Part:BBa_K2609009

mcp-1 with PelB signal peptide under T4 phage recombination cassette

This part consists of MCP-1 (a monocyte chemokine: this is the mouse equivalent - JE) attached to the PelB signal peptide at the N-terminus. The MCP-1 protein is a monocyte chemoattractant belonging to the C-C family of chemokines. The sequence is flanked by the downstream and upstream sequences of the e-gene from the T4 phage. This part is intended for recombination into the phage using the Lambda red recombination system.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 79
  Illegal NgoMIV site found at 105
  
• 1000
  COMPATIBLE WITH RFC[1000]

Biology

Monocyte Chemoattractant protein(MCP-1/CCL2) is a key member of the C-C chemokine family, involved in the regulation of the recruitment and migration of monocytes/macrophages to the site of infection^[1]. This protein has been tagged with the PelB secretion signal peptide to allow the transport of protein from the cytoplasm to the periplasmic space via the Sec-dependent pathway^[4]), where it is cleaved off by a protease before further transport of the fused protein. We believe that the small size of the protein compared to the average pore size in the cell wall will translate to a significant enough diffusion of the chemokine to the extracellular medium for an observable chemotaxis to occur.

Usage

IISc-Bangalore iGEM 2018:

This part was used for recombination into T4 phage genome, by means of the homologous sites (the 50 bp sequences upstream and downstream of the e-gene in the T4 genome), to replace the existing e-gene with the mcp-1 gene. The PelB secretion signal ensures that we have a properly folded protein in the periplasmic space, which can then diffuse out through the cell wall pores.

References

[1] Deshmane SL, Kremlev S, Amini S, Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res. 2009;29(6):313–326.

[2] Singh, Pranveer, et al. "Effect of signal peptide on stability and folding of Escherichia coli thioredoxin." PloS one 8.5 (2013): e63442.

[3] Sletta, H., et al. "The presence of N-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three tested medically important proteins during high-cell-density cultivations of Escherichia coli." Applied and environmental microbiology 73.3 (2007): 906-912.

[4] Beckwith, Jon. “The Sec-Dependent Pathway.” Research in microbiology 164.6 (2013): 497–504. PMC. Web. 12 Oct. 2018.