Difference between revisions of "Part:BBa K3378002"

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	===Usage and Biology===		===Usage and Biology===
	<p>Wild type promoter P<sub>fhuA</sub> has a significant leakage in iron rich environments when used in <i>E. coli</i>. This makes P<sub>fhuA</sub> not suitable for many conditions that require tightly regulated. By modifying Fur box in promoter sequences, the tighter promoter P<sub>fhuA1</sub> was successfully constructed (<b>Figure 1</b>). P<sub>fhuA1</sub> has a higher induction rate and stronger constitutive expression intensity than P<sub>fhuA</sub>. It can be used as a suicide switch to confine the survival range of engineered bacteria. </p>		<p>Wild type promoter P<sub>fhuA</sub> has a significant leakage in iron rich environments when used in <i>E. coli</i>. This makes P<sub>fhuA</sub> not suitable for many conditions that require tightly regulated. By modifying Fur box in promoter sequences, the tighter promoter P<sub>fhuA1</sub> was successfully constructed (<b>Figure 1</b>). P<sub>fhuA1</sub> has a higher induction rate and stronger constitutive expression intensity than P<sub>fhuA</sub>. It can be used as a suicide switch to confine the survival range of engineered bacteria. </p>
−	<img src="https://static.igem.org/mediawiki/parts/9/91/T--HZAU-China--ImF0.jpg" style="width:721px;height:191.5px">	+	<center><img src="https://static.igem.org/mediawiki/parts/9/91/T--HZAU-China--ImF0.jpg" style="width:721px;height:191.5px"></center>
		+	<br><br>
		+	<center><b>Figure 1.</b> Modified methods of Fur box.</center>
	<br>		<br>
	<span class='h3bb'>Sequence and Features</span>		<span class='h3bb'>Sequence and Features</span>

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Revision as of 06:38, 26 October 2020

Ferrous iron sensetive promoter fhuA1

P_fhuA1 is a Fe^{2+-sensitive promoter and improved from P_fhuA. 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.

<img src="" style="width:721px;height:191.5px">

Figure 1. Modified methods of Fur box.

Sequence and Features

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

[1] Yang, Hang, et al. "A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method." Scientific reports 5 (2015): 17257.
[2] Yang, Hang, et al. "Antibiofilm activities of a novel chimeolysin against Streptococcus mutans under physiological and cariogenic conditions." Antimicrobial agents and chemotherapy 60.12 (2016): 7436-7443.
[3] Xu, Jingjing, et al. "Activity of the chimeric lysin ClyR against common Gram-positive oral microbes and its anticaries efficacy in rat models." Viruses 10.7 (2018): 380.