Part:BBa_K4242000

FleQ

This part sequence originated from Pseudomonas aeruginosa encodes FelQ protein. FleQ can repress pel gene expression by a simple roadblock mechanism in which FleQ sits at the pelA promoter and prevents RNA polymerase to access DNA. The binding of c-di-GMP to FleQ induces dissociation of FleQ from the pelA promoter allowing the RNA polymerase to access DNA[1]. FleQ is not only a repressor of pel gene expression in the absence of c-di-GMP but also an activator of pel expression in the presence of c-di-GMP, which suggest two FleQ binding sites at the pel promoter are independently dedicated to activation and repression[2]. What’s more, the pelA promoter occurs distortion needs FleQ work with FleN in the presence of ATP. The DNA bending mediated by FleN occurs only when FleQ is bound to both FleQ boxes. The DNA bending stimulated by FleN occurs only when ATP is present and disappears when c-di-GMP is added[3].

FleQ protein bend with DNA

* Baraquet C et al. Nucleic Acids Res, 2012. Figure 1: Model of pel regulation. FleQ binds to two FleQ boxes on the pel promoter (A). FleQ interacts with FleN in the absence of ATP (B) as well as in the presence of ATP, but in this case, it induces a distortion of pel DNA (C). We propose that FleN forms a bridge between two FleQ bound to their binding sites. The binding of FleQ to FleQ box 2 is essential for repression. The binding of c-di-GMP to FleQ induces a conformational change of FleQ, probably propagated through FleN, which induces the relief of pel distortion and leads to pel expression (D). The binding of FleQ to FleQ box 1 is essential for activation[3].

Usage and Biology

FleQ can control flagella biosynthesis and exopolysaccharide (EPS). scientists found FleQ can activate the expression of flagella biosynthesis genes but inhibit the expression of exopolysaccharide genes. It has an important usage we can regulate FleQ to control biofilm information and explore medical research.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 868
  Illegal XhoI site found at 1321
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 261
  Illegal NgoMIV site found at 1269
  Illegal NgoMIV site found at 1281
  Illegal NgoMIV site found at 1357
  Illegal NgoMIV site found at 1442
  Illegal AgeI site found at 670
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 283
  Illegal SapI.rc site found at 1216
  Illegal SapI.rc site found at 1225
  

Functional Parameters: CUG_China's contributions

when we constructed the biobrick, we found that there is a PstⅠ enzyme cutting site in its coding sequence. Its coding sequence is not compatible with biobrick assembly, and we cannot directly use it as a part. To make this gene compatible with biobrick, we removed this cutting site by using site-directed mutagenesis. Now the new sequence of fleQ has been registered as a new basic part, and we are happy if this may be helpful to any other future iGEMers.

Blast result of our site-removed FleQ

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figure 2. Blast Result of the site-directed sequence and original sequence of fleQ, CTG and CTA code the same amino acid, Leu

References

[1]Matsuyama BY, Krasteva PV, Baraquet C, Harwood CS, Sondermann H, Navarro MV. Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 2016 Jan 12;113(2):E209-18.

[2]Hickman JW, Harwood CS. Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol. 2008 Jul;69(2):376-89.

[3]Baraquet C, Murakami K, Parsek MR, Harwood CS. The FleQ protein from Pseudomonas aeruginosa functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c-di-GMP. Nucleic Acids Res. 2012 Aug;40(15):7207-18.