Part:BBa_K5034223

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 pBBR1MCS-terminator 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.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 11
  Illegal PstI site found at 3787
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 11
  Illegal PstI site found at 3787
  Illegal NotI site found at 2828
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 11
  Illegal BglII site found at 3574
  Illegal XhoI site found at 4985
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 11
  Illegal PstI site found at 3787
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 11
  Illegal PstI site found at 3787
  Illegal NgoMIV site found at 556
  Illegal NgoMIV site found at 4238
  Illegal NgoMIV site found at 4521
  Illegal AgeI site found at 396
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3150
  Illegal SapI.rc site found at 4087
  Illegal SapI.rc site found at 4297
  

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 a double terminator rrnBT1-T7TE(BBa_B0015) 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.

Plasmid Backbone: pBBR1MCS plasmid, a standard vector used for gene expression in synthetic biology. The plasmid backbone(BBa_K5034201) of this part is a modified version of pBBR1MCS, with a double terminator(BBa_B0015) on it.

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.