Part:BBa_K5034206

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. In a sentence, it can convert Poly p to NADP. For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella.

Figure 1: Basic function of NADK

Chassis and Genetic Context

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

Construct features(only coding sequence included in basic part)

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.

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

Origin (Organism)

The NADK gene was sourced from Mycobacterium tuberculosis H37Rv strain.

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

Figure 4: statistical data on the phosphorus accumulation capacity of Shewanella with PPK2

Figure 5: 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. Sequence and Features

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]