Difference between revisions of "Part:BBa K3076100"
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<partinfo>BBa_K3076100 short</partinfo>
 
<partinfo>BBa_K3076100 short</partinfo>
  
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==Description==
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<p><br><i>Metallothioneins (MT)</i> are proteins made of 61-68 amino acids with a small molecular weight. It is found in almost every known organism, from bacteria to humans. Due to this ubiquity, it minimizes the gene toxicity effect when we choose it as the ectopic expression target.</br></p>
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This part contains the coding sequence of <i>Corynebacterium glutamicum</i> metallothionein gene (<i>CgMT</i>). This part was synthesized into pET151 expression vector as an expression construct <html>(<a href="https://parts.igem.org/Part:BBa_K3076803">BBa K3076803</a>)</html> and then transformed into BL21(DE) <i>E. coli</i> strain. The expression of this part showed a significant increase of copper absorption ability in <i>E. coli</i> and it suits our purpose of creating a bacterial absorbent of metal pollutants in the liquid medium.
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==Usage and Biology==
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Metallothioneins (MT) are proteins made of 61-68 amino acids with a small molecular weight. It is found in almost every known organism, from bacteria to humans. Due to this ubiquity, it minimizes the gene toxicity effect when we choose it as the ectopic expression target.
 +
 
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MT contains many cysteine groups which were reported to be responsible for chelating a wide range of metal ions such as cadmium, lead, copper, and mercury, etc. Nevertheless, MT from different species showed different affinity towards different metal ions.
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Literature reported that MT protein from <i>Corynebacterium glutamicum</i> (CgMT) shows strong binding affinity towards divalent cations, such as Zn2+ and Pb2+. [1] Since our project used Cu2+ as a model for the metal pollutants, we decided to explore the plausibility of using CgMT to increase the metal accumulation ability of <i>E. coli</i>.
  
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<p><br><i>MT</i> contains many cysteine groups which were reported to be responsible for chelating a wide range of metal ions such as cadmium, lead, copper, and mercury, etc. Nevertheless, <i>MT</i> from different species showed different affinity towards different metal ions.</br><p/>
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===Copper removal assay===
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The <i>E. coli</i> expressing CgMT was put into copper (II) sulphate-containing medium. The concentration of copper ions in the medium was measured at different time points.
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API copper testing reagent was used in this assay. The reagent reacts with copper ions and forms brown colour precipitate. We used a colourimeter to measure the absorbance (440nm) of the reagent mixture and calculate the concentration of copper ion by a standard curve.
  
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<p><br>Literature reported that <i>MT</i> protein from <i>Corynebacterium glutamicum (CgMT)</i> shows strong binding affinity towards divalent cations, such as Zn2+ and Pb2+. [1] Since our project used Cu2+ as a model for the metal pollutants, we decided to explore the plausibility of using <i>CgMT</i> to increase the metal accumulation ability of <i>E. coli</i>.</br></p>
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[[File:Standard curve for absorbance (440nm) against copper concentration.png|400px|thumb|center|This standard curve was constructed by using standard solutions of copper (II) sulphate. The absorbance (440nm) of the API reagent and standard solution mixture were measured by colourimeter. The linear equation of the standard curve was then used in other assays to convert the absorbance value into concentration.]]
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<p><br>Furthermore, <i>Corynebacterium glutamicum</i> belongs to the same order as <i>Mycobacterium tuberculosis</i>. Previous iGEM team Oxford 2016 demonstrated that <i>MT</i> from <i>M. tuberculosis</i> (BBa_K1980002) chelates copper ions in cells effectively, thus we expected <i>CgMT</i> also chelates copper (II) efficiently and it will increase the accumulation of copper ions inside <i>E. coli</i> expressing <i>CgMT</i>.</br></p>
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<p><br>This part was synthesized into pET151/TOPO vector for the ectopic expression in <i>E. coli BL21</i>. The results showed that the <i>CgMT</i> transformant absorbed significantly more copper ions (~15%) in the culture medium when compared with non-induced control and empty vector control.</br></p>
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We found that when compared with no IPTG induction control group and empty vector control group, the <i>CgMT</i> gene -expressing group removed significantly more copper ions inside the medium after 4 hours of incubation.
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This result indicates <i>CgMT</i> gene expression can effectively increase the copper absorption ability of <i>E. coli</i> and it serves the aim of our project which we tried to increase the metal pollutant removal ability of <i>E. coli</i>.
  
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[[File:CgMT result.png|400px|thumb|center|The <i>CgMT</i>-expressing group removed ~30% of copper ions in the medium when compared with controls removing only ~12 - 15%.]]
  
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<h3>Assay 1: Test for copper absorption ability on <i>E.coli</i> transformed with <i>CgMT</i></h3>
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===Future work===
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<h4>Abstract of experiment:</h4>
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In this project, we also studied the effect of knocking out copper exporters in <i>E. coli</i> to see if the copper absorption in the organism will be increased. We found that by knocking out <i>cusF</i> gene, the KO strain significantly removed more copper in the medium when compared with the control group. <html>(<a href="https://parts.igem.org/Part:BBa_K3076500">BBa K3076500</a>)</html>
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<p>We utilized pET-151/TOPO vector to drive the expression of <i>CgMT</i> coding sequence under T7 promoter inside <i>BL21(DE) E. coli</i> strain. Then, the bacteria transformants will be tested inside copper (II) sulphate added culture medium to measure their copper absorption efficiency along time.
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Thus, in the next step, we will combine two modifications together to see if it can further improve our metaL pollutant removal system.
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</p>
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===References===
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<h3>Results:</h3>
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[1] Jafarian, V., & Ghaffari, F. (2017). A unique metallothionein-engineered in Escherichia coli for biosorption of lead, zinc, and cadmium; absorption or adsorption? Microbiology, 86(1), 73–81. doi: 10.1134/s0026261717010064
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<br><p><u>Graph 1: Percentage decrease of copper concentration along time</u></p>
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<img src="https://static.igem.org/mediawiki/parts/1/18/T--Hong_Kong_JSS--result_for_CgMT.png" ><br>
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<p><u>Graph 1: The percentage decrease of copper concentration in the culture medium
 
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In the graph, the percentage change of copper(II) ion concentration along time is shown. The copper added media were incubated with IPTG+ (IPTG induced <i>CgMT</i> expression <i>E. coli</i>), IPTG- (<i>CgMT</i> expression <i>E. coli</i> without IPTG added) and Empty (Empty vector control <i>E. coli</i>). IPTG+ showed ~30% decrease, IPTG- showed ~18% decrease and empty vector control showed ~14% decrease.</u></p>
 
  
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<br>The result shows that IPTG+ (blue) had a significant increase in copper removal ability when compared with IPTG- control (red) and Empty control (green). The purple curve shows the blank control in which no bacteria were inoculated in the copper added medium.</br>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3076100 SequenceAndFeatures</partinfo>
  
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<br>It indicates that <i>CgMT</i> expression contributes to the copper absorption ability of the <i>E. coli strains (BL21)</i>.
 
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Although IPTG- also showed a statistical difference when compared with empty vector control, we believe it was due to the small sample size or leaky expression of the T7 promoter. Further investigation is required.
 
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This assay was repeated twice and the error bars represent standard deviation which indicates the distribution of data range.
 
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</br>
 
  
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<h3>Future work: </h3>
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<br>Since our assay method is relatively insensitive when compared with using advanced techniques such as mass spectrometry, we are going to collaborate with university labs to seek a more sensitive way of measurement and thus we can generate a model with higher precision.</br>
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===Functional Parameters===
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<br>Secondly, we aim to create an <i>E. coli</i> absorbent of heavy metals so we are going to combine <i>CgMT</i> with metal exporter knockout modification and further increase the absorption ability of <i>E. coli</i>.</br>
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<partinfo>BBa_K3076100 parameters</partinfo>
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Revision as of 13:32, 13 October 2019


Coding sequence of metallothioneins (MT) gene from Corynebacterium glutamicum (CgMT)

Description

This part contains the coding sequence of Corynebacterium glutamicum metallothionein gene (CgMT). This part was synthesized into pET151 expression vector as an expression construct (BBa K3076803) and then transformed into BL21(DE) E. coli strain. The expression of this part showed a significant increase of copper absorption ability in E. coli and it suits our purpose of creating a bacterial absorbent of metal pollutants in the liquid medium.


Usage and Biology

Metallothioneins (MT) are proteins made of 61-68 amino acids with a small molecular weight. It is found in almost every known organism, from bacteria to humans. Due to this ubiquity, it minimizes the gene toxicity effect when we choose it as the ectopic expression target.

MT contains many cysteine groups which were reported to be responsible for chelating a wide range of metal ions such as cadmium, lead, copper, and mercury, etc. Nevertheless, MT from different species showed different affinity towards different metal ions.

Literature reported that MT protein from Corynebacterium glutamicum (CgMT) shows strong binding affinity towards divalent cations, such as Zn2+ and Pb2+. [1] Since our project used Cu2+ as a model for the metal pollutants, we decided to explore the plausibility of using CgMT to increase the metal accumulation ability of E. coli.

Copper removal assay

The E. coli expressing CgMT was put into copper (II) sulphate-containing medium. The concentration of copper ions in the medium was measured at different time points. API copper testing reagent was used in this assay. The reagent reacts with copper ions and forms brown colour precipitate. We used a colourimeter to measure the absorbance (440nm) of the reagent mixture and calculate the concentration of copper ion by a standard curve.

This standard curve was constructed by using standard solutions of copper (II) sulphate. The absorbance (440nm) of the API reagent and standard solution mixture were measured by colourimeter. The linear equation of the standard curve was then used in other assays to convert the absorbance value into concentration.

We found that when compared with no IPTG induction control group and empty vector control group, the CgMT gene -expressing group removed significantly more copper ions inside the medium after 4 hours of incubation. This result indicates CgMT gene expression can effectively increase the copper absorption ability of E. coli and it serves the aim of our project which we tried to increase the metal pollutant removal ability of E. coli.

The CgMT-expressing group removed ~30% of copper ions in the medium when compared with controls removing only ~12 - 15%.

Future work

In this project, we also studied the effect of knocking out copper exporters in E. coli to see if the copper absorption in the organism will be increased. We found that by knocking out cusF gene, the KO strain significantly removed more copper in the medium when compared with the control group. (BBa K3076500) Thus, in the next step, we will combine two modifications together to see if it can further improve our metaL pollutant removal system.

References

[1] Jafarian, V., & Ghaffari, F. (2017). A unique metallothionein-engineered in Escherichia coli for biosorption of lead, zinc, and cadmium; absorption or adsorption? Microbiology, 86(1), 73–81. doi: 10.1134/s0026261717010064


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]