Team:HZAU-China/Improvement

Improvement

Ferrous iron sensetive promoter fhuA1

PfhuA1 is a Fe2+-sensitive promoter and improved from PfhuA. This promoter is strongly repressed in iron-rich environments by Fur-Fe2+ complex, but fully derepressed in absence of iron.

Click here to enter the registry page: BBa_K3378002

Usage and Biology

Wild type promoter PfhuA has a significant leakage in iron rich environments when used in E. coli. This makes PfhuA not suitable for many conditions that require tightly regulated. By modifying Fur box in promoter sequences, the tighter promoter PfhuA1 was successfully constructed (Figure 1). PfhuA1 has a higher induction rate and stronger constitutive expression intensity than PfhuA. It can be used as a suicide switch to confine the survival range of engineered bacteria.


Figure 1. Modified methods of Fur box.

Functional Parameters

To characterize this part, PfhuA1 and PfhuA 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 PfhuA1 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 PfhuA1 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.