Part:BBa_K5034225

Poly P -> NADP

Basic Description

This basic part encodes the NADK gene which is initially from Mycobacterium tuberculosis H37Rv and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the conversion of inorganic polyphosphate (PolyP) to nicotinamide adenine dinucleotide phosphate (NADP). The NADK enzyme is crucial for the phosphorylation of NAD to NADP, which is essential for various metabolic processes. NAD kinase is regarded as a key enzyme in NADP synthesis and, hence, in numerous cellular processes such as anabolic/biosynthetic pathways and protection against oxidative stress.

Figure 1: Basic function of NADK

Chassis and Genetic Context

Successfully expressed in Escherichia coli DH5α and BL21(DE3) strains.

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.

NADK Coding Sequence: Encodes the NAD kinase enzyme.

Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.

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]

Figure 2: PCR of target genes PCR before plasmids construction (The extra small fragment in the picture is primer dimer)

Figure 3: Basic construction of NADK plasmid

Figure 4: Construction of NADK plasmid

Figure 5: Bacterial PCR indicating that different plasmids can replicate in Shewanella

Origin (Organism)

The NADK gene was sourced from Mycobacterium tuberculosis H37Rv strain. The PYYDT plasmid backbone is a standard vector used for gene expression in synthetic biology applications.

Experimental Characterization and results

In our team’s previous research we found that the behavior of the modified Shewanella did not reach our expectation and the electron microscopic observation also showed an abnormal morphology of the bacterium, we postulated that too much PPK1 may lead to an abnormal charge distribution in the bacterium thus result in a decrease in the bacterium's activity and a reduction in its capacity for electricity production, so we planed to improve the situation by introducing different polyphosphate hydrolases which influence the phosphorus metabolism of Shewanella. Electricity production: Using half-cell reaction(electrochemistry) to measure the electricity production ability. Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration. The expression of NADK showed relatively high phosphorus accumulation and electricity generation ability. Also, the ATP level is considerably enhanced.

Figure 6: statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases

Figure 7: statistical data on the phosphorus accumulation capacity of Shewanella with NADK

Figure 8: ATP level in Shewanella with the introduction of different hydrolases

Potential Applications

In bioelectrochemical Systems, utilizing NADP in microbial fuel cells for improved electron transfer and energy production. Also can be utilized in metabolic engineering, stress response studies, and biotechnological applications where enhanced NADP production is beneficial.

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.