Contribution

We successfully knocked out the serine sensor protein of E. coli Nissle 1917 and introduced the lactate sensor protein from Helicobacter pylori. At the same time, we constructed two ADC drugs that can be induced at different lactate concentrations, so they will also have different expression levels. Finally, he can commit suicide under the induction of arabinose.

plldR is regulated by lldR, which allows promoter responds to lactate signal. plldR comes from the genome of E. coli, but its strength is weak and cannot meet the needs for high gene expression. We replaced the promoter region of the original plldR with the highly expressed T7 promoter to expand the usage scenarios of plldR.

Figure. 1 The structure of lactate induced promoter

This is a chemical signal sensor protein that has been tested to mediate lactate chemotaxis in E.coli Nissle1917. It is a fusion protein that combines the Tsr gene of E.coli Nissle1917 and the TlpC gene of Helicobacter pylori. Tsr chemoattractant protein was originally a chemosensory protein of E.coli Nissle1917 chemotactic serine. By changing its extramembrane ligand-binding domain and transmembrane part to TlpC, the intramembrane cytoplasmic signaling domain does not move, allowing it to pass through Cascade signaling controls flagella and mediates E. coli Nissle1917 chemotaxis to lactate.The test was conducted in E. coli Nissle1917 with the Tsr gene knocked out, because the signal cascade that drives the flagellum requires CheW and CheA to bind to the intracellular part of the chemosensory protein. Therefore, retaining the original Tsr chemosensory protein of E.coli Nissle1917 may affect the chemotactic effect of this protein.

1.Structural analysis of TlpC protein and Tsr protein

Figure. 2 The plasmid of eTlpC-A

Figure. 3 The structure of eTlpC-A

Figure. 4 The distinction of curve of deletion of Tsr1&2 and wild type

This is a protein that mediates lactate chemotaxis of E.coli Nissle1917, and its basic structure is consistent with BBa_K4617001. The difference is that its transmembrane part is the part of the Tsr protein. Fig. 5. The plasmid of eTlpC-B

Figure. 5 The plasmid of eTlpC-B

The light chain variable region and heavy chain variable region of trastuzumab are retained, and (GGGGS)3 short peptide is used to connect, and (GGGGS)3 is used to connect Pseudomonas aeruginosa exotoxin A (PE-38) at the C-terminus. constitutes a single-chain antibody conjugate drug.This protein is secreted out of the cell mediated by the signal peptide of E. coli (Signal Peptide Database ID: AFAE3_ECOLX).

Figure. 6 The sequence of GS3 ADC Drug

It is roughly the same as BBa_K4617003, except that the linker connecting PE-38 is changed to a rigid (EAAAK) 2, which may have a better function of separating the structural domains. Another type drug has same parts, but they have different order.It is roughly the same as BBa_K4617003, but its VH and VL are swapped to increase its expression.(see fig.8)

Figure. 7 The plasmid of EAK-2 ADC Drug

Figure. 8 The sequence of EAK-2-B ADC Drug

From Escherichia coli draA gene(Signal Peptide Database ID:240). A confirmed signal peptide mediated by the Sec/SPI pathway in Escherichia coli. In out project it can mediated ADC drug secreted out from bacteria.

Figure. 9 The analyze of Signal Peptids

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[2]Wadhams, G., Armitage, J. Making sense of it all: bacterial chemotaxis. Nat Rev Mol Cell Biol 5, 1024–1037 (2004). https://doi.org/10.1038/nrm1524

[3]Machuca MA, Johnson KS, Liu YC, Steer DL, Ottemann KM, Roujeinikova A. Helicobacter pylori chemoreceptor TlpC mediates chemotaxis to lactate. Sci Rep. 2017 Oct 26;7(1):14089. doi: 10.1038/s41598-017-14372-2. PMID: 29075010; PMCID: PMC5658362.

This study provides a novel solution for the modification of bacterial chemotaxis and explores the different structures and functions of chemotactic proteins. relationship, and an EcN-SY strain with lactate chemotaxis was obtained. This study only tested eTlpC from a qualitative perspective to confirm its effectiveness, and did not test it from a quantitative perspective. set out to characterize this new biological element. This may hinder the further application of eTlpC, and the next step should be to Continued to characterize the extent to which eTlpC responds to different concentrations of different types of chemoattractant molecules. and expand its application scope so that it can Act on more organisms. It can be seen from this study that chemotactic proteins are a type of modular and structured biosensor elements.