We knocked out the gene sequences of Tsr and its homologous proteins that would cause competition, reducing the
competitiveness of binding. We measured their growth curves and found that there was no difference in the growth
curves
between the knockout strain and the wild-type strain.
Deletion of Tsr and growth-curve
In the detection module, we expressed the fusion proteins eTlpC-A and eTlpC-B. This fusion protein has a gene sequence of approximately 1899 bp. The pET expression vector system is a widely used system for recombinant protein expression in E. coli. The system is equipped with a phage T7 promoter, which can regulate the transcription and translation of the target gene. However, EcN lacks the gene for T7 RNA polymerase, which makes the original pET-21a system unusable in EcN.
Therefore, we used the Trc promoter that can be expressed in EcN to replace the T7 promoter on the original pET-21a system to initiate the expression of downstream eTlpC. At the same time, we deleted the lactose operon repressor protein site on pET-21a, thereby achieving constitutive expression of eTlpC within the EcN.[1]
3D structure of TlpC
The plasmid of eTlpc
When we introduced the constructed fusion protein into the gene-edited Nissle1917, Nissle1917 became chemotactic. After it grows to a certain stage, we inoculate the bacterial liquid into a new glycerol salt medium. After it grows to od600=0.4, take 10ml of the bacterial liquid and centrifuge it at 4°C for 10 minutes.
Pour the supernatant and add 5 ml of washing medium and gently resuspend. Centrifuge again, resuspend in 5 ml of washing medium, and centrifuge again. After pouring off, resuspend gently in remaining liquid. Observed with a microscope, you can see that more than 90% of bacteria can flow. Add 10 ml of chemotaxis medium to the resuspended culture medium to bring the bacterial concentration to 6*107/ml.
Capillary method[2]
Disc test method
(Preparation of chamber) Melt and bend a 5cm long capillary tube to form a U-shape. Fix the U-shaped tube on the microscope slide and fill the chamber with 200ul of bacterial solution. Insert the capillary containing the configured attractants of different concentrations, incubate for one hour, and directly move the slide to the microscope for observation and recording.
In the ADC module, we use lactate-regulated promoter to express ADC drugs to kill tumor cells.
The lactate-regulated promoter LldR protein belongs to the GntR regulatory family and is a gene involved in L-lactate metabolism in Escherichia coli. There are two control sites, O1 and O2, at 25-54 bases upstream and downstream of the promoter. Complete inhibition of gntT is achieved through DNA circularization through the interaction between two GntR molecules, of which the upstream O1 is the key. Mutation of the regulatory site will eliminate the induction effect of L-lactate, but mutation of the O2 site will not.
When the LldR protein is not bound to lactate, the LldR protein will bind to the operator site and prevent transcription from proceeding. When LldR binds to a lactic acid molecule, it disengages from the binding site, allowing transcription to proceed smoothly.[3]
The sequence of PlldR promoter
Amino acid position 106 of the light chain of trastuzumab is the light chain variable region, and amino acid position 118 of the heavy chain is the heavy chain variable region, which are retained. Different attempts were made to connect single-chain antibodies to PE38 using (EAAAK)2 and (GGGGS)3. (EAAAK)2 is a rigid connecting peptide that will form a stable α-helical structure, while (GGGGS)3 is a flexible connection Peptides.
In order for the ADC drug to be secreted out of the cell smoothly and play its role, an E. coli signal peptide is used at the N-terminus of the protein to mediate the drug. Using a signal peptide that mediates secretion from the Sec/SPI pathway, we used an established signal peptide, Signal Peptide Website ID: 240.[4]
alphaFold2 predicted structure of scFv-EAK2-PE38
alphaFold2 predicted structure of scFv-GS3-PE3
Since the N-terminal REDLK sequence of PE38 is related to protein transport, in order to avoid damaging the C-terminal structure of the PE38 domain, a signal peptide was inserted into the N-terminal of the fusion protein. The inserted protein was analyzed by Signal P4.1 and its D score was 0.862 greater than 0.5, so it can be considered to have secretory activity. And when it is secreted out of the cell, it is hydrolyzed by leader peptidase (Lep) between positions 21 and 22.
Use Nco I and Hind III restriction sites to connect the ADC drug to the expression vector pET-28a, and transform it into E.coli BL21 strain for expression. IPTG was used to induce its expression, and SDS-PAGE was used to extract the strain fermentation supernatant and cells for testing. It was purified using ion exchange chromatography and tested using MTT staining. HER2-positive cell lines and HER2-negative cell lines were used for control tests.[5]
We cultured these strains and detected the expression of fluorescent protein with fluorescence intensity to judge the actual effect of adding function modules (leghemoglobin, laccase or both) on the intracellular oxygen concentration. We judge the concentration of bacteria in the liquid culture medium by the data of bacterial liquid absorbance (i.e. OD value) to reflect the growth of bacteria. In order to reduce the chance of the experiment, we set up three parallel experiments for each group of data, and finally took the average value of the three groups as the experimental data of the sampling time of this group.(Click here to know about result)
[1] Callahan AM, Parkinson JS. Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: cheD mutations affect the structure and function of the Tsr transducer. [J] J Bacteriol. 1985 Jan;161(1):96-104.
[2] Adler J. A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli. J Gen Microbiol. 1973 Jan;74(1):77-91. doi: 10.1099/00221287-74-1-77. PMID: 4632978.
[3] Chien T, Harimoto T, Kepecs B, Gray K, Coker C, Hou N, Pu K, Azad T, Nolasco A, Pavlicova M, Danino T. Enhancing the tropism of bacteria via genetically programmed biosensors. Nat Biomed Eng. 2022 Jan;6(1):94-104. doi: 10.1038/s41551-021-00772-3. Epub 2021 Jul 29. PMID: 34326488; PMCID: PMC8956018.
[4] Rogers LM, Veeramani S, Weiner GJ.Complement in monoclonal antibody therapy of cancer[J]. Immunol Res. 2014 Aug;59(1-3):203-10.
[5] https://bio-protocol.org/e374
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