Team:SCAU-China/R-Parts

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<div class="r hx-main">
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            <div class="hx-box hx-boxd hx-part">
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                <div class="ti"><strong>Parts</strong></div>
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                    <div class="con">
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                        <table cellpadding="0" cellspacing="0" border="0">
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                        <tr>
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                            <td>&nbsp;</td>
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                                <td>Name</td>
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                                <td>Type</td>
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                                <td>Description</td>
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                                <td width="13%">Lenth</td>
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                                <td>Designer</td>
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                            </tr>
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                            <tr>
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                            <td>&nbsp;</td>
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                                <td>BBa_K1373000</td>
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                                <td>coding</td>
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                                <td><i>nadE</i> ORF</td>
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                                <td>828 bp</td>
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                                <td>Wenxuan Chen</td>
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                            </tr>
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                            <tr class="hxtdbg">
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                            <td>❤</td>
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                                <td>BBa_K1373001</td>
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                                <td>device</td>
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                                <td><i>nadE</i> with strong promoter and strong RBS</td>
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                                <td>883 bp</td>
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                                <td>Wenxuan Chen</td>
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                            </tr>
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                            <tr class="hxtdbg">
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                            <td>❤</td>
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                                <td>BBa_K1373002</td>
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                                <td>device</td>
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                                <td><i>nadE</i> with medium prmoter and strong RBS</td>
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                                <td>875 bp</td>
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                                <td>Wenxuan Chen</td>
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                            </tr>
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                            <tr>
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                            <td>&nbsp;</td>
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                                <td>BBa_K1373003</td>
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                                <td>device</td>
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                                <td><i>nadE</i> with constitutive promoter</td>
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                                <td>883 bp</td>
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                                <td>Wenxuan Chen</td>
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                            </tr>
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                            <tr>
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                            <td>&nbsp;</td>
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                                <td>BBa_K1373004</td>
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                                <td>device</td>
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                                <td><i>nadE</i> and <i>oprF</i> co-expression</td>
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                                <td>1942 bp</td>
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                                <td>Wenxuan Chen</td>
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                            </tr>
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                            <tr>
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                            <td>&nbsp;</td>
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                                <td>BBa_K1373010</td>
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                                <td>regulatary</td>
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                                <td><i>ppsA</i> promoter</td>
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                                <td>170 bp</td>
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                                <td>Ziyan Huang</td>
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                            </tr>
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                            <tr>
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                            <td>&nbsp;</td>
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                                <td>BBa_K1373011</td>
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                                <td>reporter</td>
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                                <td><i>gfp</i> regulated by <i>ppsA</i> promoter</td>
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                                <td>890 bp</td>
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                                <td>Wenxuan Chen</td>
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                            </tr>
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                            <tr>
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                            <td>&nbsp;</td>
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                                <td>BBa_K1373012</td>
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                                <td>device</td>
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                                <td><i>arcA</i> regulated by <i>ppsA</i> promoter</td>
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                                <td>887 bp</td>
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                                <td>Ziyan Huang</td>
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                            </tr>
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                        </table>
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                        <br /><br />
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                        BBa_K1373001: Strong promoter (J23014) + strong RBS (B0034) + <i>nadE</i> (NAD synthetase open reading frame)
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                        <br />
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                        BBa_K1373002: Weak promoter + strong RBS + <i>nadE</i>
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                        <br /><br />
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                        <h3>Results</h3>
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                        <b>1.<a href="https://2014.igem.org/Team:SCAU-China/Omega-PCR" target="_blank">Ω-PCR</a> cloning results</b><br />
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                        <div class="pic">
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                        <img src="https://static.igem.org/mediawiki/2014/e/ec/Hx-part_p1.jpg" width="620" />
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                        <br />
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The <i>nadE</i> fragment was cloned into the vector pSB1C3 containing an existing part BBa_K608002 (strong promoter \with strong RBS) through Ω-PCR, resulting in a fusion of BBa_K608002 and <i>nadE</i> (BBa_K1373001). <i>nadE</i> fragment, the 1st Ω-PCR product of <i>nadE</i> ; BBa_K608002-<i>nadE</i>, the 2nd Ω-PCR product containing BBa_K1373001; Cyclic amplification, cyclic testing amplification product, with the second PCR product as template and forward target gene and reverse vector specific primers; lane D, template plamid of 2nd Ω-PCR (BBa_K608002); lane E, recombinant plasmid (BBa_K1373001); the resultant constructs BBa_K608002 and BBa_K1373001 were confirmed by  double-enzyme digestion with Xba I and Spe I.
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                        <br /><br />
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                        <b>2.Sequence feature from sequencing result of BBa_K1373001</b><br />
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                        <div class="pic">
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                        <img src="https://static.igem.org/mediawiki/2014/9/91/Hx-part_p2.jpg" width="620" />
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                        </div>
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                        <br />
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                        <b>Supportive assays</b>
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                        <br />
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Over-expression of the key enzyme NAD synthetase in energy producing metabolism, physicochemical and biochemical characteristics of fuel cells were analyzed. We have performed semi-quantitative RT-PCR analysis, SDS-PAGE to confirm the expression of transgene in genetically modified <i>E.coli</i> and applied resultant transgenic <i>E.coli</i> to MFC and MDC system to examine its effect on power output.
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                        <br /><br />
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                        <b>Semi-quantitative RT-PCR analysis</b><br />
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mRNA level of nadE was quantified by RT-PCR with, 16s ribosomal RNA rrsA as control. (Fig.1) Transcriptional level of nadE driven by constitutive promoters at different strength can be distinguished according to the brightness of related bands in agarose gel electrophoresis.
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                        <br />
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                        <div class="pic">
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                        <img src="https://static.igem.org/mediawiki/2014/d/dc/Hx-part_p3.jpg" width="620" />
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                            <strong>Fig. 1  Semi-quantitative assay of nadE in modified <i>E.coli</i> strains using reverse transcription polymerase chain reaction (RT-PCR). <i>nadE</i> was driven by strong promoter in BBa_K1373001 and weak promoter in BBa_K1373002. 16S gene serves as loading amount control.</strong>
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                        </div>
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                        <br />
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                        The graph demonstrates that <i>nadE</i> gene express weakly in wild type strain, while modified strains with BBa_K1373001 and BBa_K1373002 present obvious higher nadE level than the wild type. Over-expression level is also consistent with the strength of promoter. These results indicate that our parts BBa_K1373001 and BBa_K1373002 are properly function in modified <i>E.coli</i> for our MFC. <br /><br />
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                        <b>SDS-PAGE</b>
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                        <br />
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In order to make sure whether NAD synthetase was translated into specific protein, we also conducted an SDS-PAGE to detect the target protein in the modified strains. <Br />
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                        <div class="pic">
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                        <img src="https://static.igem.org/mediawiki/2014/c/ca/Hx-part_p4.jpg" width="620" />
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                        </div>
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                        <br />
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Total proteins of each strain were sperated with the SDS-PAGE. An obvious extra band presents in the strains with BBa_K1373001 and BBa_K1373002 compair to wild type <i>E.coli MG1655</i>. This result is consistent with the RT-PCR analysis because it shows higher NadE protein level in BBa_K1373001 (stronge promoter) than that of BBa_K1373002 (weak promoter).
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                        <br /><br />
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                        <b>Microbial Fuel Cell Performance Measurement</b>
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                        <br />
 +
The principles and results above support that our parts have potential to enhance intracellular redox state of fuel cells to improve its power generating performance. This hypothesis was verified by measurement of power generation features in wild type <i>E.coli</i> and <i>nadE</i> overexpressed lines BBa_K1373001 and BBa_K1373002.<br />
 +
As results, overexpression of <i>nadE</i> may enhance the amount of NAD<sup>+</sup>(H) in cells and lead to a higher electricity output at the end. (Figs. 2 and 3 )
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<br />
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                        <div class="pic">
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                        <img src="https://static.igem.org/mediawiki/2014/8/86/Hx-part_p5.jpg" width="620" />
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                            <strong>Fig. 2 Microbial Fuel Cells wild type <i>E.coli MG1655</i> and transgenic <i>MG1655</i> carrying <i>nadE</i> over-expression vector (BBa_K1373001) was cultivated to same concentration (OD 600nm= 2.0), electricity power was monitored every minute up to 700 min. PBS buffer containing 2 g/L glucose and 100 μM riboflavin was used in MFC. Measurement was conducted over a resistance of 1 kΩ.</strong>
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                        </div>
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                        <div class="pic">
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                        <img src="https://static.igem.org/mediawiki/2014/5/51/Hx-part_p6.png" width="620" />
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                            <strong>Fig. 3 Electric charge yield in wild type, <i>nadE</i>-overexpressed BBa_K1373002 and nBBa_K1373001 in 700 minutes.</strong>
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                        </div>
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                        <br /><br />
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                        ◇Fuel cells carrying nadE overexpression vector (BBa_K1373001) shows 1 fold higher maximal voltage output than the wild type one with a peak value 172.09 mV. <br />
 +
◇ Microbial Fuel Cell with our device BBa_K1373001 can obviously produce approximately 738.60% more electric charge than the wild type while the one with medium promoter (BBa_K1373002) perform better than the wild type with an increase of 433.30% electrical energy.
 +
                        <br /><br /><br />
 +
                        <h3>Conclusion</h3>
 +
Experimental results proved that BBa_K1373001 and BBa_K1373002 are functional parts that can overexpress the target gene <i>nadE</i>, which encodes the NAD synthetase, in the modified bacteria. Moreover, overexpression of <i>nadE</i> gene can significantly improve the electrogenic capacity of MFC, supporting the possibility that increase of releasable intracellular electrons in this strategy will promote electric output from fuel cells.
 +
                        <br /><br /><br />
 +
                        <h3>References </h3>
 +
                        [1] Chen L, Wang F, Wang X, Liu YG. Robust one-tube Ω-PCR strategy accelerates precise sequence modification of plasmids for functional genomics. Plant Cell Physiology. 2013, 54: 634-642.<Br />
 +
[2] Yong, X.-Y. et al. Enhancement of bioelectricity generation by cofactor manipulation in microbial fuel cell.
 +
Biosensorsand Bioelectronics. 2014, 56: 19-25.
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Latest revision as of 03:35, 18 October 2014

Parts
  Name Type Description Lenth Designer
  BBa_K1373000 coding nadE ORF 828 bp Wenxuan Chen
❤ BBa_K1373001 device nadE with strong promoter and strong RBS 883 bp Wenxuan Chen
❤ BBa_K1373002 device nadE with medium prmoter and strong RBS 875 bp Wenxuan Chen
  BBa_K1373003 device nadE with constitutive promoter 883 bp Wenxuan Chen
  BBa_K1373004 device nadE and oprF co-expression 1942 bp Wenxuan Chen
  BBa_K1373010 regulatary ppsA promoter 170 bp Ziyan Huang
  BBa_K1373011 reporter gfp regulated by ppsA promoter 890 bp Wenxuan Chen
  BBa_K1373012 device arcA regulated by ppsA promoter 887 bp Ziyan Huang


BBa_K1373001: Strong promoter (J23014) + strong RBS (B0034) + nadE (NAD synthetase open reading frame)
BBa_K1373002: Weak promoter + strong RBS + nadE

Results

1.Ω-PCR cloning results

The nadE fragment was cloned into the vector pSB1C3 containing an existing part BBa_K608002 (strong promoter \with strong RBS) through Ω-PCR, resulting in a fusion of BBa_K608002 and nadE (BBa_K1373001). nadE fragment, the 1st Ω-PCR product of nadE ; BBa_K608002-nadE, the 2nd Ω-PCR product containing BBa_K1373001; Cyclic amplification, cyclic testing amplification product, with the second PCR product as template and forward target gene and reverse vector specific primers; lane D, template plamid of 2nd Ω-PCR (BBa_K608002); lane E, recombinant plasmid (BBa_K1373001); the resultant constructs BBa_K608002 and BBa_K1373001 were confirmed by double-enzyme digestion with Xba I and Spe I.

2.Sequence feature from sequencing result of BBa_K1373001

Supportive assays
Over-expression of the key enzyme NAD synthetase in energy producing metabolism, physicochemical and biochemical characteristics of fuel cells were analyzed. We have performed semi-quantitative RT-PCR analysis, SDS-PAGE to confirm the expression of transgene in genetically modified E.coli and applied resultant transgenic E.coli to MFC and MDC system to examine its effect on power output.

Semi-quantitative RT-PCR analysis
mRNA level of nadE was quantified by RT-PCR with, 16s ribosomal RNA rrsA as control. (Fig.1) Transcriptional level of nadE driven by constitutive promoters at different strength can be distinguished according to the brightness of related bands in agarose gel electrophoresis.
Fig. 1 Semi-quantitative assay of nadE in modified E.coli strains using reverse transcription polymerase chain reaction (RT-PCR). nadE was driven by strong promoter in BBa_K1373001 and weak promoter in BBa_K1373002. 16S gene serves as loading amount control.

The graph demonstrates that nadE gene express weakly in wild type strain, while modified strains with BBa_K1373001 and BBa_K1373002 present obvious higher nadE level than the wild type. Over-expression level is also consistent with the strength of promoter. These results indicate that our parts BBa_K1373001 and BBa_K1373002 are properly function in modified E.coli for our MFC.

SDS-PAGE
In order to make sure whether NAD synthetase was translated into specific protein, we also conducted an SDS-PAGE to detect the target protein in the modified strains.

Total proteins of each strain were sperated with the SDS-PAGE. An obvious extra band presents in the strains with BBa_K1373001 and BBa_K1373002 compair to wild type E.coli MG1655. This result is consistent with the RT-PCR analysis because it shows higher NadE protein level in BBa_K1373001 (stronge promoter) than that of BBa_K1373002 (weak promoter).

Microbial Fuel Cell Performance Measurement
The principles and results above support that our parts have potential to enhance intracellular redox state of fuel cells to improve its power generating performance. This hypothesis was verified by measurement of power generation features in wild type E.coli and nadE overexpressed lines BBa_K1373001 and BBa_K1373002.
As results, overexpression of nadE may enhance the amount of NAD+(H) in cells and lead to a higher electricity output at the end. (Figs. 2 and 3 )
Fig. 2 Microbial Fuel Cells wild type E.coli MG1655 and transgenic MG1655 carrying nadE over-expression vector (BBa_K1373001) was cultivated to same concentration (OD 600nm= 2.0), electricity power was monitored every minute up to 700 min. PBS buffer containing 2 g/L glucose and 100 μM riboflavin was used in MFC. Measurement was conducted over a resistance of 1 kΩ.
Fig. 3 Electric charge yield in wild type, nadE-overexpressed BBa_K1373002 and nBBa_K1373001 in 700 minutes.


◇Fuel cells carrying nadE overexpression vector (BBa_K1373001) shows 1 fold higher maximal voltage output than the wild type one with a peak value 172.09 mV.
◇ Microbial Fuel Cell with our device BBa_K1373001 can obviously produce approximately 738.60% more electric charge than the wild type while the one with medium promoter (BBa_K1373002) perform better than the wild type with an increase of 433.30% electrical energy.


Conclusion

Experimental results proved that BBa_K1373001 and BBa_K1373002 are functional parts that can overexpress the target gene nadE, which encodes the NAD synthetase, in the modified bacteria. Moreover, overexpression of nadE gene can significantly improve the electrogenic capacity of MFC, supporting the possibility that increase of releasable intracellular electrons in this strategy will promote electric output from fuel cells.


References

[1] Chen L, Wang F, Wang X, Liu YG. Robust one-tube Ω-PCR strategy accelerates precise sequence modification of plasmids for functional genomics. Plant Cell Physiology. 2013, 54: 634-642.
[2] Yong, X.-Y. et al. Enhancement of bioelectricity generation by cofactor manipulation in microbial fuel cell. Biosensorsand Bioelectronics. 2014, 56: 19-25.

Retrieved from "http://2014.igem.org/Team:SCAU-China/R-Parts"