Our project mainly consists of two parts, namely the detection of chemotaxis module and ADC killing drug module. The chemotaxis module causes the bacteria to swim to the high lactic acid area of the tumor. The ADC drug module expresses ADC drugs under the control of different lactic acid concentrations to achieve the tumor killing effect.
We proposed two ideas for the lactate chemotaxis module, one is to retain the extramembrane sensor part of the H. pylori lactate sensor, and the other is to retain the extramembrane and transmembrane parts of the H. pylori lactate sensor protein.
In the initial design of the experiment, we planned to directly introduce the fused sensor protein into Nissle1917, express this protein through a plasmid, and combine it with the corresponding signaling protein to induce flagellar movement. One of our fusion proteins is a fusion of the extramembrane sensor part of the lactic acid sensor protein of Helicobacter pylori and the transmembrane and intramembrane parts of the serine sensor protein of Nissle1917. The second solution is to retain only the intramembrane part of Nissle1917, and the rest are from Helicobacter pylori.
However, we noticed that the native sensor protein of Nissle1917 may bind itself better, which will cause our fusion protein to compete for ligands, which makes it difficult for our fusion protein to operate normally. Our initial idea was to let the bacteria overexpress these signal transduction proteins again, but this would cause greater pressure on the bacteria. Therefore, after many opinions, we decided to knock out the gene for the serine sensor protein of Nissle1917.
When knocking out the serine sensor protein, we found that Nissle1917 also has a homologous protein of the serine sensor protein. Because it is not clear which type of biological membrane this protein is on, we knocked it out again. The whole bacteria after knocking out the two sensor proteins did not show any difference in the growth status of the wild type.
Figure 1: Deletion of Tsr and Tar
Nissle1917, which contains a plasmid containing lactate sensor protein, has obvious lactate chemotaxis, but the wild-type strain does not show this property. However, we noticed that the plasmid would be lost during the passage, and the offspring Nissle1917 would lose its ability to be deoxidized. Therefore, we decided to knock the fusion protein directly into the genome of Nissle1917, which can ensure the stability of passage.
Figure 2: Plasmid expression
Figure 3: Genome insertation
Finally, we preliminarily proved the role of lactate chemotactic protein through the disk method and capillary method.
At first we decided to directly use a complete antibody for drug design, but we noticed that the complete antibody did not express well in the prokaryotic expression system, and its penetration ability was also poor, so we decided to use a single-chain antibody. The reduced molecular weight ensures tissue penetration and immunogenicity.
At the same time, we noticed that the single-chain antibody and PE38 of Pseudomonas aeruginosa chlorotoxin can achieve complementary advantages and offset each other's disadvantages.
Considering that there is no T7RNA polymerase in Nissle1917, we knocked in the T7RNA polymerase gene in the genome and directly expressed it through the Trc strong promoter. At first, we wanted to directly use the T7 promoter to control drug expression, but the T7 promoter is a strong promoter, which would cause overexpression and risk of leaky expression. Therefore, we hope to have sufficient expression intensity but also sufficient induction sensitivity, so that T7 RNA polymerase is also expressed under the induction of PlldR. And finally achieve the induction effect of plldR-T7 promoter.
Figure 4: Characterization of promoter effects
We plan to develop a complete set of sensor protein components that can be used in different scenarios. And it can be expanded from therapy to more areas of life.
Figure 5: A complete of Biosensors[1]
During the experiment, we discovered that the promoter used was not ideal. Initially, we used the default T7 promoter, but leaky expression was severe. For convenient control, we redesigned the promoter PlldR-T7 lactate-regulated promoter. The LldR protein belongs to the GntR regulatory family and is a gene involved in L-lactate metabolism in Escherichia coli.
There are two operating 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.The upstream O1 is a key regulatory site. Its mutation will eliminate the induction effect of L-lactate, but the mutation of the O2 site will not.
The PlldR-T7 promoter is a hybrid promoter of lldR protein and T7 promoter. The inducer is lactic acid, which is universal and its mechanism is relatively clear. However, due to insufficient experimental time, our web page has not been able to display specific experimental data. We hope to show the results live in a subsequent presentation.
Figure 6: Exact data of promoter
Figure 7: A series of Promoters [2]
This project focuses on lactate tropism, and the drug is also expressed under the regulation of lactate. At the same time, we noticed that high lactate may also be caused by strenuous anaerobic exercise.Therefore, the regulation of future drugs can be expressed under a double AND gate logic, and another regulatory factor can be hypoxia, which is also a characteristic of the tumor microenvironment.In other words, under the expression regulated by lactate, whether there are other inhibitory factors that can prevent its expression in other high-lactate environments, this is also something that needs to be explored in the future.
Figure 8: Future And Gate induced Express
[1]Stock AM. A nonlinear stimulus-response relation in bacterial chemotaxis. Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):10945-7. doi: 10.1073/pnas.96.20.10945. PMID: 10500102; PMCID: PMC34220.
[2]Meyer AJ, Segall-Shapiro TH, Glassey E, Zhang J, Voigt CA. Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors. Nat Chem Biol. 2019 Feb;15(2):196-204. doi: 10.1038/s41589-018-0168-3. Epub 2018 Nov 26. PMID: 30478458.