Difference between revisions of "Team:NYMU-Taipei/Basic Part"

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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2017.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2017.igem.org/Judging/Awards"> award listed above</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2017.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<h1>Basic Parts</h1>
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A <b>basic part</b> is a functional unit of DNA that cannot be subdivided into smaller component parts. <a href="http://parts.igem.org/wiki/index.php/Part:BBa_R0051">BBa_R0051</a> is an example of a basic part, a promoter regulated by lambda cl.
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<p>Most genetically-encoded functions have not yet been converted to BioBrick parts. Thus, there are <b>many</b> opportunities to find new, cool, and important genetically encoded functions, and refine and convert the DNA encoding these functions into BioBrick standard biological parts. </p>
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<h3>Best Basic Part Special Prize</h3>
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<p>Most genetically-encoded functions have not yet been converted to BioBrick parts. Thus, there are *many* opportunities to find new, cool, and important genetically encoded functions, and refine and convert the DNA encoding these functions into BioBrick standard biological parts. To be eligible for this award, this part must adhere to <a href="http://parts.igem.org/DNA_Submission">Registry sample submission guidelines</a> and have been sent to the Registry of Standard Biological Parts. If you have a part you wish to nominate your team for this <a href="https://2017.igem.org/Judging/Awards">special prize</a>, make sure you add your part number to your <a href="https://2017.igem.org/Judging/Judging_Form">judging form</a> and delete the box at the top of this page.
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<b>Please note:</b> Judges will only look at the first part number you list, so please only enter ONE (1) part number in the judging form for this prize. </p>
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        <h1> Basic Part</h1>
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<h4>Note</h4>
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        <h4><a href="http://parts.igem.org/Part:BBa_K2350004">K2350004</a></h4>
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<p>This page should list all the basic parts your team has made during your project. You must add all characterization information for your parts on the Registry. You should not put characterization information on this page. Remember judges will only look at the first part in the list for the Best Basic Part award, so put your best part first!</p>
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<h4>NrtA (a part of nitrate channel protein from <i>Synechocystis</i> sp. PCC 6803)</h4>
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        <p>　　This part produces a part of nitrate channel protein from <i>Synechocystis</i> sp. PCC 6803 which plays the role of catching nitrate ion from the environment.</p>
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        <h4>Usage and Biology</h4>
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        <p>　　NrtA is a high-affinity nitrate/nitrite-binding lipoprotein .On <i>Synechocystis</i> sp. PCC 6803, it is tethered to the cell membrane by a lipidated cysteine and a flexible linker rich in glycine and serine. The two domains of it are both composed of five-stranded mixed sheets surrounded by helices. When catch occurs, the nitrate ion is on the middle region of two domains. The resonance state of the nitrate during binding distributes evenly among the three oxygen atoms. However, the second oxygen atom and its interactions with the affinity protein will be absent in the case of nitrite. The second oxygen atom in the bound nitrate molecule also has relatively few interactions. It is the possible answer that nitrate and nitrite have almost the same affinity on NrtA protein.
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        <h4> Analysis</h4>
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        <p>　　To test whether NrtA protein could capture nitrate and nitrite, we used French Pressure Press to lyse the cell. After dialysis of the cell lysates containing NrtA protein, we used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration in the medium.</p>
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<p style="font-size:18px;">Figure 1. Nitrate absorbance of different cell lysate.<br>　　　 Blank: nitrate concentration assay kit assay buffer.<br>　　　 CC: competent cell. <br>　　　 BG11: microalgae culture medium buffer. <br>　　　 NrtA: NrtA producing E.coli.</p>
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<p style="font-size:18px;">Table 1. Dunnett's T3 Test.<br>　　　Blank: nitrate concentration assay kit assay buffer.<br>　　　CC: competent cell.<br>　　　BG11: microalgae culture medium buffer. <br>　　　NrtA: NrtA producing E.coli.</p>
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<p>　　The nitrate and nitrite concentrations of competent cell and NrtA were significantly different. The results indicated that NrtA protein can capture nitrite and/or nitrate.</p>
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<h4></h4>
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<p>　　Then we wanted to know whether the engineered E.coli could decrease nitrate and nitrite in the medium. We used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration of the supernatant of the engineered and native E.coli liquid culture.</p>
  
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<p style="font-size:18px;">Figure 2. Nitrate absorbance of cell.<br>　　　 Blank: nitrate concentration assay kit assay buffer. <br>　　　 CC: competent cell <br>　　　 BG11: microalgae culture medium buffer. <br>　　　 NrtA: NrtA producing E.coli.</p>
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        <img src="https://static.igem.org/mediawiki/2017/8/81/T--NYMU-Taipei--NrtA-Figure2.jpeg"><br>
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<p style="font-size:18px;">Table 2. Dunnett's T3 Test.<br>　　　 Blank: nitrate concentration assay kit assay buffer. <br>　　　 CC: competent cell. <br>　　　 BG11: microalgae culture medium buffer. <br>　　　 NrtA: NrtA producing E.coli.</p>
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        <img src="https://static.igem.org/mediawiki/2017/9/90/NYMU_2017_awards_basicpart_4.jpg" ><br><br>
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<p>　　The nitrate and nitrite concentrations of NrtA and competent cell had slight but not significant difference. The result implied that NrtA protein could capture nitrate and nitrite while the engineered E.coli with NrtA gene could not. The engineered E.coli with NrtA gene could express NrtA protein, and the NrtA protein might be inside the cell, so nitrate and nitrite concentration outside the cell did not change.</p>
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Latest revision as of 15:06, 1 November 2017

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AFFILIATIONS & ACKNOWLEDGMENT

IGEM 2017
National Yang-Ming University
Special Thanks

Basic Part

K2350004

NrtA (a part of nitrate channel protein from Synechocystis sp. PCC 6803)

　　This part produces a part of nitrate channel protein from Synechocystis sp. PCC 6803 which plays the role of catching nitrate ion from the environment.

Usage and Biology

　　NrtA is a high-affinity nitrate/nitrite-binding lipoprotein .On Synechocystis sp. PCC 6803, it is tethered to the cell membrane by a lipidated cysteine and a flexible linker rich in glycine and serine. The two domains of it are both composed of five-stranded mixed sheets surrounded by helices. When catch occurs, the nitrate ion is on the middle region of two domains. The resonance state of the nitrate during binding distributes evenly among the three oxygen atoms. However, the second oxygen atom and its interactions with the affinity protein will be absent in the case of nitrite. The second oxygen atom in the bound nitrate molecule also has relatively few interactions. It is the possible answer that nitrate and nitrite have almost the same affinity on NrtA protein.

Analysis

　　To test whether NrtA protein could capture nitrate and nitrite, we used French Pressure Press to lyse the cell. After dialysis of the cell lysates containing NrtA protein, we used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration in the medium.

Figure 1. Nitrate absorbance of different cell lysate.
　　　 Blank: nitrate concentration assay kit assay buffer.
　　　 CC: competent cell.
　　　 BG11: microalgae culture medium buffer.
　　　 NrtA: NrtA producing E.coli.

Table 1. Dunnett's T3 Test.
　　　Blank: nitrate concentration assay kit assay buffer.
　　　CC: competent cell.
　　　BG11: microalgae culture medium buffer.
　　　NrtA: NrtA producing E.coli.

　　The nitrate and nitrite concentrations of competent cell and NrtA were significantly different. The results indicated that NrtA protein can capture nitrite and/or nitrate.

　　Then we wanted to know whether the engineered E.coli could decrease nitrate and nitrite in the medium. We used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration of the supernatant of the engineered and native E.coli liquid culture.

Figure 2. Nitrate absorbance of cell.
　　　 Blank: nitrate concentration assay kit assay buffer.
　　　 CC: competent cell
　　　 BG11: microalgae culture medium buffer.
　　　 NrtA: NrtA producing E.coli.


Table 2. Dunnett's T3 Test.
　　　 Blank: nitrate concentration assay kit assay buffer.
　　　 CC: competent cell.
　　　 BG11: microalgae culture medium buffer.
　　　 NrtA: NrtA producing E.coli.



　　The nitrate and nitrite concentrations of NrtA and competent cell had slight but not significant difference. The result implied that NrtA protein could capture nitrate and nitrite while the engineered E.coli with NrtA gene could not. The engineered E.coli with NrtA gene could express NrtA protein, and the NrtA protein might be inside the cell, so nitrate and nitrite concentration outside the cell did not change.