Part:BBa_K3378002

Ferrous iron sensetive promoter fhuA1

P_fhuA1 is a Fe²⁺-sensitive promoter and improved from P_fhuA (https://parts.igem.org/Part:BBa_J100385). This promoter is strongly repressed in iron-rich environments by Fur-Fe²⁺ complex, but fully derepressed in absence of iron.

Usage and Biology

Wild type promoter P_fhuA has a significant leakage in iron rich environments when used in E. coli. This makes P_fhuA not suitable for many conditions that require tightly regulated. By modifying Fur box in promoter sequences, the tighter promoter P_fhuA1 was successfully constructed (Figure 1). P_fhuA1 has a higher induction rate and stronger constitutive expression intensity than P_fhuA. It can be used as a suicide switch to confine the survival range of engineered bacteria.

Figure 1. Modified methods of Fur box.
Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

To characterize this part, P_fhuA1 and P_fhuA were cloned into pUC57 vector separately. We chose mCherry as the reporter. Plasmids were transferred into E. coli DH5α. The E. coli strain was cultured overnight, and then inoculated into different mediums. The relative fluorescence unit (RFU) in different culture conditions was determined by Synergy H1 microplate reader. In this experiment, LB medium is regarded as a representative of normal eutrophic culture conditions, M9 medium is regarded as an iron-limiting environment, and LB medium containing 500 μM FeSO₄ is regarded as an iron-rich environment. As shown in Figure 2, the constitutive expression intensity of P_fhuA1 is slightly higher than WT, and it shows extremely low leakage under the conditions of LB and LB+500 μM FeSO₄. As detailed by Kalantari et al., E. coli growth will reduce when the Fe²⁺ concentration exceeds 500 μM. However, the WT promoter shows a significantly leaky expression even in LB+500 μM FeSO₄.

These results indicate that the affinity of modified Fur box for Fur protein has been significantly enhanced in E. coli, and improved P_fhuA1 is a tighter promoter.

Figure 2. mCherry expressions under different promoters in different culture conditions. A. RFU curve in M9 medium. B. RFU curve in LB medium. C. RFU curve in LB+500 μM FeSO₄. D. OD₆₀₀ corrected RFU values after 12 h culturing. The error bars indicate standard errors (SEM) of three independent biological replicates.

Reference

[1] Guan, Lingyu, et al. "Development of a Fur-dependent and tightly regulated expression system in Escherichia colifor toxic protein synthesis." BMC biotechnology 13.1 (2013): 25.
[2] Kalantari, N., and S. Ghafari. "Evaluation of toxicity of heavy metals for Escherichia coli growth." (2008): 173-178.