe-by-design supervisor. A short discussion with Cecile from RIVM about the risk assessment of Forbidden FRUITS was held.
  • NEMO kennislink blog (Kelly): Writing the draft version of the third blog.
  • Micropia workshop: We continued with our workshops in Micropia this week. The visitors and staff from Micropia were enthusiastic about the workshops.

Wetlab

  • Producing salicylic acid with Synechocystis (Samira): It was confirmed that the gRNA we had designed for the CRISPR-Cpf1 experiment to knockout pyk2 was not functional. Last week we already designed new gRNA and ligated it into a pWD199 plasmid. We have transformed E.coli with the necessary plasmids for CRISPR-Cpf1 and conjugation to synechocystis and finalized the synthesis of the DNA template fragments for the CRISPR-Cpf1 experiment.
  • Producing Mannitol with Synechocystis (Dennis): Cells are not segregated yet. We will continue the growth experiment under various conditions.
  • Producing lactate with Synechocystis (Kelly): Due to illness, experiments were on hold. The reparation of the non-synonymous mutations in the PcpcBA_NAPD_ME2_Kan_Cm plasmid was confirmed by sequencing.
  • Producing salicylic acid with E.coli (Joris): The insertion of the pFL-irp9 plasmid int E. coli strain JW6 (Wang, et al. 2019, University of Georgia) was successful. We are trying to grow this strain independent of pyruvate.
  • Multiwell cultivator (Vicky): The fourth growth experiment was executed. Meanwhile, we are analysing the results from the first growth experiments and making plots to draw some preliminary conclusions.
  • Producing lactate with Synechococcus UTEX 2973 (Robin): The plasmid containing all fragments of template DNA to knock in the mlth and me genes.

Drylab

  • Merging Databases (Dennis): The merger was successfully implemented in the working version of Forbidden FRUITS.
  • Global Plumber (Samira):Validation of the Plumber using the older synechocystis model was continued. The program is slow and there are still some things going on which need further investigation.
  • Cheap lunches (Vicky): Before we can validate the cheap lunch strategy, the code needs to be debugged and wait for the final adjustments in the database file.
  • GPRA network (Mingdong): We polished and improved the code.
  • Network Builder (Mingong): The network builders were polished and improved.
  • Optimizing the modification strategy (Kelly): We adjusted the current code such that the reactions containing metabolites upstream of the coupling metabolite will be allowed to make part of the new path. The code is working on toy models, but should be expanded to real models of microorganism.
Forbidden FRUITS

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