Results
Wetlab
The goal of the wetlab part was to construct two sensors, specifically kaempferol and vanillin ratiometric biosensors, along with the matching inducible two-step pathway plasmids for metabolite production.
Sensors
The vanillin and kaempferol sensor subgroups completed various plasmids, including:
| Plasmid Name | Function |
|---|---|
| GH_J23114_QdoR | Transcription factor for pqdoI |
| GH_J23114_VanR | Transcription factor for pVan |
| EF_pqdoI_sfGFP | Promoter for kaempferol sensor plasmid |
| EF_pVan_sfGFP | Promoter for vanillin sensor plasmid |
| IJ_J23100_mCherry | Background fluorophore for both sensor plasmids |
A level 2 assembly was attempted but when verifying the cloned constructs via sequencing, we only got the sequences of the spectinomycin resistance fragments and the mCherry L1 plasmid in the sequencing results.
After multiple attempts, revisions to isolate the plasmids, and various troubleshooting efforts including testing different molar ratios, changing ligation conditions, and repeated transformations, no strategy succeeded in assembling the full level 2 sensor plasmids. Further approaches like reducing the spectinomycin resistance cassette amount and proceeding with equimolar fragment ratios did not solve the observed problems.
Due to limited time, more elaborate troubleshooting like ccdB negative selection or gel purification of the assembled plasmid could not be attempted, and the plasmid assembly remained unsuccessful.
Pathways
The complete assembly of the Vanillin pathway plasmid was attempted, progressing from verified Level 0 parts through Level 1 constructs to a final Level 2 assembly. All Level 1 plasmids were successfully assembled and sequence-verified.
Multiple MoClo assembly optimizations were put in place to generate the Level 2 plasmid. These optimizations included varying the molar ratio of DNA fragments and vector, and attempting a classical restriction-digestion and gel extraction of the pure DNA fragments. Although no colonies were ultimately obtained, the troubleshooting attempts have given us insight into construct design and host strain limitations. Our initial theory was that excessive metabolic burden from dual antibiotic resistance genes might have hindered successful transformation. After redesigning the plasmid to include only a single resistance gene, all attempts remained unsuccessful, indicating that the expression of the enzymes and antibiotic resistance was too overbearing for the cell or that the assembly was inefficient.
The successful assembly of all but one Level 1 construct was achieved for the Kaempferol pathway group. The last assembly gave our group significant problems and, after improvements to our methods, like gene amplification to ensure fragment purity and replacing a promoter gene. Due to time constraints we were ultimately unable to modify our cloning strategy successfully.
Through these experiments, we identified aforementioned factors (molar ratio and techniques to extract pure DNA fragments) influencing complex MoClo assemblies, and valuable optimization strategies were established for future work. These findings provide a foundation for refining the plasmid design and theoretically reducing metabolic load by using solely promoter-inducer-fusions instead of burdening the assembly with a separate Level 1 plasmid containing only the transcription factor.
Drylab
This year's project was heavily focused on computational work and of course the construction of our chemostat. We present a fully connected system that integrates hardware, sensors, and software to enable continuous microbial cultivation. For more details and to get a better understanding of the results, please follow the links to the pages. This page serves as a first impression and summary.
Hardware
The star of the show: The chemostat. Fully customizable, open-access, and a beauty on its own - our chemostat is designed to make continuous bioproduction accessible to everyone. Equipped with all essential experiment sensors, it communicates wirelessly with our control software, enabling real-time monitoring and control. Its modular design allows complete flexibility for different experimental setups, while the low-cost and scalable construction means you can easily build multiple units for parallel or scale-up experiments. We use it to add inducers to our cultures - this stabilizes production and keeps the cells happy!
Software
Our second gold candidate: The software. It is open-source, easy to operate and the perfect companion for the chemostat. With its two modes you can have it all: In live mode, the software controls your laboratory setup, sending commands to pumps, reading sensor data, and providing accurate, real-time feedback from a functioning microbial culture. In demo mode, the software allows you to explore, analyze, and learn - without handling live organisms.
Model
All good things come in threes: The model. It provides you with a mathematical representation of what is actually going on in the bioreactor. You can test parameters and their effects, and see future trajectories. We use the model to test the effect of the inducer we add. To avoid unnecessary metabolic burden and the overuse of inducers, an integrated optimization algorithm analyzes stochastic simulations to determine the best time points and dosages for inducer addition - all tailored to your experimental parameters.