Difference between revisions of "Team:ECUST/Fur Inverter"

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<h1 class="box-heading">Plasmid construction</h1>
 
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<h1 class="box-heading">Result</h1>
 
<h1 class="box-heading">Result</h1>
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<p>Vector pET-28a(T7 promoter is replaced by J23119) is cut by BamH1 and Xho1, mCherry is amplified by PCR. Exogenous sequence（promoters with fur-box） and linearized vector are ligated by Ezmax. </p>
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<p>The plasmid was transformed into <i>E.coli</i> DH5α, positive bacteria is cultured in LB medium adding with 0.1% kanamycin. Fluorescence intensity is measured at excitation wavelength of 587nm and emission wavelength of 610nm. </p>
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<p>We design to insert fur box in different site of promoter to characterize different fur box response to ferric iron concentration. We use mCherry as reporter to characterize strength of promoter. </p>
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<p>Fe-deficit medium is added 2,2’-bipyridine to final concentration of 200uM. </p>
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<p>Fe-excess medium is added FeCl3 to final concentration of 100uM. </p>
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<p>Bacteria is inoculated at 1% seed culture medium in Fe-deficit medium and Fe-excess medium and cultured for 12 hours. Then fluorescence intensity is measured. </p>
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<img src="https://static.igem.org/mediawiki/2018/c/ce/T--ECUST--fur1.jpg" class="zoom">
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<p>Because fur2 has the lowest leakage expression of lacI under low Fe concentration and the highest expression under high Fe concentration, we choose fur2 promoter in our system. </p>
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<figcaption><b>Figure 1. The fur-box is inserted into three different positions of the promoter ,named Pfur1(a),Pfur2(b) and Pfur3(c)</b></figcaption>
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<p>Under conditions of high(10<sup>-5<sup>M) and low(10<sup>-7<sup>M) concentration of ferric iron, fur1,fur2 and fur3 has different repression efficiency. So we want to select a fur box, when there is low Fe concentration, mCherry has the most fluorescence intensity, and when there is high Fe concentration , mCherry has the least fluorescence. </p>
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<p>Bacteria is cultured in LB culture medium adding with different concentration of Fe over night, and expression of mCherry is measured. </p>
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<figure>
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<figure class="makeresponsive" style="width: 50%;">
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<img src="https://static.igem.org/mediawiki/2018/3/38/T--ECUST--fur2.jpg" class="z2om">
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<figcaption><b>Figure 2.  (a) Relative expression of mCherry under the regulate of different fur box with Fe concentration of 10<sup>-5<sup>M
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(b)Relative expression of mCherry under the regulate of different fur box with Fe concentration of 10<sup>-7<sup>M
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</b></figcaption>
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<p>Click here to see the complete data(Supplementary Table 1). </p>
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<p>From the figure we can see that, fur2 has the least relative expression. </p>
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<p>When Fe concentration is low in medium, Fur can not bind with fur box. mCherry can be successfully expressed. So we need to choose fur box downstream which, under this condition , mCherry can have high expression. </p>
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<p>After adding with 10-7M Fe in medium ,Fur2 has the highest relative expression.So finally we choose fur2. </p>
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<figure>
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<figure class="makeresponsive" style="width: 50%;">
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<img src="https://static.igem.org/mediawiki/2018/e/ea/T--ECUST--fur3.jpg" class="zo3om">
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<figcaption><b>Figure 3. Fluorescence intensity of mCherry under regulate of 10<sup>-5<sup>M Fe.</b></figcaption>
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</figure>
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<p>After lacI is constructed into our system, it becomes fur invertor system. That is to say, when Fe concentration is high, mCherry can express. So we measure fur2, under Fe concentration is 10-5 M, mCherry can express. </p>
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Revision as of 01:06, 18 October 2018

Fur Inverter System

Description

Fur is a transcription factor that binds to fur box using Fe2+ as a helper repressor to inhibit gene transcription. The inhibition mechanism is shown in Figure 1. The Fe3+ is transferred into the cells by siderophore, and then Fe3+ is reduced to Fe2+. The Fe2+ binds to the Fur protein, and Fur-Fe2+ complex binds to the fur box to inhibit the expression of the export gene.

Design

As the siderophore is continuously secreted outside the cell, the concentration of the extracellular siderophore-Fe3+ complex is continuously increased. We want bacteria to be able to perceive the signal of high iron concentration (because this signal represents the degree of damage to the microenvironment that bacteria are living - rust), and the rust is converted from solid to ferric ions that are soluble and absorbed by the engineered E.coli.

That is to say, when the external Fe3+ concentration is high, the gene is in a condition of being suppressed. This is exactly the opposite of what we hope to be when the external Fe3+ concentration is high to trigger gene expression, so we need to introduce a reversal system. We reversed by introducing a lacI gene. As shown in Figure 2, when the concentration of external Fe3+ is high, the expression of the lacI gene is inhibited. At this time, the lacI gene cannot suppress the output, that is, the opening of the output gene is achieved.

Among them, the Fur protein binds to the 19-bp inverted repeat Fur box (GATAATGATAATCATTATC) with high affinity. There may be different positional relationships between the Fur box and the promoter, and this positional relationship results in a change in the iron concentration response threshold. In order to be able to adjust this threshold, we have designed three different positional relationships, as shown.

Result

We design to insert fur box in different site of promoter to characterize different fur box response to ferric iron concentration. We use mCherry as reporter to characterize strength of promoter.

Figure 1. The fur-box is inserted into three different positions of the promoter ,named Pfur1(a),Pfur2(b) and Pfur3(c)

Under conditions of high(10-5M) and low(10-7M) concentration of ferric iron, fur1,fur2 and fur3 has different repression efficiency. So we want to select a fur box, when there is low Fe concentration, mCherry has the most fluorescence intensity, and when there is high Fe concentration , mCherry has the least fluorescence.

Bacteria is cultured in LB culture medium adding with different concentration of Fe over night, and expression of mCherry is measured.

Figure 2. (a) Relative expression of mCherry under the regulate of different fur box with Fe concentration of 10-5M (b)Relative expression of mCherry under the regulate of different fur box with Fe concentration of 10-7M

Click here to see the complete data(Supplementary Table 1).

From the figure we can see that, fur2 has the least relative expression.

When Fe concentration is low in medium, Fur can not bind with fur box. mCherry can be successfully expressed. So we need to choose fur box downstream which, under this condition , mCherry can have high expression.

After adding with 10-7M Fe in medium ,Fur2 has the highest relative expression.So finally we choose fur2.

Figure 3. Fluorescence intensity of mCherry under regulate of 10-5M Fe.

After lacI is constructed into our system, it becomes fur invertor system. That is to say, when Fe concentration is high, mCherry can express. So we measure fur2, under Fe concentration is 10-5 M, mCherry can express.

Reference

1. PNAS November 28, 2017. 114 (48) 12785-12790 Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis Hualiang

2. Chu et al. Microbial Cell Factories (2015) 14:37 A quorum sensing-based in vivo expression system and its application in multivalent bacterial vaccine Teng Chu, Chunshan Ni