Part:BBa_K5034231
Poly P -> Poly P(smaller) or Pi
Basic Description
This basic part encodes the PPN1 gene which is initially from Saccharomyces cerevisiae and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the conversion of long-chain inorganic polyphosphate (PolyP) into shorter fragments without completely degrading it to inorganic phosphate (Pi). The PPN1 enzyme exhibits both exopolyphosphatase and endopolyphosphatase activities, depending on the presence of specific metal ions. Inactivation of the PPN1 gene encoding another protein, which exhibited exopolyPase activity in the yeast (CRN and CNX strains), resulted in almost total elimination of the nuclear exopolyPase activities in both growth phases.
Figure 1: Basic function of PPN1
Construct features
Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. PPN1 Coding Sequence: Encodes the polyphosphatase 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)
Figure 3: Basic construction of PPN1 plasmid
Figure 4: Construction of PPN1 plasmid
Figure 5: Bacterial PCR indicating that different plasmids can replicate in Shewanella
Origin (Organism)
The PPN1 gene was sourced from Saccharomyces cerevisiae. 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. Conducting molybdate assays to determine Pi concentration and found PPN1 a bad capacity to polymerize phosphorus.
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 PPN1
Figure 8: ATP level in Shewanella with the introduction of different hydrolases
References
1.Lichko, L. P., Kulakovskaya, T. V., & Kulaev, I. S. (2006). Inorganic polyphosphate and exopolyphosphatase in the nuclei of Saccharomyces cerevisiae: dependence on the growth phase and inactivation of the PPX1 and PPN1 genes. Biochemistry (Moscow), 71(11), 1171-1175.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 4981
Illegal suffix found in sequence at 1 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4981
Illegal BglII site found at 3580 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 4981
Illegal suffix found in sequence at 2 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
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