Difference between revisions of "Part:BBa K5034223"

 
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<partinfo>BBa_K5034223 short</partinfo>
 
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===Basic Description===
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This composite part includes the PPK2 gene from Pseudomonas aeruginosa and the NADK gene from Mycobacterium tuberculosis H37Rv, both we performed codon optimization on and are expressed in the PYYDT plasmid together. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), while the NADK enzyme converts PolyP to NADP. Importing them separately was successful, thus we intend to
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proceed with continued optimisation by their combination.
  
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===Construct features===
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Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
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RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment.
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PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 enzyme.
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NADK Coding Sequence: Encodes the NAD kinase enzyme.
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Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.
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Figure 1: Basic construction of PPK2-NADK plasmid
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Figure 3: Construction of PPK2-NADK plasmid
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Figure 2: Bacterial PCR indicating that PPK2-NADK plasmid can replicate in Shewanella
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===Origin (Organism)===
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PPK2 Gene: Pseudomonas aeruginosa PAO1 strain.
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NADK Gene: Mycobacterium tuberculosis H37Rv strain.
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===Experimental Characterization and results===
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Students from dry lab group using mathematical modelling to introduce the two enzymes and found an enhancement in the polyphosphate and electroproduction capabilities of Shewanella.
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Using half-cell reaction(electrochemistry) to measure the electricity production ability and conducting molybdate assays to determine Pi concentration, we also found a notable enhancement in the polyphosphate, ATP and electroproduction capabilities.
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The results of the half-cell experiments indicated an elevated electron transfer activity, with currents of 137.4 ± 16.34 µA/cm² for the SPPK2 strain, 134.56 ± 17.01 µA/cm² for the SNADK strain, and 164.2 ± 17.64
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µA/cm² for the SPPK2-NADK strain.
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Figure 3: statistical data on phosphorus accumulation capacity of Shewanella with the introduction of PPK2-NADK
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Figure 4: statistical data on electricity production capacity of Shewanella with the introduction of PPK2-NADK
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Figure 5: statistical data on ATP level of Shewanella with the introduction of PPK2-NADK
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===Potential Applications===
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In bioelectrochemical Systems, utilizing PolyP and NADP in microbial fuel cells for further improved electron transfer and energy production.
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===References===
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1.Mori S, Yamasaki M, Maruyama Y, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K. Crystallographic studies of Mycobacterium tuberculosis polyphosphate/ATP-NAD kinase complexed with NAD. J Biosci Bioeng. 2004;98(5):391-3.
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2. Zhang, H., Ishige, K., & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26), 16678-16683.
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3. Neville N, Roberge N, Jia Z. Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence. Int J Mol Sci. 2022 Jan 8;23(2):670.
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 06:39, 29 September 2024


Pi <-> Poly P Poly P -> NADP

Basic Description

This composite part includes the PPK2 gene from Pseudomonas aeruginosa and the NADK gene from Mycobacterium tuberculosis H37Rv, both we performed codon optimization on and are expressed in the PYYDT plasmid together. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), while the NADK enzyme converts PolyP to NADP. Importing them separately was successful, thus we intend to proceed with continued optimisation by their combination.

Construct features

Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment. PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 enzyme. NADK Coding Sequence: Encodes the NAD kinase enzyme. Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.


Figure 1: Basic construction of PPK2-NADK plasmid





Figure 3: Construction of PPK2-NADK plasmid





Figure 2: Bacterial PCR indicating that PPK2-NADK plasmid can replicate in Shewanella


Origin (Organism)

PPK2 Gene: Pseudomonas aeruginosa PAO1 strain. NADK Gene: Mycobacterium tuberculosis H37Rv strain.

Experimental Characterization and results

Students from dry lab group using mathematical modelling to introduce the two enzymes and found an enhancement in the polyphosphate and electroproduction capabilities of Shewanella. Using half-cell reaction(electrochemistry) to measure the electricity production ability and conducting molybdate assays to determine Pi concentration, we also found a notable enhancement in the polyphosphate, ATP and electroproduction capabilities. The results of the half-cell experiments indicated an elevated electron transfer activity, with currents of 137.4 ± 16.34 µA/cm² for the SPPK2 strain, 134.56 ± 17.01 µA/cm² for the SNADK strain, and 164.2 ± 17.64 µA/cm² for the SPPK2-NADK strain.



Figure 3: statistical data on phosphorus accumulation capacity of Shewanella with the introduction of PPK2-NADK


Figure 4: statistical data on electricity production capacity of Shewanella with the introduction of PPK2-NADK


Figure 5: statistical data on ATP level of Shewanella with the introduction of PPK2-NADK

Potential Applications

In bioelectrochemical Systems, utilizing PolyP and NADP in microbial fuel cells for further improved electron transfer and energy production.

References

1.Mori S, Yamasaki M, Maruyama Y, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K. Crystallographic studies of Mycobacterium tuberculosis polyphosphate/ATP-NAD kinase complexed with NAD. J Biosci Bioeng. 2004;98(5):391-3. 2. Zhang, H., Ishige, K., & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26), 16678-16683. 3. Neville N, Roberge N, Jia Z. Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence. Int J Mol Sci. 2022 Jan 8;23(2):670. Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 4981
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4981
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 2834
    Illegal NotI site found at 4987
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4981
    Illegal BglII site found at 3580
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 4981
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 4981
    Illegal XbaI site found at 4996
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 562
    Illegal NgoMIV site found at 4244
    Illegal NgoMIV site found at 4527
    Illegal AgeI site found at 402
  • 1000
    COMPATIBLE WITH RFC[1000]