Difference between revisions of "Part:BBa J100385"
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===HZAU-China 2019's Improvement===
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<h1>HZAU-China 2019's Improvement</h1>
 
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<p style="text-align:center;font-size:12px;line-height:12px;font-weight:bold;">The improved part can be found here:<a href="https://parts.igem.org/Part:BBa_K3378002">https://parts.igem.org/Part:BBa_K3378002</a></p>
 
<p style="text-align:center;font-size:12px;line-height:12px;font-weight:bold;">The improved part can be found here:<a href="https://parts.igem.org/Part:BBa_K3378002">https://parts.igem.org/Part:BBa_K3378002</a></p>
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===Background===
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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:855.9px;height:227.4px">
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<center><b>Figure 1.</b> Modified methods of Fur box.</center>
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===Functional Parameters===
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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:369px">
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<img src="https://static.igem.org/mediawiki/parts/e/ec/T--HZAU-China--ImFig3.1.jpg" style="width:448px;height:369px">
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<p><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] Guan, Lingyu, et al. "Development of a Fur-dependent and tightly regulated expression system in Escherichia colifor toxic protein synthesis." <i>BMC biotechnology</i> 13.1 (2013): 25.
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[2] Kalantari, N., and S. Ghafari. "Evaluation of toxicity of heavy metals for Escherichia coli growth." (2008): 173-178.
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Revision as of 08:19, 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


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]


HZAU-China 2019's Improvement

无标题文档

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

Background

无标题文档

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

无标题文档

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.




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.