Difference between revisions of "Team:HBUT-China/Improve"

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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://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</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://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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                <h1 style="font-size:350%;">
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                        Improve
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                <p class="subtitle">
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                        <!-- Wuhan China -->
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                        <!-- <strong>Bulma</strong>! -->
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                    </p>
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            </center>
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            <div class="content" style="width:80%;margin-left: auto;margin-right: auto;">
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                <center>
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                <h5 style="margin-left: auto;margin-right: auto; font-size: 1.5em">
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                    Background
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                </h5>
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                </center>
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                <div class="content">
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                    <p> </p>
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                    <p style="text-justify: inter-ideograph;text-align: justify; ">
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                            The 2017 HBUT-China team designed the nickel ion detection recombinant strain - Nickel Hunter. They first found an operon model found in the <i>Leptospirillum ferriphilum</i> UBK03 strain, whose repressor NcrB binds to the promoter pncrA to block the expression of the downstream gene <i>ncrA</i>, nickel ions can specifically bind to it to eliminate this repression. So they thought that they could replace the <i>ncrA</i> gene with a reporter gene <i>mRFP</i>, and use the interaction between NcrB and nickel ions to design a recombinant plasmid (BBa_K2304002) and transform it into <i>E. coli</i>, which was the birth of Nickel Hunter.
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                    <p> </p>
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                    <center>
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                        <img src="https://static.igem.org/mediawiki/2018/1/11/T--HBUT-China--improve_1.png" width="80%">
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                        <img src="https://static.igem.org/mediawiki/2018/a/aa/T--HBUT-China--improve_2.png" width="80%">
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                    </center>
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                    <p> </p>
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                    <p style="text-justify: inter-ideograph;text-align: justify; ">
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                            Although this system can detect the presence of nickel ions, it has two limitations that make the detection hard to meet the quantitative requirements, so we designed Nickel Hunter 2.0 this year to make the following improvements.
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                    </p>
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                <br/>
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                <h5 style="margin-left: auto;margin-right: auto; font-size: 1.5em">
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                        Two limitations
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                </h5>
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                </center>
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                <div class="content">
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                    <center>
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                    <p>
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                        <span class="tag is-info is-medium">Unreliable results due to natural fluorescence of DH5α</span>
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                    </p>
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                    <p>
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                        <span class="tag is-info is-medium">Insufficient sensitivity of mRFP</span>
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                    </p>
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                    <p> </p>
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                    </center>
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                </div>
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                <br/>
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                <center>
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                <h5 style="margin-left: auto;margin-right: auto; font-size: 1.5em">
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                        Methods
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                </h5>
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                </center>
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                <div class="content">
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                    <p> </p>
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                    <p style="text-justify: inter-ideograph;text-align: justify; ">
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                            Replacing the gene <i>mRFP</i> with <i>luxCDABE</i>, the <i>mRFP</i> fluorescent protein requires excitation light, while LuxCDABE is a biological autoluminescent protein. Because E. coli itself emits red fluorescence, the use of bioluminescent proteins complegtely avoids the interference of <i>E. coli</i> that version 1.0 suffered from.
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                    </p>
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                    <p></p>
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                    <center>
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                        <img src="https://static.igem.org/mediawiki/2018/0/04/T--HBUT-China--improve_3.png" width="80%">
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                    </center>
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                    <p></p>
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                </div>
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                <br/>
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                <center>
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                <h5 style="margin-left: auto;margin-right: auto; font-size: 1.5em">
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                        Result
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                </h5>
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                </center>
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                <div class="content">
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                    <p> </p>
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                    <p style="text-justify: inter-ideograph;text-align: justify; ">
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                            After replacing the gene <i>mRFP</i> with <i>luxCDABE</i>, we got BBa_K2652004.
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                    </p>
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                    <p></p>
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                    <p style="text-justify: inter-ideograph;text-align: justify; ">
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                            1) We used a nickel ion solution at a concentration of 10 mmol/L to induce their carriers and <i>E. coli</i>. After 3 to 4 hours, we detected the fluorescence intensity of <i>E. coli</i> and the BBa_ K2304002 and the luminescence intensity of <i>E. coli</i> and the BBa_K2652004.
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                    </p>
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                    <center>
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                        <img src="https://static.igem.org/mediawiki/2018/e/ee/T--HBUT-China--improve_4.png" width="80%">
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                        <img src="https://static.igem.org/mediawiki/2018/9/98/T--HBUT-China--improve_6.png" width="80%">
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                    </center>
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                    <p> </p>
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                    <p style="text-justify: inter-ideograph;text-align: justify; ">
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                            It can be seen from the figure that the fluorescence intensity of DH5α itself is not negligible compared to the fluorescence of mRFP, and it may fluctuate depending on the environmental conditions, which will affect our final detection. However, LuxCDABE is luminescent; completely avoiding the issue of self-fluorescence in DH5α.
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                    </p>
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                    <p> </p>
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                    <p style="text-justify: inter-ideograph;text-align: justify; ">
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                            2) Comparison of BBa_K2304002 and BBa_K2652004 response to nickel ions concentration
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                    </p>
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                    <p></p>
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                    <p>
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                            BBa_K2304002:
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                    </p>
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                    <center>
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                        <img src="https://static.igem.org/mediawiki/2018/1/14/T--HBUT-China--improve_7.png" width="80%">
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                    </center>
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                    <p style="text-justify: inter-ideograph;text-align: justify; ">
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                            We induced the BBa_K2652004 with 0, 10<sup>-6</sup>, 10<sup>-5</sup>, 10<sup>-4</sup> mmol/L NiCl<sub>2</sub> solution.
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                    </p>
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                    <p></p>
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                    <p>BBa_K2652004:</p>
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                    <center>
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                        <img src="https://static.igem.org/mediawiki/2018/9/96/T--HBUT-China--improve_8.png" width="80%">
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                    </center>
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                    <p>When c<sub>(Ni2+)</sub>=0, LI/OD<sub>600</sub>= 1096308</p>
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                    <p style="text-justify: inter-ideograph;text-align: justify; ">
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                        The fluorescence range detected by BBa_K2304002 is very small, as shown in the figure, only 0.2 phase difference, there are some external factors such as temperature, which will cause a large deviation of the measured value. After our new optimization, however, BBa_K2652004 produces a wide range of luminescence intensity, so some small deviations can be ignored, thus improving accuracy.
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<h1>Improve</h1>
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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<h3>Gold Medal Criterion #2</h3>
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement. Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
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The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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Latest revision as of 02:49, 17 October 2018

Improve



Background

The 2017 HBUT-China team designed the nickel ion detection recombinant strain - Nickel Hunter. They first found an operon model found in the Leptospirillum ferriphilum UBK03 strain, whose repressor NcrB binds to the promoter pncrA to block the expression of the downstream gene ncrA, nickel ions can specifically bind to it to eliminate this repression. So they thought that they could replace the ncrA gene with a reporter gene mRFP, and use the interaction between NcrB and nickel ions to design a recombinant plasmid (BBa_K2304002) and transform it into E. coli, which was the birth of Nickel Hunter.

Although this system can detect the presence of nickel ions, it has two limitations that make the detection hard to meet the quantitative requirements, so we designed Nickel Hunter 2.0 this year to make the following improvements.


Two limitations

Unreliable results due to natural fluorescence of DH5α

Insufficient sensitivity of mRFP


Methods

Replacing the gene mRFP with luxCDABE, the mRFP fluorescent protein requires excitation light, while LuxCDABE is a biological autoluminescent protein. Because E. coli itself emits red fluorescence, the use of bioluminescent proteins complegtely avoids the interference of E. coli that version 1.0 suffered from.


Result

After replacing the gene mRFP with luxCDABE, we got BBa_K2652004.

1) We used a nickel ion solution at a concentration of 10 mmol/L to induce their carriers and E. coli. After 3 to 4 hours, we detected the fluorescence intensity of E. coli and the BBa_ K2304002 and the luminescence intensity of E. coli and the BBa_K2652004.

It can be seen from the figure that the fluorescence intensity of DH5α itself is not negligible compared to the fluorescence of mRFP, and it may fluctuate depending on the environmental conditions, which will affect our final detection. However, LuxCDABE is luminescent; completely avoiding the issue of self-fluorescence in DH5α.

2) Comparison of BBa_K2304002 and BBa_K2652004 response to nickel ions concentration

BBa_K2304002:

We induced the BBa_K2652004 with 0, 10-6, 10-5, 10-4 mmol/L NiCl2 solution.

BBa_K2652004:

When c(Ni2+)=0, LI/OD600= 1096308

The fluorescence range detected by BBa_K2304002 is very small, as shown in the figure, only 0.2 phase difference, there are some external factors such as temperature, which will cause a large deviation of the measured value. After our new optimization, however, BBa_K2652004 produces a wide range of luminescence intensity, so some small deviations can be ignored, thus improving accuracy.