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~~__NOTOC__~~

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~~<partinfo>BBa_K3076300 short</partinfo>~~

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~~==Description==~~

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~~This part is designed to use as double-stranded (ds) DNA substrate for knocking out CopA gene in E. coli by Lambda Red recombineering system.~~

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~~==Usage and Biology==~~

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This part contains homology sequences of 40 bp flanking a double terminator <html>(<a href="https://parts.igem.org/Part:BBa_B0015">BBa B0015</a>)</html>and a kanamycin resistance gene. The recombination site is at the 76 bp to 156 bp of the CopA gene. If the recombination succeeded, the kanamycin resistance gene will be inserted in between the CopA gene and disrupting the expression. Meanwhile, the kanamycin resistance gene can be used as a selection marker for successful recombination. The double terminator <html>(<a href="https://parts.igem.org/Part:BBa_B0015">BBa B0015</a>)</html> was added at the 5’ to ensure the termination of gene expression.

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To use this substrate, simply amplify this part by PCR. The PCR product is ready to be transformed and recombined. The E. coli strain used should express lambda red recombineering genes. On the other hand, the lambda red recombineering system can be introduced to the E. coli strain by transforming the E. coli with plasmids containing lambda red genes, such as pKD46.

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~~Due to the time constraint, we obtained the knockout strain from Keio knockout strain library directly to carry out the functional study. This dsDNA substrate has not been tested yet.~~

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~~===Functional assay of knockout strain===~~

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CopA, cusA, cutA and cusF are four major genes involved in the copper ions exportation in E. coli. Therefore, we hypothesize that after KOing these genes, the E. coli will accumulate more copper ions intracellualrly. This assay aims to test if this hypothesis was correct.

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~~The KO E. coli was put into copper (II) sulphate-containing medium and the concentration of copper ions in the medium was then 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 (Fig. 1).

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[[File:Standard curve for absorbance (440nm) against copper concentration.png|400px|thumb|center|Fig 1. 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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The results showed that among four KO strains, only cusF KO strain group showed a significant decrease in copper amount after 4 hours when compared with the control group. (Fig. 2 and 3) This result indicates knocking out cusF gene may serve the aim of our project which we tried to increase the metal pollutant removal ability of E. coli.

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[[File:T--Hong Kong JSS--KO test1.jpeg||400px|thumb|center|Fig. 2 CopA, cusA and cutA knockout strains showed no significant increase in copper removal ability when compared with control group]]

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~~[[File:T--Hong Kong JSS--cusF KO result.jpeg|400px|thumb|center|Fig. 3 cusF knockout significantly increased the copper removal by the E. coli when compared with control group]]~~

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~~===Future work===~~

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Another part of our project studied if ectopically expressing the metallothionein protein from Corynebacterium glutamicum (CgMT) <html>(<a href="https://parts.igem.org/Part:BBa_K3076803">BBa K3076803</a>)</html> can increase the metal absorption ability of E. coli and the result was positive. 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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~~<partinfo>BBa_K3076300 SequenceAndFeatures</partinfo>~~

@@ Line 1: / Line 1: @@
-__NOTOC__
-<partinfo>BBa_K3076300 short</partinfo>
-==Description==
-<p>This part is designed to use as double-stranded (ds) DNA substrate for knocking out <i>CopA</i> gene in <i>E. coli</i> by Lambda Red recombineering system.</p>
-==Usage and Biology==
-<p>This part contains homology sequences of 40 bp flanking a double terminator <html>(<a href="https://parts.igem.org/Part:BBa_B0015">BBa B0015</a>)</html>and a kanamycin resistance gene. The recombination site is at the 76 bp to 156 bp of the <i>CopA</i> gene. If the recombination succeeded, the kanamycin resistance gene will be inserted in between the <i>CopA</i> gene and disrupting the expression. Meanwhile, the kanamycin resistance gene can be used as a selection marker for successful recombination. The double terminator <html>(<a href="https://parts.igem.org/Part:BBa_B0015">BBa B0015</a>)</html> was added at the 5’ to ensure the termination of gene expression.</p>
-<p>To use this substrate, simply amplify this part by PCR. The PCR product is ready to be transformed and recombined. The <i>E. coli</i> strain used should express lambda red recombineering genes. On the other hand, the lambda red recombineering system can be introduced to the <i>E. coli</i> strain by transforming the <i>E. coli</i> with plasmids containing lambda red genes, such as pKD46.</p>
-<p>Due to the time constraint, we obtained the knockout strain from Keio knockout strain library directly to carry out the functional study. This dsDNA substrate has not been tested yet.</p>
-===Functional assay of knockout strain===
-<i>CopA, cusA, cutA</i> and <i>cusF</i> are four major genes involved in the copper ions exportation in <i>E. coli</i>. Therefore, we hypothesize that after KOing these genes, the <i>E. coli</i> will accumulate more copper ions intracellualrly. This assay aims to test if this hypothesis was correct.
-The KO <i>E. coli</i> was put into copper (II) sulphate-containing medium and the concentration of copper ions in the medium was then 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 (Fig. 1).
-[[File:Standard curve for absorbance (440nm) against copper concentration.png|400px|thumb|center|Fig 1. 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.]]
-The results showed that among four KO strains, only <i>cusF</i> KO strain group showed a significant decrease in copper amount after 4 hours when compared with the control group. (Fig. 2 and 3) This result indicates knocking out <i>cusF</i> gene may serve the aim of our project which we tried to increase the metal pollutant removal ability of <i>E. coli</i>.
-[[File:T--Hong Kong JSS--KO test1.jpeg||400px|thumb|center|Fig. 2 <i>CopA, cusA</i> and <i>cutA</i> knockout strains showed no significant increase in copper removal ability when compared with control group]]
-[[File:T--Hong Kong JSS--cusF KO result.jpeg|400px|thumb|center|Fig. 3 <i>cusF</i> knockout significantly increased the copper removal by the <i>E. coli</i> when compared with control group]]
-===Future work===
-Another part of our project studied if ectopically expressing the metallothionein protein from <i>Corynebacterium glutamicum</i> (CgMT) <html>(<a href="https://parts.igem.org/Part:BBa_K3076803">BBa K3076803</a>)</html> can increase the metal absorption ability of <i>E. coli </i> and the result was positive. Thus, in the next step, we will combine two modifications together to see if it can further improve our metaL pollutant removal system.
-<partinfo>BBa_K3076300 SequenceAndFeatures</partinfo>