CsgA is a bacteriophage protein of E. coli that can assemble extracellularly to grow bacteriophages; Mfp3S is a mussel foot filament mucin; and pep can adsorb ions from the culture environment, which results in mineralization. According to the research results of Living materials fabricated via gradient mineralization of light-inducible biofilms[1], the fusion and expression of these three proteins can fix the host to form a biofilm on the solid surface and adsorb calcium ions to form a mineralized layer. To test the function of this protein, we placed the protein downstream of the T7 promoter, constructed an expression system on pSB1A3, and transformed it into BL21(DE3) host bacteria and performed induction tests. As a control we placed amilCP downstream of the T7 promoter in the same manner, constructed the expression system on pSB1A3, and tested it simultaneously with the CsgA-Mfp3s-pep fusion protein for induction.
Fig. 1 CsgA-Mfp3S-pep sequence
Fig.2 amilCP sequence
The experimental results demonstrated the feasibility of this expression system and the performance of the CsgA-Mfp3S-pep fusion protein could be performed as expected. Finally, to facilitate the simultaneous expression of CsgA-Mfp3S-pep fusion protein and amilCP, we used an enzymatic ligation to join the CsgA-Mfp3S-pep fusion protein sequence downstream of the amilCP sequence to form a recombinant plasmid.
Fig.3 Diagram of recombinant plasmid
The T7RNAP produced by this system is regulated by blue light, and T7RNAP is inactivated when there is no blue light irradiation and T7RNP is active when there is blue light irradiation. We co-transformed it with our recombinant plasmid with above into DH5α, constructed the expression system and assayed it.
Fig.4 Sensory system p70-VVD-VVD-T7R-179 sequence
Fig.5 Schematic diagram of the function of the light control system
Above we demonstrated the performance of CsgA-Mfp3S-pep expression with amilCP and the performance of the photoreceptor system, which was cotransferred into DH5α, constituting our final expression system, and tested.
Fig.6 Schematic diagram of the Co-transferring
The results of the experimental tests demonstrated the effectiveness of the photoreceptor system in controlling the CsgA-Mfp3S-pep and amilCP recombinant plasmids, i.e. the feasibility of co-transferring into the expression system.
In order to improve our project, we wanted to have more kinds of colors. A photo sensitive promoter based RGB color vision system [2] may contribute to the realization of this idea. In this system, a key device is the PhlF based NOT gate which controlling the expression of T7RNAP, and indirectly controlling the activity of T7 promoter. Here, in order to obtain more accurate developmentight, we need a more tightly control for T7 promoter. Referring to the structure of BL21(DE3)+ pET28 protein expression system, we decided to make the T7 promoter and T7RNAP both under the control of PhlF at the same time to reduce the leakage activity:
Fig.7 Improve the photosensing system by using a PhlF-controlable T7 promoter.
So we added the PhlO sequence downstreamed the T7 promoter thus created a novel T7 promoter (BBa_K4055525) which can be repressed by PhlF.
For characterizing the improved promoter, we had designed a kind of device using GFP translational unit(BBa_K3254024):
Fig.8 Characterization system for pT7-PhlO
We attached each of these two test devices to the pSB4C5 backbone and transformed them into an E. coli that constitutively expresses T7RNAP. We in turn transferred a pSB1K3 plasmid which harbored a PhlF generator (BBa_K2525007).
The results showed that for pT7-phlO, PhlF significantly inhibited its activity by more than 70-fold.
[1]Wang Y, An B, Xue B, et al. Living materials fabricated via gradient mineralization of light-inducible biofilms[J]. Nature Chemical Biology, 2021, 17(3): 351-359. [2]Fernandez-Rodriguez J, Moser F, Song M, et al. Engineering RGB color vision into Escherichia coli[J]. Nature chemical biology, 2017, 13(7): 706-708.
