Part:BBa_K1373001

nadE with strong promter and strong RBS

nadE overexpression

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

Principle

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]

Informational Contribution

Group: iGEM21_NU_Kazakhstan
Author: Arsen Orazbek
iGEM21_NU_Kazakhstan team also worked with nadE genes as iGEM14_SCAU-China did. However, our nadE gene was extracted from Pseudomonas putida, thus, there are some differences in nucleotide sequence (BBa_K4083004). Nonetheless, the main function of NAD synthethase expressed from nadE is mostly universal for different organisms. Therefore, our team considered to contribute the informational data of NAD/NADH importance. The NAD+ is important for metabolism in organisms. NAD+ can reduce into NADH during cell digestion like glucolysis or Krebs cycle. Thus, more available NAD+ can lead to faster substrate catabolism. Moreover, NADH interacts with the electron transport chain where it releases one electron and one proton. As an electron moves, more protons exit the bacterial membrane which increases the proton gradient. Finally, to reach equilibrium, protons enter the cell by ATP synthase, and one proton can generate up to 3 ATP molecules this way. Thus, one NADH that releases one proton can generate 3 ATP molecules. [1]
Reference:
[1]Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular biology of the cell. New York: Garland Science.

Fig.1 De novo synthesis and salvage pathway of NAD biosynthesis.

Features

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⁺(H) pool size in E.coli there by enhancing electricity performance of Microbial Fuel Cells (MFC).

1.Construction procedures of BBa_K1373001 through Ω-PCR

2.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.

3.Sequence feature from sequencing result

4.Part uses

The nadE gene was amplified from E.coli and cloned into pSB1C3 resulting BBa_K1373000

nadE(BBa_K1373000)	Promoter/RBS	Introduction
BBa_K1373001	BBa_K608002	Strong Promoter and Strong RBS
BBa_K1373002	BBa_K608010	Medium Promoter and Strong RBS + GFP (substituted)

Results

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.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.

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.

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.3 and 4 )

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.

Fig.4 Electric charge yield in wild type, nadE-overexpressed BBa_K1373002 and BBa_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.Yong, X.-Y. et al. Enhancement of bioelectricity generation by cofactor manipulation in microbial fuel cell. Biosensorsand Bioelectronics 56, 19-25, doi:http://dx.doi.org/10.1016/j.bios.2013.12.058 (2014).
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. Plant Cell Physiology