This year, NAU-CHINA has designed and characterized 37 parts in total, including 11 new basic parts, 9 new composite parts and 5 new improved parts.

Traditional textile manufacturing processes, which separate fiber production from dyeing, are highly water-intensive and incompatible with the principles of sustainable fashion. To address these challenges, our project developed an integrated system for bacterial cellulose synthesizing and dyeing using E. coli. We have achieved one-step synthesis of colored bacterial cellulose through temperature regulation with CI857 (BBa_K2816006) and FourU (BBaK115002). At 25°C, bacterial cellulose was produced in large quantities through cascade amplification. When the temperature was raised to 37°C, natural pigments were expressed to realize customizable and pollution-free dyeing. We added hydrophobic proteins to the surface of bacterial cellulose to form a hydrophobic coating to enhance the fabric's waterproof performance. We showed how we designed and tested the parts in Design and documented them in part registry. Please click the names for more details.

Table 1. Total Parts

This year, NAU-CHINA designed 11 new basic parts to produce colored bacterial cellulose and increase the dynamic range of the light response in the BBa_K4192130 and BBa_K4613462 in our improvement project.

BcsA (BBa_25AFSLF6), BcsB (BBa_259XRYXS), BcsC (BBa_256970Q1) and BcsD (BBa_254VX0ZI) all participated in the synthesis of bacterial cellulose.When BcsA is activated by c-di-GMP, it incorporates glucose units into a cellulose chain in the cytoplasm using UDP-glucose as a substrate. BcsB guides the glucan chain through the periplasm; BcsD crystallizes four glucan chains in the periplasm, and finally, BcsC exports the bacterial cellulose micro-fibrils into the extracellular space.

In the part of pigment synthesis, TyrBm (Tyrosinase from Bacillus megaterium) (BBa_254QJJQT) converts L-Tyrosine into L-DOPA and subsequently transforms L-DOPA into Dopaquinone. Eventually, eumelanin is formed through oxidation. Indole is converted into indigo under the catalysis of CYP102A indigoidine synthetase (BBa_25GPCH5Q).

Besides the colourful appearance, practical functions such as hydrophobicity attach the same importance. Thus, we engineered BslA-dCBM (BBa_253K8RUJ) from Bacillus subtilis to connect the recombinant protein with the cellulose. Once induced by IPTG, T7 promoter starts the expression of BslA-dCBM, hence forming a waterproof coat over our bio-based textiles.

Under blue light irradiation, OptoLacI(D) (BBa_25BHXP3N) can bind to the operator sequence lacO3 (BBa_25V87BRR), effectively blocking the RNA polymerase from transcribing the target genes and achieving the goal of expanding the dynamic range of the light response. In this process, gfp1-11 (BBa_25KXWUKK) is used as a reporter gene for detection.

Table 2. Basic Parts

To optimize the system and better implement our expected design, we designed 9 new composite parts.

In order to provide a solution for the sustainable fashion industry, we constructed a temperature-controlled one-pot production platform which consisted of four parts: temperature regulation module (T7 promoter-RBS-CI857-T7 ter-R promoter-FourU-PhlF-mRFP1-T7 ter-PhlF promoter-RBS-sfGFP-T7 ter: BBa_25FDWALN), cellulose synthesis module (T5 promoter-RBS-bcsAB-lambda t0 terminator: BBa_25KBLKIT, T5 promoter-RBS-bcsCD-lambda t0 terminator: BBa_255DIYN6), dyeing module (T7 promoter-RBS-TyrBm-T7 terminator: BBa_25ECYPMT, T7 promoter-CYP102A-T7 ter: BBa_25PMMXD6) and waterproof module (T7 promoter-RBS-BslA-dCBM-T7 terminator: BBa_25K8XDBD).

In the improved part, we constructed the devices of lacI promoter-OptoLacI(D)-T7 promoter-RBS-YF1-FixJ-Fixk2-LacO3-GFP-T7 terminator(BBa_25SYBTV0) and lacI promoter-LacI-T7 promoter-RBS-YF1-FixJ-Fixk2-GFP-T7 terminator(BBa_252QAUN3) to verify the feasibility of increasing the dynamic range of the light response.

Table 3. Composite Parts

Our improvement project aims to address the issue of the dynamic range of the light response in the blue light-regulated system based on the YF1, FixJ and Fixk2 promoter^[1]. Practical implementation of this optogenetic circuit has faced challenges. For instance, CAU-China 2022 (BBa_K4192130) was unable to achieve normal expression and detect mCherry reporter signals due to its weak promoter. Additionally, the blue light-regulated system composed of YF1, FixJ and Fixk2 promoter exhibited significant leakage issues, which results in a relatively small dynamic range of blue light response for this optogenetic circuit. Although NAU-CHINA 2023 (BBa_K4613462) improved this by using a T7 promoter and a gfp reporter gene, the key PAL sequence was not verified due to time constraints.

To enhance the system's ability to distinguish between blue light and dark signals, we integrated OptoLacI^D (BBa_25BHXP3N) into the plasmid and embedded the operator sequence lacO3 (BBa_25V87BRR) between the Fixk2 promoter and gfp1-11 (BBa_25KXWUKK) reporter gene^[2]. Therefore, we constructed lacI promoter-OptoLacI(D)-T7 promoter-RBS-YF1-FixJ-Fixk2-LacO3-GFP-T7 terminator (BBa_25SYBTV0) and lacI promoter-LacI-T7 promoter-RBS-YF1-FixJ-Fixk2-GFP-T7 terminator (BBa_252QAUN3) devices and transformed them into E. coli BL21(DE3) to achieve the desired functionality.

Our improvement achieved the optimization of signal-to-noise ratio by significantly enhancing the maximum output expression of the system in the dark (the "on" state). Future work can focus on integrating the powerful expression enhancement module (OptoLacI^D and LacO3) that we have developed this time with the next-generation inhibition module that can more thoroughly "switch off" the system, ultimately achieving an ideal system that simultaneously possesses ultra-high expression and ultra-low background.

Table 4. Improved Parts

• [1]Ohlendorf R, Vidavski R R, Eldar A, et al. From dusk till dawn: one-plasmid systems for light-regulated gene expression[J]. Journal of Molecular Biology, 2012, 416(4): 534-542.
• [2]Liu M, Li Z, Huang J, et al. OptoLacI: optogenetically engineered lactose operon repressor LacI responsive to light instead of IPTG[J]. Nucleic Acids Research, 2024, 52(13): 8003-8016.