Part:BBa_K2016005

Superfolder GFP under control of strong constitutive promoter and RyhB siRNA domain

BBa_K2016000), followed by an incorporated RyhB binding site contaning the ribosomal binding site, followed by a fully functioning superfolder GFP. Our superfolder GFP was cloned to be operated by RyhB on the RNA interference level. RhyB is an srRNA that is expressed in E.coli and several other bacteria under low iron conditions, it is therefore responsive to iron concentrations within the cell and provides us with a viable tool needed for iron detection. RyhB causes downregulation of sfGFP in low iron conditions, which we see in the form of lowered florescence. Strong or medium promoters were used for expression of gfp on different levels. However, due to difficulties with cloning, only the GFP under control of strong promoter was submitted to the iGEM registry (Medium-RyhB-GFP cloned into pSB1C3 between EcoRI and SpeI sites available upon request). The gene was cloned into pSB1C3 vector as shown on the right.

Usage and Biology

Our composite biobrick was used for detection of changes in concentration of intracellular iron in E. coli. Our part showed to be responsive to changes in iron concentrations in WT (W3110) E. coli, as well as siderophore-production deficient mutant - JC28 (∆entC). In the figure below we can see differences in the GFP fluorescence when expressed using a medium promoter (J23106 derivative) or strong promoter (J23100 derivative) in two different strains: W3110 (WT) and JC28 (∆entC).

In another experiment we showed changes in GFP fluorescence after addition or iron and followed its expression for 1.5h. In a figure below we show that GFP is significantly overexpressed after addition of iron in all RyhB-GFP strains. Read in detail about our experiments.

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 XhoI site found at 514
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 38