Part:BBa_K2715114

Constitutive E.coli promoter BBa_J23114, strong RBS and GFP reporter

Usage and Biology

BBa_J23114 is in a family of strong constitutive promoters tested by a previous iGEM team. We wanted to establish whether these promoters would be suitable for use in our non-model organism chassis Clostridium difficile, and also wanted to put the strength of these promoters into context by characterising fluorescence using the iGEM Interlab calibration curves, and also compare their strengths to the Interlab positive and negative controls. This construct allows us to characterize these promoters more extensively in E. coli.

Characterisation

In this composite part we've added a strong RBS BBa_K2715009, shown to function in Gram-positive and Gram-negative organisms, downstream of the BBa_J23114 promoter, driving expression of GFP taken from BBa_E0040. The construct is part of a family of composite parts which all share the same strong RBS and GFP gene and were all characterised in the same plasmid backbone and in parallel in a fluorescence assay, the results of which can be seen below. The positive and negative controls are parts BBa_I20270 and BBa_R0040 respectively, used in the Interlab 2018 study. The composite parts tested in this assay under the same conditions using a range of alternative promoters are as follows:

BBa_K2715106
BBa_K2715114
BBa_K2715119
BBa_K2715001
BBa_K2715002
BBa_K2715003
BBa_K2715004

These composite parts were assembled in the shuttle cloning vector pMTL84151, and characterised in E. coli within this plasmid. Additionally these promoters were characterised in C. difficile using the gusA biobrick BBa_K330002 as a reporter gene in place of GFP, as GFP requires oxygen in order to function, and C. difficile is an anaerobic organism. The gusA containing composites used to assay the promoter activities in C. difficile are listed below.

BBa_K2715025
BBa_K2715026
BBa_K2715027
BBa_K2715028
BBa_K2715029
BBa_K2715030
BBa_K2715031

The plasmid used for this characterisation in E. coli is displayed below.

Plasmid pMTL84151 containing the biobrick BBa_K2715114

Conclusions

This composite part has enabled a more standardised characterisation of BBa_J23114 when used in conjugation with a strong RBS shown to function in both Gram-positive and Gram-negative organisms, and its strength can be quantified using the iGEM 2018 interlab units of fluorescence.

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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 706
  

References

Heap, J.T., Pennington, O.J., Cartman, S.T. and Minton, N.P., 2009. A modular system for Clostridium shuttle plasmids. Journal of microbiological methods, 78(1), pp.79-85.

Davis, D.F., Ward, W.W. and Cutler, M.W., 1994. Posttranslational chromophore formation in recombinant GFP from E. coli requires oxygen. In Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. Proceedings of the 8th International Symposium on Bioluminescence and Chemiluminescence, Cambridge. Wiley, New York, NY (pp. 569-599).

Chiu, N.H. and Watson, A.L., 2017. Measuring β‐Galactosidase Activity in Gram‐Positive Bacteria Using a Whole‐Cell Assay with MUG as a Fluorescent Reporter. Current protocols in toxicology, 74(1), pp.4-44.