Difference between revisions of "Part:BBa K3378002"

 
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PfhuA1 is sensitive to ferrous ions. Ferrous ions can bind to Fur protein, making it an active repressor, thereby shutting down the transcription of the PfuhA1.  
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P<sub>fhuA1</sub> is a Fe<sup>2+<sup>-sensitive promoter and improved from P<sub>fhuA</sub>. This promoter is strongly repressed in iron-rich environments by Fur-Fe<sup>2+</sup> complex, but fully derepressed in absence of iron.
  
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===Usage and Biology===
 
===Usage and Biology===
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<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>
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<img src="https://static.igem.org/mediawiki/parts/9/91/T--HZAU-China--ImF0.jpg" style="width:721px;height:191.5px">
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3378002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3378002 SequenceAndFeatures</partinfo>
  
  
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3378002 parameters</partinfo>
 
<partinfo>BBa_K3378002 parameters</partinfo>
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<p>To characterize this part, P<sub>fhuA1</sub> and P<sub>fhuA</sub> were cloned into pUC57 vector separately, mCherry as a reporter. Plasmids were transferred into <i>E. coli</i> DH5α. The <i>E. coli</i> 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 FeSO<sub>4</sub> is regarded as an iron-rich environment. As shown in <b>Figure 2</b>, the constitutive expression intensity of P<sub>fhuA1</sub> is slightly higher than WT, and it shows extremely low leakage under the conditions of LB and LB+500 μM FeSO<sub>4</sub>. As detailed by Kalantari et al., <i>E. coli</i> growth will reduce when the Fe<sup>2+</sup> concentration exceeds 500 μM. However, the WT promoter shows a significantly leaky expression even in LB+500 μM FeSO<sub>4</sub>. These results indicate that the affinity of modified Fur box for Fur protein has been significantly enhanced in <i>E. coli</i>, and improved P<sub>fhuA1</sub> is a tighter promoter.</p>
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<img src="https://static.igem.org/mediawiki/parts/8/8f/T--HZAU-China--ImFig1.1.jpg" style="width:448px;height:336px">
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<img src="https://static.igem.org/mediawiki/parts/e/e7/T--HZAU-China--ImFig2.1.jpg" style="width:448px;height:336px">
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<p align="center"><b>Figure 2.</b> mCherry expressions under different promoters in different culture conditions. <b>A.</b> RFU curve in M9 medium. <b>B.</b> RFU curve in LB medium. <b>C.</b> RFU curve in LB+500 μM FeSO<sub>4</sub>. <b>D.</b> OD<sub>600</sub> corrected RFU values after 12 h culturing. The error bars indicate standard errors (SEM) of three independent biological replicates. </p>
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<p style="font-size:20px"><b>Reference</b></p>
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[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.<br>
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[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.<br>
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[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.<br>
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Revision as of 06:32, 26 October 2020


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.

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.

<img src="T--HZAU-China--ImF0.jpg" style="width:721px;height:191.5px">
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, PfhuA1 and PfhuA 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 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.

无标题文档

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.