Part:BBa_K2486005

Diphtheria Toxin Repressor (dtxR) codon optimized E. coli

Introduction

This part was used in our project as an exogenous iron sensor for E. coli and P. agglomerans. The repressor should detect an increase in Fe2+ levels in the midgut of anophelines fed with blood and regulate the expression of antiparasitic peptides (against Plasmodium spp., as a proof of concept). As dtxR is a repressor in the presence of Fe2+ we have coupled it to a logic inverter (TetR) which ultimately regulates the expression of our system in combination with a lactate detector (LldR). We have selected this regulator instead of the endogenous Fur (ferric uptake regulator) in order to avoid metabolic interferences as there are no DNA binding motifs for dtxR in the genome of our chassis ([http://www.pnas.org/content/87/15/5968.short Boyd, 1998]).

"Diphtheria toxin is synthesized by Corynebacterium diphtheriae lysogenic for one of a family of corynebacteriophages that carries the structural gene for the toxin, tox. Optimal yields of tox gene products have long been known to be obtained only from C. diphtheriae grown under conditions where iron becomes the growth-rate-limiting substrate. In 1936, Pappenheimer and Johnson showed that adding iron in low concentration to the growth medium inhibited the production of diphtheria toxin. Both biochemical and genetic evidence support the hypothesis that the corynebacteriophage tox gene is regulated by a corynebacterial-determined iron-binding repressor as postulated by Murphy et al. This model predicted an aporepressor that in the presence of iron forms a complex; this complex then binds to the tox operator and blocks transcription. Under conditions of iron limitation, the iron-repressor complex dissociates, derepressing the tox gene. The nucleic acid base sequence of tox revealed a 9-basepair (bp) inverted repeat that overlaps the " -10" region of the promoter. Because many operators exhibit dyad symmetry and are positioned near their respective promoters, this region was designated the putative tox operator." ([http://www.pnas.org/content/87/15/5968.short Boyd, 1998])

"DtxR is the prototype of a family of metal-dependent regulatory proteins that have been identified in numerous gram-positive and gram-negative bacteria. The physiological role of DtxR in C. diphtheriae is similar to that of the ferric uptake regulator, Fur. However, the two proteins have very little amino acid homology. The DtxR protein is known to function as a global regulator of metabolism in C. diphtheriae. It is not only involved in the regulation of expression of diphtheria toxin but also responsible for regulating the synthesis and production of the C. diphtheriae siderophore, corynebacteria, as well as at least seven other promoters." ([http://jcm.asm.org/content/43/1/223.short De Zoysa, 2005])

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 509
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Our experiments with part