Plasmid

Part:BBa_K5034229

Designed by: Zongyu Guo   Group: iGEM24_Nanjing-China   (2024-09-26)
Revision as of 17:02, 29 September 2024 by Ji-yf (Talk | contribs)


Poly P -> Pi

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


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]


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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