The YF1, FixJ and pFixK2 can constitute a blue light-regulated system . In dark conditions, the phosphoryl group is transferred from the YF1 protein to the FixJ protein, with phosphorylated FixJ activating the pFixK2 promoter and subsequently inducing expression of downstream genes . Under blue light induction, phosphorylation of FixJ is blocked, inhibiting expression of genes regulated by the pfixK2 promoter .

In comparison to existing light-regulated gene expression systems in bacteria, the PAL system achieves optical genetic control at the RNA level .The light-oxygen-voltage receptor PAL binds to small RNA aptamers with sequence specificity upon blue-light illumination .By embedding the responsive aptamer in the ribosome-binding sequence of genes of interest,their expression can be downregulated by light .As PAL exerts light-dependent control at the RNA level,it can be combined with other optogenetic circuits that control transcription initiation .By integrating regulatory mechanisms operating at the DNA and mRNA levels,optogenetic circuits with emergent properties can thus be devised .

The BBa_K4192130 construct 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 pfixK2 exhibited significant leakage issues . The NAU- CHINA aimed to address these problems by enabling normal expression of BBa_K4192130 and resolving leakage in this system .

The improvements of NAU-CHINA this year is to address the issues of normal expression and leakage of the BBa_K4192130 system . Firstly, to enable normal expression of this system, we integrated it into the pet29a(+) plasmid containing the T7 promoter . Due to problems with the mcherry reporter sequence, we replaced the reporter with the GFP from BBa_K608011. To prevent leakage of the system, we integrated the LOV receptor PAL into the plasmid . Additionally, an aptamer sequence capable of forming a hairpin structure was added upstream of the reporter gene. Binding of PAL and the aptamer sequence allows optical genetic control at the RNA level . Under blue light irradiation, the LOV receptor PAL can cause the aptamer sequence to form a hairpin structure, thereby blocking translation of downstream genes and achieving the goal of preventing leakage .

We used pET-29a(+) as the vector and replaced the reporter BBa_K1065310 with the GFP BBa_K608011 to form BBa_K4613735, enabling it to function properly . Furthermore, we constructed BBa_K4613891 and BBa_K4613508 in pET-29a(+) as control groups for BBa_K4613462 . These plasmids were transformed into E. coli BL21 (DE3) respectively and grown in LB medium at 37℃ until the optical density at 600nm (OD600) reached 0.6 . After that, the bacteria culture fluid was incubated in 50mL LB medium with IPTG induction at 37℃ for 12 hours, and measured the fluorescence intensity .

We designed BBa_K4613735, BBa_K4613891, BBa_K4613508, BBa_K4613462, and have already accomplished BBa_K4613735, BBa_K4613891, achieving the normal operation of the blue light system . However, unfortunately we encountered issues synthesizing the pal gene . The synthesis of this gene was significantly delayed, and after waiting for a month the gene was still not synthesized . Therefore, we were unable to continue resolving the leakage issue of the system .

Ranzani A T, Wehrmann M, Kaiser J, et al. Light-dependent control of bacterial expression at the mRNA level[J]. ACS Synthetic Biology, 2022, 11(10): 3482-3492.

Weber A M, Kaiser J, Ziegler T, et al. A blue light receptor that mediates RNA binding and translational regulation[J]. Nature chemical biology, 2019, 15(11): 1085-1092.

Dietler J, Gelfert R, Kaiser J, et al. Signal transduction in light-oxygen-voltage receptors lacking the active-site glutamine[J]. Nature Communications, 2022, 13(1): 2618.