Part:BBa_K845009

paraBAD_gfp_cyaA

In order to create a biological AND gate the 2012 Grenoble team worked on the cAMP-CRP complex and arabinose inductible promoter.

We assembled this AND gate a reporter and a enzyme (cyaA gene) to create an amplification loop.

Usage and Biology

paraBAD is a promoter which can be activated by 2 molecules : CRP-cAMP complex and the AraC protein. It has two states; in absence of L-arabinose the paraBAD promoter is repressed by AraC, whereas the paraC promoter is activated (unless an excess of AraC is present). In presence of L-arabinose and the CRP-cAMP complex, the promoter is activated thus enabling the transcription of the downstream elements. This AND gate provides a filter to biological noise.

In Escherichia coli, cAMP is involved in carbon catabolite repression [1] and binds to the cAMP receptor protein (CRP). The corresponding complex (CRP-cAMP) is a transcriptional factor controlling the expression of more than 220 operons [2]. It has been known for a long time that E. coli actively exports cAMP into the growth medium [3][4]. The transcription of the cyaA gene and the crp gene is negatively regulated by CRP-cAMP [5] [6]. The translation of adenylate cyclase mRNA is ineffective as to prevent excessive synthesis of adenylate cyclase. This can be attributed to the fact that overproduction of cAMP is lethal to Escherichia coli possibly due to an accumulation of methylglyoxal [7] [8]. As we want to use paraBAD like an AND Gate and because high a concentration of cAMP is lethal for E. Coli we make our device in a BW25113 ΔcyaA.

http://www.uniprot.org/uniprot/P00936

http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10170

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 500
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 344
  Illegal BamHI site found at 1538
  Illegal XhoI site found at 494
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 500
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 500
  Illegal AgeI site found at 179
  Illegal AgeI site found at 484
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2172
  Illegal BsaI.rc site found at 1169
  Illegal SapI site found at 161
  Illegal SapI.rc site found at 1662
  Illegal SapI.rc site found at 1902