Part:BBa_K5034229
Poly P -> Pi
Contents
Basic Description
This plasmid is the expression vector of PPX gene(BBa_K5034210).
The basic part(BBa_K5034210) encodes the PPX gene which is initially from Escherichia coli and we performed codon optimization on. The basic part(BBa_K5034210) is designed to facilitate the complete conversion of inorganic polyphosphate (PolyP) to inorganic phosphate (Pi). The PPX enzyme, also known as exopolyphosphatase, is crucial for degrading PolyP into Pi, which is essential for various cellular processes. Inactivation of PPX had no effect on the PolyP level in nuclei in the stationary phase, PolyP level in the nuclei increased 1.5- and 2-fold in the exponential phase in the parent strain and PPX mutant, respectively.
Figure 1: Basic function of PPX
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]
Construct features
Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
PPX Coding Sequence: Encodes the exopolyphosphatase 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 PPX plasmid
Figure 4: Construction of PPX plasmid
Figure 5: Bacterial PCR indicating that different plasmids can replicate in S. oneidensis
Origin (Organism)
The PPX gene was sourced from S. 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 S. oneidensis 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 S. oneidensis.
Electricity production: Using half-cell reaction(electrochemistry) to measure the electricity production ability.
Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration.
We conducted molybdate assays to determine Pi concentration and found that PPX has a bad capacity to polymerize phosphorus.
Figure 6: statistical data on electricity production capacity of S. oneidensis with the introduction of different hydrolases
Figure 7: statistical data on the phosphorus accumulation capacity of S. oneidensis with PPX
Figure 8: ATP level in S. oneidensis with the introduction of different hydrolases
Chasis and genetic context
This part can be normally expressed and function properly in S. oneidensis.
Potential applications
PPX can hydrolyze inorganic polyphosphate (PolyP) to inorganic phosphate (Pi), which can be a crucial part in phosphate metabolism.
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.
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