Intracellular redox state of electricity active cells (EAC) is one of the most important physiological traits of extracellular electron transfer efficiency. In particular, the NAD<sup>+</sup>(H) pool size plays a central role of most metabolic pathways. By overexpressing the NAD synthetase, encoded be gene nadE and catalyzes the final step in de novo synthesis and salvage pathway of NAD biosynthesis (Fig. 1), the NAD+ level is increased thereby up-regulating genes whose products catalyze NADH synthesis. Therefore the augmented pool size of NAD<sup>+</sup>(H) result in promotion of NADH(the carrier of electrons)level, leading to high generation of intracellular releasable electrons and better electricity performance of EAC. <sup>[1]</sup>
Intracellular redox state of electricity active cells (EAC) is one of the most important physiological traits of extracellular electron transfer efficiency. In particular, the NAD<sup>+</sup>(H) pool size plays a central role of most metabolic pathways. By overexpressing the NAD synthetase, encoded be gene nadE and catalyzes the final step in de novo synthesis and salvage pathway of NAD biosynthesis (Fig. 1), the NAD+ level is increased thereby up-regulating genes whose products catalyze NADH synthesis. Therefore the augmented pool size of NAD<sup>+</sup>(H) result in promotion of NADH(the carrier of electrons)level, leading to high generation of intracellular releasable electrons and better electricity performance of EAC. <sup>[1]</sup>
<strong>Fig. 1 De novo synthesis and salvage pathway of NAD biosynthesis.</strong>
<strong>Fig. 1 De novo synthesis and salvage pathway of NAD biosynthesis.</strong>
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<h4>HUST-China 2023</h4>
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<h3>Features </h3>
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We codon optimized the sequence of nadE for <i>S. oneidensis</i> MR-1 and determined its effect on intracellular NAD(H/+) concentration when co-expressed with nadD[BBa_K4595014], nadM[BBa_K4595015], and the experimental results proved that it could effectively increase the intracellular NAD(H/+) concentration.
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This part describes construction of nadE overexpression plasmid with Ω-PCR, a robust in vitro molecular modification strategy. It contains subparts K608002 (J23014 + B0034, designed by team iGEM11_Freiburg) and nadE open reading frame from genome of Escherichia coli str.K-12 substr.MG1655. The nadE encodes a NAD synthetase, which catalyze the final step of NAD biosynthetic pathway, resulting the augmentation of NAD<sup>+</sup>(H) pool size in E.coli there by enhancing electricity performance of Microbial Fuel Cells (MFC).
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1.Construction procedures of BBa_K1373001 through <a href="http://2014.igem.org/Team:SCAU-China/Omega-PCR" target="_blank">Ω-PCR</a>
<figcaption>Fig.2 The NAD(H/+) concentration of <i>S. oneidensis</i> MR-1, <i>S. oneidensis</i> MR-1(ycel-pncB), <i>S. oneidensis</i> MR-1(nadE-nadD-nadM).</figcaption>
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2.Ω-PCR cloning results<br />
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We Compared to the wild type, the total amount of NAD(H/+) in <i>S. oneidensis</i> MR-1(nadD-nadE-nadM) increased by 27.34%. This indicates that, <i>S. oneidensis</i> MR-1(nadD-nadE-nadM) facilitates more efficient electron transfer.
he 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.
<td>Medium Promoter and Strong RBS + GFP (substituted)</td>
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<h3>Results </h3>
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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 E coli and applied resultant transgenic E coli to MFC and MDC system to examine its effect on power output.
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<b>semi-quantitative RT-PCR analysis</b>
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mRNA level of nadE was quantified by RT-PCR with, 16s ribosomal RNA rrsA as control. (Fig. 2) 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.
<strong>Fig. 2 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.</strong>
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<b>SDS-PAGE</b><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.
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).
he 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.<br />
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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. 3 and 4 )
<strong>(Fig. 3 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 )
<strong>(Fig. 4 Electric charge yield in wild type, nadE-overexpressed BBa_K1373002 and BBa_K1373001 in 700 minutes.)</strong>
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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 />
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◇ 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.
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<h3>Conclusion </h3>
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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.
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<h3>References </h3>
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1.Yong, X.-Y. et al. Enhancement of bioelectricity generation by cofactor manipulation in microbial fuel cell.
2.Chen L, Wang F, Wang X, Liu YG. (2013) Robust one-tube Ω-PCR strategy accelerates precise sequence modification of plasmids for functional genomics. <b>Plant Cell Physiology</b>
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In addition, we also determined the effect of nadE on the electroproduction capacity of <i>S. oneidensis</i> MR-1 when co-expressed with nadD and nadM, and the results showed that the three co-expression could effectively increase the electroproduction efficiency of<i>S. oneidensis</i> MR-1
<figcaption>Fig.3 The out put voltage of S.oneidensis MR-1, S.oneidensis MR-1(ycel-pncB), S.oneidensis MR-1(nadD-nadE-nadM) when the anoditic solution is M9 buffer and 18mM lactate.</i>
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The results showed that <i>S. oneidensis</i> MR-1(nadD-nadE-nadM) significantly higher discharge peak and prolonged high-efficiency discharge duration compared to the wild type. The highest out put voltage was up to 150.7 mV, with a 42.32% increase in the hightest power output . It is speculated that this could be attributed to the ability of<i>S. oneidensis</i> MR-1(nadD-nadE-nadM) to accelerate intracellular NADH synthesis, resulting in a higher power output.
Latest revision as of 14:45, 12 October 2023
This part encodes NAD synthetase
Open reading frame of nadE
Intracellular redox state of electricity active cells (EAC) is one of the most important physiological traits of extracellular electron transfer efficiency. In particular, the NAD+(H) pool size plays a central role of most metabolic pathways. By overexpressing the NAD synthetase, encoded be gene nadE and catalyzes the final step in de novo synthesis and salvage pathway of NAD biosynthesis (Fig. 1), the NAD+ level is increased thereby up-regulating genes whose products catalyze NADH synthesis. Therefore the augmented pool size of NAD+(H) result in promotion of NADH(the carrier of electrons)level, leading to high generation of intracellular releasable electrons and better electricity performance of EAC. [1]
Fig. 1 De novo synthesis and salvage pathway of NAD biosynthesis.
HUST-China 2023
We codon optimized the sequence of nadE for S. oneidensis MR-1 and determined its effect on intracellular NAD(H/+) concentration when co-expressed with nadD[BBa_K4595014], nadM[BBa_K4595015], and the experimental results proved that it could effectively increase the intracellular NAD(H/+) concentration.
Fig.2 The NAD(H/+) concentration of S. oneidensis MR-1, S. oneidensis MR-1(ycel-pncB), S. oneidensis MR-1(nadE-nadD-nadM).
We Compared to the wild type, the total amount of NAD(H/+) in S. oneidensis MR-1(nadD-nadE-nadM) increased by 27.34%. This indicates that, S. oneidensis MR-1(nadD-nadE-nadM) facilitates more efficient electron transfer.
In addition, we also determined the effect of nadE on the electroproduction capacity of S. oneidensis MR-1 when co-expressed with nadD and nadM, and the results showed that the three co-expression could effectively increase the electroproduction efficiency ofS. oneidensis MR-1
Fig.3 The out put voltage of S.oneidensis MR-1, S.oneidensis MR-1(ycel-pncB), S.oneidensis MR-1(nadD-nadE-nadM) when the anoditic solution is M9 buffer and 18mM lactate.
The results showed that S. oneidensis MR-1(nadD-nadE-nadM) significantly higher discharge peak and prolonged high-efficiency discharge duration compared to the wild type. The highest out put voltage was up to 150.7 mV, with a 42.32% increase in the hightest power output . It is speculated that this could be attributed to the ability ofS. oneidensis MR-1(nadD-nadE-nadM) to accelerate intracellular NADH synthesis, resulting in a higher power output.
Fig. 1 De novo synthesis and salvage pathway of NAD biosynthesis.
