Part:BBa_K3718010

Demonstrative circuit of tool kit part 2

An example product of our toolkit part II after cloning

After the experiments of the Modular Receptor Platform (MRP) expression , and the transformation of the arcA and iclR genes into E. coli, which has its arcA and iclR knocked out before the transformation, have proven to be successful. We will use caffeine-sensitive MRP as a testing part to model the system. Caffeine will be used to homodimerize the receptors, and is expected to induce the expression of downstream genes. This model will be used to conduct three experiments to confirm the functionality of the tool. By cloning in an antibody VHH gene against caffeine and green fluorescent protein (GFP) as a desired gene to be expressed, we expect the platform is activated in the presence of caffeine to express GFP, arcA and iclR, which will result in an increase in growth rate and a visible green color under UV.

Reference

H. Waegeman, J. Beauprez, H. Moens, J. Maertens, M. De Mey, M. R. Foulquié-Moreno, J. J. Heijnen, D. Charlier, and W. Soetaert, “Effect of ICLR and ARCA knockouts on biomass formation and metabolic fluxes in escherichia coli K12 and its implications on understanding the metabolism of escherichia coli BL21 (DE3),” BMC Microbiology, vol. 11, no. 1, p. 70, 2011.

H.-J. Chang, P. Mayonove, A. Zavala, A. De Visch, P. Minard, M. Cohen-Gonsaud, and J. Bonnet, “A modular receptor platform to expand the sensing repertoire of bacteria,” ACS Synthetic Biology, vol. 7, no. 1, pp. 166–175, 2017.

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 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 120
  Illegal BglII site found at 994
  Illegal BglII site found at 3417
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 686
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1974