Plasmid

Part:BBa_K5291032:Design

Designed by: Haosheng Lin   Group: iGEM24_BNUZH-China   (2024-09-26)


pBBR1MCS2-nadM-nadK-pntA-pntB


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

During the design phase, our emphasis was on the engineering of metabolic pathways that augment intracellular levels of NADPH. Consequently, taking into account the de novo biosynthesis of NAD+ and the metabolic pathway for NADPH production, we have incorporated the NadM, NADK, and PntAB proteins into the genetically engineered bacteria. This strategic introduction is aimed at facilitating the biosynthesis of NADPH while concurrently mitigating any disruptive effects on the bacteria's physiological functions.

Source

nadM: Francisella tularensis strain Schu S4
nadK: Rhodopseudomonas palustris strain RCB100
pntA,pntB: Escherichia coli str. K-12 substr. MG1655
VtmoJ: Velvet tobacco mottle virus
BCD1,BCD2:The sequence of BDC1,BCD2 is derived from the article Front Bioeng Biotechnol:Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency.
BCD5:The sequence of BDC5 is derived from the article Precise and reliable gene expression via standard transcription and translation initiation elements.
RiboJ: robacco ringspot virus
RNase Ⅲ R1.1,RNase Ⅲ R0.5:early region of bacteria T7 phage
Bi term:The sequence of BiTerm is derived from the article Nature methods:Characterization of 582 natural and synthetic terminators and quantification of their design constraints.
T7:T7 bacteriophage

References

[1] Sorci L, Martynowski D, Rodionov DA, Eyobo Y, Zogaj X, Klose KE, Nikolaev EV, Magni G, Zhang H, Osterman AL. Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis. Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3083-8. doi: 10.1073/pnas.0811718106. Epub 2009 Feb 9. PMID: 19204287; PMCID: PMC2651253.
[2] Oka SI, Titus AS, Zablocki D, Sadoshima J. Molecular properties and regulation of NAD+ kinase (NADK). Redox Biol. 2023 Feb;59:102561. doi: 10.1016/j.redox.2022.102561. Epub 2022 Dec 5. PMID: 36512915; PMCID: PMC9763689.
[3] Clarke DM, Loo TW, Gillam S, Bragg PD. Nucleotide sequence of the pntA and pntB genes encoding the pyridine nucleotide transhydrogenase of Escherichia coli. Eur J Biochem. 1986 Aug 1;158(3):647-53. doi: 10.1111/j.1432-1033.1986.tb09802.x. PMID: 3525165.
[4] Shi A, Zhu X, Lu J, Zhang X, Ma Y. Activating transhydrogenase and NAD kinase in combination for improving isobutanol production. Metab Eng. 2013 Mar;16:1-10. doi: 10.1016/j.ymben.2012.11.008. Epub 2012 Dec 14. PMID: 23246519.
[5] Spielmann A, Baumgart M, Bott M. NADPH-related processes studied with a SoxR-based biosensor in Escherichia coli. Microbiologyopen. 2019 Jul;8(7):e00785. doi: 10.1002/mbo3.785. Epub 2018 Dec 25. PMID: 30585443; PMCID: PMC6612552.
[6] Neves D, Vos S, Blank LM, Ebert BE. Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency. Front Bioeng Biotechnol. 2020 Jan 24;7:458. doi: 10.3389/fbioe.2019.00458. PMID: 32039175; PMCID: PMC6993053.
[7] Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, Mai QA, Tran AB, Paull M, Keasling JD, Arkin AP, Endy D. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods. 2013 Apr;10(4):354-60. doi: 10.1038/nmeth.2404. Epub 2013 Mar 10. PMID: 23474465.