In this year, Guangxi-U-China has conducted the engineering of 3 parts including scFv-Fc, SIRPαD1-Fc and acid inducible P-asr promoter for the designed “TuTaBa” bacteria. It is such a joyful journey in which we continuously discover new things and obtain better understanding of the world. The three engineered parts are designed for specific targeting, phagocytic promotion, and acid inducible expression of intended functional genes. Two problems have been solved and two experimental protocols have been established. We have completed the extraction of full-length RNA from breast cancer tumor tissues, reverse transcriptase-polymerase chain reaction (RT-PCR), generation of cDNA for gene cloning, construction of recombinant plasmid and translation in E. coli. Nissle 1917. Their full functions will be characterized in 2023.

AGTGTCCGTGCTGGGAAACTGGCGGGGAACTTTTAGAGATAACCCTCCGAATTGCTGGCAAATTTCTGATGAAATTTCTCCGCGAAGCCCACATGAACTACCCCCGTTTACCCTCAAAATAAGCCCTGTGACACACATAACACCCCCTAATCGTACCCGCTCACACGCTATTTTCGAGGTGTGGTTCGCCTTCGGAAACGAATGCCCCCGCCCCACTTGGATAAAAGACGAATTCACCTGTTAGTCTATAACGCGGGTTGAACCGAGAAACCCCTCAAGGCAGCAGACAATAGCCGCAAGGGGTTTTGCGGAGCACGTCCCCTGTGATCGTTGCGCTGATGTGCGACGGAGTCCGG

CGATCAAGACTACTATTATTGGTAGCTAAATTTCCCTTAAGTCACAATACGTTATTATCAACGCTGTAATTTATTCAGCGTTTGTACATATCGTTACACGCTGAAACCAACCACTCACGGAAGTCTGCCATTCCCAGGGATATAGTTATTTCAACGGCCCCGCAGTGGGGTTAAATGA

AGGTTTGGCGAGTACATAGACCTCTGTTCCCCCTCCAGATTGTATTTCTGTGTCAGGCTCTGATGATCCTTTCTGGAACTTCACACAGTAGTAGATGCCAGCATCTGCTGGGGTGACATTACTGATACGGATGGAAAAGTCCATATTGTTTCTCTTAGTAGTATCTGAAACATTTCTAATTCGAGGAACGTATTCTCCTGCGAAACTGTAGATCAACAGCCGGCTTGGCCCTACTCCTCTGTACCACCTAATGGGTCCCACCGGCAACAAGGAGGTCAAAGTGCAGTTCAGAACGGTCGAATCCCCAGCAGCAACAGACACTGATTTCTCAGGCTGAGTCACCTTCAGTTCCTTCGGATCCG

Catttaccaggaggctccttgggaggtggaatggtgtacacctgtggagccttcggtctgcctttggt tttggagatggttttctcgatgggggcagggaaagctgcactgttgaccctgcatttgaactccttgccattgagccagtcctggtgcatgatgggaagttcactgactgagcggaaagtgctgttgaactgctcctcccggggttgcgtctgagctgtgtgcaagtgggagaggctcttctcagtatggtggttgtgcaggccctcatgtaacacagagcaggtgaaagtatttcctgcctcccagttgctcttctgcacattgagcttgctgtagacgaagtaagagccatttgtgttcatgatgggctgagtgttcttgtagttctccgctggctgcccattccactgccactccacagtaatgtcttcagggaagaagtctgttatcatgcaggtcagactgactttatccttggccatctcctccacatcatctacaaaccagctgaactggacctcgggatcatccttgctgatgtctaccacaacacacgtgaccttaggagtcagagtaatggtgagcacatccttgggctttggggggaagatgaagacagatgatacttctgggactgtacatatgcaaggcttacaaccacaatccctgggcac

ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTACAGTGCTCTAGACAGGTTCAGCTGGTGCAGTCTGGCAGCGAGCTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCTAGCGGCTACACATTCACCGACTACAGCATGCACTGGGTCCGAAAGGCCCCTGGACAGGGACTTGAATGGATGGGCTGGATCAACACCGCCACAGGCGAGCCTACCTACGCCGATGGCTTCACAGGCAGATTCGTGATCAGCCTGGACACCAGCGCCAGAACCGTGTACCTGCAGATCAGCTCTCTGAAGGCCGAGGATACCGCCGTGTACTTCTGCTTCAGCTACTACGACTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGATCCGACATCGTGCTGACACAGAGCCCTCTGAGCCTGCCTGTTACACCTGGCGAGCCTGCCAGCATCAGCTGTAGAGCCAGCCAGACCATCTACAAGAACCTGCACTGGTATCTGCAGAAGCCCGGCCAGTCTCCTCAGCTGCTGATTAAGTACGGCAGCGACAGCATCTCCGGCGTGCCAGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTACACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGGCTACAGCACCCCTTGGACATTCGGCCAGGGCACCAAGCTGGAAATCAAG

Table 1. Primers used in gene cloning
Name	Sequence
F-EagI- SIRPaD1	CGAGCTCCTCATTTACCAGGAGAG
R-BamHI-SIRPaD1	AAACCTGTGCCCAGGGATTGTGG
F-IgG1(Fc)	GGGCACAGGTTTGGCGAGTACATAG
R-IgG1(Fc)	CGGGATCCGAAGGAACTGAAGGTG
F-P-asr	CGGGATCCGCGATCAAGACTAC
R-p-asr	CCCAAGCTTGTCATTTAACCCCACTG
F-P-atp2-HinDIII	AAGCTTAGTGCCGTGCTGGGAAACT
F-P-atp2-BamH	GGATCCCGGACTCCGTCGACATCAG
F-pET-32b-T7	TAATACGACTCACTATAGGG
R-pET-32b-T7	GCTAGTTATTGCAGCGG

Female nude model mice BALB/c that carry with transplanted MCF7 or 4T1 mammary carcinomas have been used for the extraction of full-length RNA samples. Then resultant RNA samples are further subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and generate cDNA for gene cloning. We have experienced difficulties in isolating RNA from BALB/c mice with general experimental protocols. It seems there are strong activities of enzymes that can destroy RNA. The underlying mechanism and roles of RNases in normal and metastatic triple breast cancer have been investigated. We have conducted literature investigation on the functions of RNases and the determination of activities in tumors. Early in 1979, Peterson⁶ reported serum RNase in the diagnosis of pancreatic carcinoma. Figure 2 illustrates the electrophoretic images of RNA with regular and optimized experimental protocol. The coordination of negative phosphate group in physiological condition with Ti⁴⁺ions and the protection of RNA from the degradation has been explored. Figure 3 shows the electrophoretic image of obtained DNA and cloned bacteria.

Figure 2. Gel electrophoretic imaging of extracted RNA with regular(A) and optimized protocol (B).

Figure 3. The electrophoretic image of obtained DNA and engineered bacteria.

The co-culture of epithelial cells and bacteria for investigating host–pathogen interactions was reported in 2009.⁷ We have established a co-culture protocol of bacteria and breast epithelial cells that can be used for the screening of the colonization and interaction of bacteria to cancer cells. Figure 4 (A)-(C) represent optical images of MDA-MB-231, MCF 7 and MCF 10A that were cultured separately and their co-culture with E. coli Nissle 1917 (non-engineered) were shown in Figure 5 (A)-(C), respectively.

Figure 4. Optical images of MDA-MB-231 (A), MCF 7 (B) and MCF 10A (C).

Figure 5. Optical images of MDA-MB-231 (A), MCF 7 (B) and MCF 10A (C) with E. coli. Nissle 1917 (non-engineered).