Part:BBa_K1820014

CP8_RBS_sfGFP(Bs)_Terminator

Usage and Biology

• This has only been tested in E. coli thus far.

This is a composite part designed as a report protein sequence for use in Lactococcus lactis. The promoter is a mid-range promoter, part BBa_K1033220 from the 2013 Uppsala team, from Peter Ruhdal Jensen and Karin Hammer's library of synthetic promoters for Lactococcus lactis followed by a popular ribosome binding site (Elowitz 1999), a super folded green fluorescent protein, part BBa_K1365020 from the 2014 Groningen team, codon optimized for Bacillus subtilis (sfGFP(Bs)), and a popular double-stop terminator. We further characterized this promoter with the addition of a fluorescent protein. Although it was designed for L. lactis, it has displayed function in Escherichia coli as well. The sfGFP(Bs) has worked well in a variety of bacteria, such as L. lactis, B. subtilis, Escherischia coli and Streptococcus pneumoniae and there are indications that the promoter is functional in a large number of prokaryotic organisms. As such, it is likely that this construct will be functional in a variety of prokaryotic organisms.

We created and tested this construct in Escherichia coli in the pSB1C3 plasmid. It was tested for fluorescence relative to non-transformed E. coli cells (see Figure 1).

This part improves upon CP8 (https://parts.igem.org/Part:BBa_K1033220) by the addition of a RBS, fluorescent protein, and a terminator, as well as further characterization of the promoter.

Figure 1. Fluorescence levels from BBa_K1820014 in E. coli excited at 485/20 with emissions read at 528/20

Figure 2. sfGFP constructs with three different constitutive promoters. BBa_K1820014 (CP8-this construct) is the top left plate, BBa_K1820015 (CP11) is the top right plate, BBa_K1820016 (CP44) is the bottom plate

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 SapI.rc site found at 99
  

References

Jensen, P. R., Hammer, K. (1998). The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters. Appl Environ Microbiol. 1998 Jan; 64(1): 82–87. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124675/

Overkamp, W. et al. (2013) Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl. Environ. Microbiol. 79: 6481-6490