Our tiny robot TuTaBa bacteria are engineered from E. coli nissle 1917 strain that has intrinsic antagonistic activity against pathogenic bacteria.1 It may induce innate or adaptive immune responses, secret toxins and form biofilms that are helpful for the treatment of tumors. Figure 1 shows the major parts and functions of the robot TuTaBa.
Figure 1. Engineered parts in TuTaBa made from E. coli Nissle 1917.
Figure 2 shows the procedures used for the construction of engineered E. coli Nissle 1917. Female nude model mice BALB/c that carry with transplanted 4T1 mammary carcinomas have been used for the extraction of full-length RNA samples that are further subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and generate cDNA for gene cloning.
Figure 2. The process for engineering bacteria
Because mouse models with in situ tumors have great similarities with human tumor microenvironment, they provide a feasible way to assess the antitumor effects of engineered bacteria. We choose BALB/c mouse as breast cancer models because it is among the most widely used inbred models in biomedical research, particularly in immunology and infectious disease research. This mouse strain is an exceptional responder to immunization. With BALB/c mice, Th2 cells can be easily triggered by immunization.2 They are able to produce plasma cell tumors within soft tissue. Nude mouse models BALB/c that carry with transplantable 4T1 or MCF7 mammary carcinomas were purchased. The 4T1 tumor is a suitable experimental animal model for human mammary cancer.3 It is not only easily transplanted into the mammary gland but also spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone. The progressive spread of 4T1 metastases to the draining lymph nodes and other organs is very similar to that of human mammary cancer. Female BALB/c nude mice carrying with tumor cells of MCF-7 line that are inoculated in the subcutaneous tissue were used for the comparison with metastatic 4T1 tumors.4
Quality control (QC) refers to the process through which the team seeks to ensure that the quality of the whole experiments and activities are under strict control. The significant aspect of quality control in this project is the establishment of well-defined controls that help standardize both experimental protocols and products to quality issues.
Carefully-designed experiments. In order to be in accordance with national and institutional safety rules and guidelines, only safe materials including cell lines, reagents or chemicals are chosen for the experiments. All experiments are designed so as to minimal risk of dangerous operations and exposures. PIs, instructors and advisors have different educational backgrounds in chemistry, microbiology and medicine that can provide student members with various supports including techniques, animals, instrumental operations and data mining.
Trained personnel. Team members are trained not only in techniques but also in general label safety, biosafety, laser safety, norms, and animal ethics.
Standardized experimental protocols. Experimental procedures including step-by-step reactions, instrumental operations are optimized and finalized as written protocols so that the optimized protocols can be shared in the lab.
Safe cell cultures and animal work. The growth status of cell cultures is checked under a reversed optical microscope. The metabolic and proteomic changes under the treatment of different inhibitors are monitored with chromatographic and mass spectrometric techniques.
Characterization of intermediate and final products. Starting from the extraction of full-length RNA of tumor tissues, intermediate products and final products are tested with electrophoretic, chromatographic and mass spectrometric analysis. Activities of RNases in normal and cancerous tissues or cell lines are determined and RNA extractions are performed with modified protocols.
1.Zhou, S.; Gravekamp, C.; Bermudes, D.; Liu, K. Nat. Rev. Cancer 2018, 18, 727-743.
2.Cleator, S.; Heller, W.; Coombes, R. C. Lancet Oncol. 2007, 8, 235-244.
3.Schmid, P. et al. N. Engl. J. Med. 2018, 379, 2108–2121.
4.Sekine, H.; Yamamoto, M.; Motohashi, H. Nat. Immunol. 2018, 19, 1281–1283.