Part2: Benzopyrene degradation system

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In the benzo[a]pyrene degradation system, we primarily designed a red-light system to regulate the degradation of benzo[a]pyrene by engineered yeast, controlling the duration of their work. Through comparing different photosensitive pigments and their interaction factors, we ultimately established a comprehensive fungal red-light sensing system based on PhyA-Fhy1. We hope that this system will provide greater convenience for future teams that require red-light sensing and use fungi as their base organisms.

Within the range of BBaK4711030 to BBaK4711038 and BBaK4711050 to BBaK471104, we meticulously documented various parts of the red-light sensing system and constructed composite parts required for practical applications. Additionally, we enriched the structural information of these parts, greatly facilitating the future teams in their utilization.

Part3: Kill system

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Today, in 2023, OUC-China has designed a novel suicide switch - the NeuAc Riboswitch. The purpose of designing a suicide switch is often to ensure that an organism can only survive in a specific environment, such as a culture medium or fermentation broth, and cannot continue to exist outside of that environment. This is typically achieved through inducible promoters, auxotrophic strains, or riboswitches. Inducible promoters often require inducers that are either toxic, can be absorbed and utilized, or involve antibiotics. Auxotrophic strains are difficult to control in terms of the quantity of nutrients consumed in the fermentation broth, as it's challenging to predict when they need replenishing. Riboswitches involving compounds like thiamine pyrophosphate (TPP), S-adenosyl-L-methionine (SAM), purines and their derivatives, amino acids, phosphorylated sugars, and metal ions are often problematic since many of these compounds are toxic or can be utilized by the organism.

Therefore, we ultimately chose the NeuAc riboswitch. Its ligand, N-acetylneuraminic acid (NeuAc), cannot be synthesized within Escherichia coli and is non-toxic and non-utilizable by this bacterium (there have been studies involving E. coli strains with key NeuAc degradation genes knocked out). We designed and meticulously documented various parts of this suicide switch, along with their performance characteristics. Additionally, we used modeling to determine that the optimal dosage of NeuAc should be around 10g/L.

This NeuAc riboswitch represents an innovative approach to suicide switches, ensuring safety and control in engineered organisms.

Experiences with existing BioBricks

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The part BBa_M45424 documents the catalytic reaction using the alkB in the alkane hydroxylase system of Pseudomonas putida GPo1. Based on our experimental results, we have supplemented the optimal conditions for inducing the expression of this protein.Additionally, considering that the yeast fermentation process results in a turbid broth, which affects the transmission of red light, we conducted modeling to calculate the optimal red light intensity, taking into account the partial weakening of red light. This calculation was done to assist in the experiment, and the results were added to part BBa_K801043.While verifying the NeuAc ribose switch, we measured that approximately 2-3 hours after induction with IPTG, φX174 was able to completely kill Escherichia coli. This provides additional experimental data to validate the functionality of part BBa_K4156083.