Team:UANL/Circuit

GENETIC CIRCUIT

Our system is composed of 2 plasmids (pEHV01 y pCMR01) (Figure 1) , both plasmids co-transform using E. Coli as chasis creating a protein complex that serves as a "microbioreactor" which when in contact with vanilate activates, starting its transformation in cis, cis-muconic acid. The process uses bacterial microcompartments, which when confined a group of reactions potentially toxic in its interior, allows the bacteria to have a higher efficiency in this metabolic process.The process is regulated by pVanCC y p3B5C (promoters), which respond to initial phenolic compounds or derivatives of the metabolic route selected, this allows a regulation in phases which adapts to each enzyme specs.

Figure 1. Set of plasmids designed in a modular way, together they form a system destined to the transformation of vanillato into cis-muconic acid using bacterial micro-compartments (BMC) to increase the efficiency of the metabolic reactions while reducing the toxicity of the compounds generated by it. A) Plasmid pEHV01, generation module of modified BMCs to increase their permeability under the control of a protocatechuate sensitive promoter. B) Plasmid pCMR01, module of generation of a metabolic pathway composed of three enzymes which transform vanillate into cis-muconic acid. C) Set of BMC forming proteins pduABJKNU under the control of the protocatechuate-sensitive p3B5C promoter. D) A group of proteins belonging to a metabolic pathway which converts vanillate into catechol and finally cis-muconic.

pEHV01 transports an operon which encodes a group of 6 proteins derived from S. enterica LT2 (<partinfo>K3317033</partinfo>) responsible for the formation of the Bacterial microcompartments (BMCs), structures formed by a thin protein shell (1). The final construct is under the regulation of the promoter p3B5C (<partinfo>K3317007</partinfo>), such promoter has operator sites to which the repressor protein pcaU (<partinfo>K3317005</partinfo>) binds, when interactings with protocatechuic acid (PCA) the protein allows the transcription (2). The final operon has a modification in the protein pduU(<partinfo>K3317014</partinfo>), the deletion of the first 17 amino acid residues leads to in an increase of the permeability of the Bacterial microcompartments without altering their stability.

Figure 2. Doble standard fragment , contains the suffix and prefix of biobricks which allows to use 3A Assembly to clone it into a compatible plasmid. It carries a protocatechuate-sensitive p3B5C promoter and an RBS designed for the pduA protein that will then be cloned with the rest of the operon responsible for generating the BMCs, has 2 hybridization sites designed to carry out a homologous recombination cloning

The assembly of the plasmid pEHV01 (Figure 1A) began by cloning a fragment 543 bp (Figure 2) in a plasmid pSB3K3(<partinfo>K3317007</partinfo>), the fragment contains the promoter p3B5C a ribosome binding site (RBS) designed for the protein pduA. Inside the resulting plasmid an insertion can be performed of a group of 6 proteins (psuABJKNU) (Figure 3A) of S. enterica LT2 (NC_003197.1) of the plasmid pMCY30 provided by Ph.D Mimi C. Yung (Figure 3) through a cloning by homologous recombination as described by Jacobus et al 2015(3). The resulting plasmid (containing pSB3K3 promoter and psuABJKNU proteins) contains sites that can be amplified by PCR to perform a second homologous recombination (Figure 3B).

Figure 3. Homologous recombination scheme, A)insertion of the generator operon of BMC-forming proteins into a pSB3K3 vector which carries the p3B5C promoter, was linearized by PCR, B) insertion of the modified pduU protein together with its RBS in a vector pSB3K3 which contains the BMC-forming proteins (except for pduU) under the p3B5C promoter, the vector was linearized by means of PCR.

pCMR01 (Figure 1B) is a plasmid responsible for transport the metabolic pathway responsible of transform vanillic acid to muconic acid containing 3 enzymes: LigM , aroY y CatA. Furthermore, it contains the regulatory proteins VanR and pCau. The first enzyme, LigM , is regulated by the promoter PVanCC which allows its expression in the presence of vanilate which is the substrate of LigM transforming it in protocatechuic acid in the cytoplasm. The second and third enzyme which are regulated by a promoter sensitive to protocatechuic acid contain a signal peptide which is responsible for their internalization in the BMCs making the transformation of protocatechuic acid to catechol and afterwards to a cis, cis-muconic acid.

The assembly of the plasmid pCMR01 can be carried out through the method 3A Assembly, binding the generator of the regulatory proteins VanR and pCaU (<partinfo>K3317029</partinfo>) along with the operon responsible for the metabolic pathway (<partinfo>K3317030</partinfo>) within a vector pSB6c5.

A third plasmid named pCMR02 (Figure 4) was designed, through an enzymatic digestion in the sites Pvul of pCMR01 eliminating the protein LigM , its RBS, the corresponding terminator and the promoter pSB3K3. Performing a digestion in the sites HindeIII and XhoI the protein LigM carrying a signal peptide in its carboxil terminus which internalizes the enzyme in the BMCs can be inserted, leading to 3 enzymes ( LigM , aroY and CatA) (<partinfo>K3317032</partinfo>) regulated by the promoter pVanCC.

Figure 4. Alternative plasmid pCMR02, modified version of pCMR01 in which the LigM enzyme and the p3B5C promoter were removed, a modified LigM enzyme with a signal peptide at its C terminal, was added to the CatA enzyme, leaving it under the control of the pVanCC promoter.


Bibliography

1:Jorda, J., Liu, Y., Bobik, T. A., & Yeates, T. O. (2015). Exploring bacterial organelle interactomes: a model of the protein-protein interaction network in the Pdu microcompartment. PLoS computational biology, 11(2), e1004067.

2:Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature chemical biology, 15(2), 196.

3:Jacobus, A. P., & Gross, J. (2015). Optimal cloning of PCR fragments by homologous recombination in Escherichia coli. PLoS One, 10(3), e0119221.