Latest revision as of 09:08, 26 October 2020

Pfhua Strategy 0

This promoter, Pfhua, is a relatively high-efficient expression vector in e. Coli based on the principle of iron uptake. This promoter is repressed by the increased presence of iron and induced by iron chelator. When inserted into the pClone Red plasmid, this promoter will cause the bacteria to express red fluorescent protein.

Sequence and Features

HZAU-China 2020's Improvement

The improved part can be found here:https://parts.igem.org/Part:BBa_K3378002

Background

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.

Functional Parameters

To characterize this part, P_fhuA1 and P_fhuA were cloned into pUC57 vector separately, mCherry as a 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