Part:BBa_K257023

fec promoter + RFP

Inducible promoter controled by the FecARI pathway, ligated in BBa_J61002.

Usage and Biology

pfec is the promoter of the fecABCDE operon involved in ferric citrate uptake. FecARI are responsible for ferric citrate dependent induction of the ferric citrate uptake system. When the inducer, ferric citrate, binds FecA, which is also responsible for transport of ferric citrate across the outer membrane, a signal is transmitted across the outer membrane to the cytoplasmic membrane protein FecR. FecR transmits the signal across the cytoplasmic membrane and activates the σ70 family protein FecI in the cytoplasm. FecI directs RNA polymerase to express fecABCDE, which encodes the ferric citrate outer membrane receptor and the ferric citrate ABC transporter, as well as fecIR ( [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7729419&dopt=Abstract Harle95] , [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10633096&dopt=Abstract Enz00] ). Expression of fecIR is repressed by Fe2+ bound to Fur ( [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16333751&dopt=Abstract Braun05 ]).

Source: EcoCyc [http://biocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=CPLX-3944]

Caracterisation of BBa_K257023

Overnight cultures of BBa_K257023 were diluted 1/100 in LB or M9 medium supplemented with varing amont of FeNO3 and Na Citrate. Fluorescence and OD were assessed in a Tecan plate reader. Fluorescence divided by OD is ploted. The low Fluo/OD at high FeNO3 concentration can be explained by the toxicity of FeNO3. Note that the best response to Na Citrate is obtained with 1mM FeNO3.

Experience

pFec-RFP on LB medium

pFec-RFP on medium with Na citrate (30mM)

pFec-RFP on medium with Fe citrate (30mM)

We've got a control on LB medium : fluorescence is weak. With a concentration of 30mM (Na citrate or Fe citrate), we have the same level of induction of pFec. We can explain this because of the relative toxicity of the Fe(NO3)3 we use to get Fe citrate. Besides, for this amount of citrate, we can expect Fe to be entirely chelated by the citrate. Nevertheless, we demonstrate the induction of Fec promoter.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2919
  Illegal XbaI site found at 2934
  Illegal PstI site found at 877
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2919
  Illegal PstI site found at 877
  Illegal NotI site found at 870
  Illegal NotI site found at 2925
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2919
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2919
  Illegal XbaI site found at 2934
  Illegal PstI site found at 877
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2919
  Illegal XbaI site found at 2934
  Illegal PstI site found at 877
  Illegal AgeI site found at 573
  Illegal AgeI site found at 685
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1958