Part:BBa_K4212011

GFPmut3b

Green fluorescent protein functional in B.subtilis. Used for screening the colonies to test if the bacteria successfully take in the cloned construct.

Usage and Biology

Indeed, if plasmids exhibited self-digesting activity, spores generated from successful transformants would not contain any DNA code to express GFPmut3b. Meaning, once germination is induced and the spores turn to cells, they should no longer present fluorescence. Consequently, our new revisited pipeline would still involve assembly of a self digesting circuit, and evaluation of its efficacy using a reporter gene. In the interest of time, we opted to utilize a GFPmut3b transcriptional unit present in the toolkit, featuring constitutive promoter STK034, RBS sequence RBS_3, GFPmut3b CDS and terminator t13s3p43.

Figure 1. The schematic design of GFPmut3b

References

[1] Andersen, J.B., Sternberg, C., Poulsen, L.K., Bjørn, S.P., Givskov, M. & Molin, S. (1998) New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria. Applied and Environmental Microbiology. 64 (6), 2240–2246. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC106306/.

[2]Gc, F., S, D., D, M., Cj, W., I, S., M, C., Jk, K., Ma, H., M, A. & H, R. (2007) Expression and functional characterization of gfpmut3.1 and its unstable variants in Staphylococcus epidermidis. Journal of microbiological methods. 71 (2). doi:10.1016/j.mimet.2007.08.015.

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

  Illegal BsaI.rc site found at 732