This year, our aim was to overcome the physical, emotional and financial burden of Type 2 Diabetes mellitus. We targeted our objectives by designing a new, affordable therapy strategy, characterising our chassis organism, doing in silico work, as well as developing a new biocontainment feature for safe application of living organisms as therapeutics. Accordingly, we chose the tracks i) Therapeutics, ii) Software and iii) New Application

Targets reached:

• design and synthesis of a ready to express therapeutic
  (Incretin,
  Composite Part)
• evaluation of the public's perception of GMOs as therapeutics
  (Survey)
• education on GMOs and Diabetes prevention
  (Public_Engagement, Experimenta)
• development of a viable business strategy for the entry of a live GMO into the market
  (Entrepreneurship)
• design of a test format and the compounds to test the delivery of exendin-4 via cell penetrating peptides
  (Software)
• characterisation and data generation on chassis organism E. coli Nissle 1917
  (Nissle)

Next steps:

• glucose-dependent expression of the therapeutic
  (Incretin)
• proof of concept in vitro
  (Outlook,
  Incretin)
• integration of a biocontainment module
  (KillSwitch)
• designing features for the optimized growth of engineered E. coli Nissle 1917
  (Entrepreneurship)

Targets reached:

• design and synthesis of all parts related to the CRISPR/Cas3 kill switch
  (KillSwitch,
  Basic Part)
• isolation of Cas3 nuclease and the Cascade sequence
  (KillSwitch)
• cloning of the subunits of the kill switch
  (Notebook)
• development of reporters for CRISPR-module activity
  (Notebook)
• development of a design which allows the usage of live GMOs as therapeutics
  (Human Practice,
  KillSwitch)

Next steps:

• finalisation of the cloning of regulating modules
  (Outlook,
  KillSwitch)
• testing of all modules via the reporters
• integration of the parts into one system
• proof of concept of the kill switch: degradation and killing of bacterium in prohibitive conditions
  (Outlook)
• integration into the E. coli Nissle genome
  (Outlook)
• assembly of the biocontainment system and the therapeutic modules
• experimental confirmation in vitro
  (Outlook)
• providing a service to integrate our new biocontainment switch into other applications to allow for the safe use of live GMOs
  (Entrepreneurship)

Targets reached:

• Determined optimal growth medium for E. coli Nissle and validated it in the lab
  (Model,
  Nissle)
• Predicted interaction and competition of different gut microbe compositions
  (Model)
• Examined and modelled E. coli Nissle transcriptomic response to various stress factors
  (Nissle)

Next steps:

• Analyze NCCT RNA-Seq dataset
• optimisation of our chassis organism E. coli Nissle and growth conditions according to the metabolic model
  (Nissle,
  Model,
  Entrepreneurship)
• publication of data acquired during our characterisation
  (Outlook)
• Extend the Nissle metabolic model with more reactions suitable for our engineered system and predict its interaction with the gut microbiome
  (Model)

Targets reached:

• Optimisation of our project by determining the superior penetrability of cell penetrating peptides by C3 Pred: Penetratin
  (Software,
  Incretin)
• Develop an easy to use interface and allow for the simple integration of our software into workflows
  (Software)

Next steps:

• Include additional training data to improve our model
• Improve cell penetrating peptide design support by automatically evaluating point mutations

While we reached many subordinate targets of our project, we still require proof of concept for the designs of our therapeutic and kill switch. Nonetheless, we demonstrated that our software tool, as well as metabolic modeling, worked very well and supplied us with useful data for the optimization of our project. Moreover, we have made foundational advances in characterising E. coli Nissle 1917 and by providing a new application for Cas3 to build a basis for the organism to be used as live therapeutic. We’re excited to see how scientists will make use of our data and implement their own probiotics based on our research.