• Defined subprojects, got introduced to labs.

Wet Lab

• Amplified fragments of promoters believed to be effective in cold temperatures from Pseudomonas fluorescens SBW25.

• Obtained the Pseudomonas fluorescens etHAn strain with a functional type III secretion system from the Department of Plant and Environmental sciences of the University of Copenhagen in Denmark.

Dry Lab

• Learned how to code in NetLogo for the agent-based model.

• First trial of generating output of florigen protein change from the plant model neural network.

Wet Lab

• Linearized pSEVA 64 plasmids and ordered a GFP fragment and type III secretion signal tag.

• Root exudates and plasmids containing root exudate sensitive parts were ordered.

• A list of nine interesting antiflorigen candidates was generated.

Dry Lab

• An ordinary differential equation (ODE) model of Escherichia coli was converted from MATLAB to Python and ran successfully for the first time.

Wet Lab

• Performed Golden-Gate assemblies of the cold-responding promoters into pSEVA 23 sfGFP plasmid backbone.

• Performed Golden-Gate assemblies of the pSEVA 64 plasmid with a GFP insert. One with and one without a type III secretion signal.

• Primers for putting root exudate-sensitive parts in pSEVA plasmids were designed and ordered. Stock solutions of root exudates were made.

• The first attempt to transform P. fluorescens SBW25 with a plasmid.

• Designed primers and antiflorigen inserts arrived and were put into pET-16b plasmids using restriction digestion and ligation. The resulting plasmids were transformed into E. coli Dh5α strains.

Dry Lab

• Validated the Python ODE model with the MATLAB ODE model.

Wet Lab

• Improve P. fluorescens etHAn transformants with a spacer between the ribosome binding site and the start codon of sfGFP for enhanced protein production.

• Recieved the plasmids containing root exudate-sensitive parts from which individual gene fragments were amplified and purified.

• The first successful colony PCR of an E. coli Dh5α strain with a constructed plasmid containing an antiflorigen gene was obtained.

Wet Lab

• Amplification of homology arms for the barcode construct from the P. fluorescens SBW25 genome.

• First plant growth experiment with Arabidopsis thaliana Col-0 on different growth media.

• Performed growth and toxicity assays for P. fluorescens SBW25 on rhamnose and cuminic acid.

Dry Lab

• Added a temperature response to the Python ODE model.

• Added a temperature dependency for florigen production to the plant model.

Wet Lab

• Multiple transformation methods were attempted from which one that works was found for P. fluorescens SBW25.

• Transformed P. fluorescens SBW25 with ‘cold’-promoter plasmids.

• Transformed P. fluorescens etHAn with GFP and type III secretion signal containing plasmids.

• Isolation of the pCym promoter fragment from its host plasmid.

• Performed a Golden-Gate assembly of the dapB overexpression gene in a pSEVAb23 plasmid.

Dry Lab

• The first version of the agent-based model (ABM) for microbial communities was built and tested.

• The influence of different temperatures on simulated florigen concentrations was tested and the temperature dependency was tuned to reflect results from literature.

Wet Lab

• Plant growth experiments of A. thaliana Col-0 in Fahrraeus slides.

• All root exudate fragments were rearranged and inserted in their respective pSB1C3 plasmids compatible with BioBrick assembly. These plasmids were transformed in E. coli Dh5α and sequenced.

Dry Lab

• Redesigned and debugged the ABM for microbial communities.

Wet Lab

• The transformed P. fluorescens SBW25 with the ‘cold’-promoter plasmids were confirmed via sequencing.

• First colonisation experiment of P. fluorescens SBW25 on A. thaliana Col-0 roots.

• Most of the root exudate sensing modules that were cloned with a GFP-reporter gene in SEVA plasmids and transformed in E. coli Dh5α were confirmed via sequencing.

• Seven of the nine antiflorigen plasmids now showed successful colony PCR results

Dry Lab

• Obtained the weather data of 2015-2022 from a weather station in Wageningen.

Wet Lab

• Performed a Gibson assembly on the barcode fragments.

• Performed a plate reader experiment to get data on the secretion of sfGFP from the P. fluorescens etHAn strain.

• Most of the roots exudate sensing plasmids transformed into P. fluorescens SBW25 were confirmed via sequencing.

• The salicylic acid sensing module in P. fluorescens SBW25 was characterized and proven responsive.

• Seven of the nine antiflorigen plasmids were confirmed via sequencing and the plans to keep on trying to create the two remaining plasmids were scrapped.

• Transformed the first E. coli BL21 production strains with the antiflorigen plasmids.

Dry Lab

• Ribosome concentration for different temperatures validated between the ODE Python model and in vivo measurements.

Wet Lab

• Characterized colonisation behaviour of P. fluorescens SBW25 under the fluorescence microscope.

• The arabinose sensing module in P. fluorescens SBW25 was characterised and proven responsive.

• The creation of seven different antiflorigen producing E. coli BL21 strains were confirmed via colony PCR and the first antiflorigen production and purification run was tried.

Dry Lab

• The ccdA-ccdB toxin-antitoxin system of the ODE Python model validated against the measurements of the previous Wageningen iGEM team Colourectal.

Wet Lab

• Transformed the barcode construct into E. coli Dh5α.

• Creation of P. fluorescens etHAn double transformation with the sfGFP secretable and RFP non-secretable counterstaining.

• Transformation of P. fluorescens SBW25 with the pGNW plasmid.

• The cuminic acid sensing module in P. fluorescens SBW25 was characterised and proven responsive.

• Performed a Golden-Gate assembly of the rhamnose inducible system into a pSEVAb23 plasmid.

Dry Lab

• Tested the ABM and started to collect data for data analysis.

Wet Lab

• The barcode construct assembly was confirmed via sequencing.

• First try of working with a vibratome to visualise sfGFP injection into A. thaliana Col-0 roots.

• The naringenin sensing module in P. fluorescens SBW25 was characterised and proven responsive.

• Introduction of the previously characterised RNA toehold switch in P. fluorescens SBW25 and proven to be functional.

• Produced and purified the TFL1 and ATC/CEN antiflorigens in E. coli BL21 production cultures.

Wet Lab

• Included growth rate and substrate consumption in the ODE Python model.

Wet Lab

• Cross-sectional detection of sfGFP inside A. thaliana Col-0 using a vibratome.

• Produced and purified the CsAFT and PsTFL1 antiflorigens in E. coli BL21 production cultures.

Dry Lab

• Added real life temperature and day length in the plant model to simulate daily florigen concentrations and flowering time.

Wet Lab

• Performed a tri-parental conjugation of the barcode construct into P. fluorescens SBW25.

• First try of sfGFP production induction by A. thaliana root exudates.

• Produced and purified the BvFT1 and CEN-like 1 antiflorigens in E. coli BL21 production cultures.

Dry Lab

• The plant model is able to generate florigen concentrations and the flowering time for the 2000 days between 1st of January 2015 and 31st of December 2022.

Wet Lab

• Performed a buffer exchange to get the antiflorigens from their elution buffer into PBS buffer.

• Plate reader assay of incubation of ‘cold’-promoter-GFP bearing P. fluorescens SBW25 strains in different temperatures.

Dry Lab

• Introduced the rhamnose and cuminic acid sensing systems in the ODE Python model for different temperatures.

• Constructed a neural network to predict daily florigen concentrations, flowering time and minimum temperature.

Wet Lab

• Confirmation of recombination of the barcode construct in P. fluorescens SBW25.

• The quorum sensing module from Vibrio fischeri responsive to AHL was introduced in P. fluorescens SBW25.

Dry Lab

• Built and tested the cell level ABM.

• Tested multiple data transformation techniques for the plant model to predict future florigen concentrations, flowering time and minimum temperature.

Wet Lab

• The introduced quorum sensing module was characterised in P. fluorescens SBW25 for a wide range of AHL concentrations.

Dry Lab

• Data collecting and analysis of the cell level ABM.

Wet Lab

• Assessed the primer sets for the LAMP assay.

• Plasmids containing the RNA toehold switch combination of induction by the quorum sensing module and the salicylic acid sensing module were introduced in P. fluorescens SBW25.

Dry Lab

• Collected data and performed data analysis on the interaction of the microbial community ABM model and the cell level ABM model.

Wet Lab

• Started the in vivo A. thaliana Col-0 experiment to assess the efficacy of the heterologously produced antiflorigens on flowering delay.

• Characterisation of the introduced RNA toehold switch induced by AHL and salicylic acid in P. fluorescens.

Dry Lab

• Constructed a separate neural network to predict future temperature from current temperature time series.

Dry Lab

• Combined the temperature prediction neural network with the plant model neural network.