Part:BBa_K4375017

anti-CEA Nanobody Display System

This construction consists of 4 described parts. These are: J23101* (BBa_K4375003), anti-CEA-specific Nanobody, (BBa_K4375006 ), Intimin (BBa_K4375012), Myc (BBa_K4375014). They allow the bacterium to bind to the cancer cell surface, thus allowing its visualization and colonization on the tumor surface.

Usage and Biology

Figure 1: Intimin and CEA nanobody conjugation scheme.

This construct is meant for detecting cancer cells that express CEA, carcioembrional antigen by bacterial cells. It is expressed by the bacterial cells on their surface and can be used to detect the tumor by as it causes the bacteria to bind to cancer cells. This device contains two different parts whose production is controlled by a constitutive strong promoter J23101* (BBa_K4375003). The anti-CEA Nanobody (BBa_K4375006) is at the N-terminal, tagged with a c-myc for detection. The Nanobody is fused to the Intimin part, which is a 12-stranded β-barrel protein found in bacterial adhesins (BBa_K4375012). Specific interaction can be detected by two tags: Myc (BBa_K4375014) and anti-E, or via the fluorescence of the sfGFP at the C-terminal.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 29
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2043
  Illegal BamHI site found at 4059
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2064
  Illegal NgoMIV site found at 4080
  Illegal AgeI site found at 4146
  Illegal AgeI site found at 4278
  Illegal AgeI site found at 4678
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

Leo, J. C.; Oberhettinger, P.; Schütz, M.; Linke, D. The Inverse Autotransporter Family: Intimin, Invasin and Related Proteins. International Journal of Medical Microbiology 2015, 305 (2), 276–282. https://doi.org/10.1016/j.ijmm.2014.12.011.

Wentzel, A.; Christmann, A.; Adams, T.; Kolmar, H. Display of Passenger Proteins on the Surface of Escherichia Coli K-12 by the Enterohemorrhagic E. Coli Intimin EaeA. Journal of Bacteriology 2001, 183 (24), 7273–7284. https://doi.org/10.1128/jb.183.24.7273-7284.2001.

Rutherford, N., Mourez, M. Surface display of proteins by Gram-negative bacterial autotransporters. Microb Cell Fact 5, 22 2006. https://doi.org/10.1186/1475-2859-5-22

Salema, V.; Marín, E.; Martínez-Arteaga, R.; Ruano-Gallego, D.; Fraile, S.; Margolles, Y.; Teira, X.; Gutierrez, C.; Bodelón, G.; Fernández, L. Á. Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. Coli Cells with Two β-Domains of Opposite Topologies. PLoS ONE 2013, 8 (9), e75126. https://doi.org/10.1371/journal.pone.0075126.

Lwin, T. M.; Turner, M. A.; Nishino, H.; Amirfakhri, S.; Hernot, S.; Hoffman, R. M.; Bouvet, M. Fluorescent Anti-CEA Nanobody for Rapid Tumor-Targeting and Imaging in Mouse Models of Pancreatic Cancer. Biomolecules 2022, 12 (5), 711. https://doi.org/10.3390/biom12050711.

De Meyer, T.; Muyldermans, S.; Depicker, A. Nanobody-Based Products as Research and Diagnostic Tools. Trends in Biotechnology 2014, 32 (5), 263–270. https://doi.org/10.1016/j.tibtech.2014.03.001.

Turner, M. A.; Lwin, T. M.; Amirfakhri, S.; Nishino, H.; Hoffman, R. M.; Yazaki, P. J.; Bouvet, M. The Use of Fluorescent Anti-CEA Antibodies to Label, Resect and Treat Cancers: A Review. Biomolecules 2021, 11 (12), 1819. https://doi.org/10.3390/biom11121819.

Lee, J. H.; Lee, S.-W. The Roles of Carcinoembryonic Antigen in Liver Metastasis and Therapeutic Approaches. Gastroenterology Research and Practice 2017, 2017, 1–11. https://doi.org/10.1155/2017/7521987.

Grunnet, M.; Sorensen, J. B. Carcinoembryonic Antigen (CEA) as Tumor Marker in Lung Cancer. Lung Cancer 2012, 76 (2), 138–143. https://doi.org/10.1016/j.lungcan.2011.11.012.

Boon L., Yutong Y.; Reprogramming Synthetic Cells for Targeted Cancer Therapy 2022; 11 (3), 1349-1360 DOI: 10.1021/acssynbio.1c00631