Part:BBa_K5034229

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 sourced from E. 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

Plasmid backbone: pBBR1MCS-terminator. The Lac promoter and the double terminator are on the plasmid backbone.

Promoter: We use Lac promoter in our experiment. Since there is no LacI protein on plasmid backbone, the gene expression is constitutive. Since the plasmid backbone does not encode the regulatory gene lacI for the repressor protein, the Lac promoter can be used as a constitutive promoter. This allows the subsequent genes to be constantly expressed.

RBS: Ribosome binding site for efficient translation. We use BBa-B0034 which shows the relatively strongest translation in our experiments.

PPX Coding Sequence: Encodes the exopolyphosphatase enzyme.

Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.

Figure 2: Basic construction of PPX plasmid

The results of our amplification of this enzyme are shown in the figure below.(Fig.3)

Figure 3: PCR of target genes before plasmids construction (The extra small fragment in the picture is primer dimer)

The plasmid map of this part is shown in the figure below.(Fig.4)

Figure 4: Construction of PPX plasmid

Origin (Organism)

The PPX gene was sourced from S. cerevisiae.

The pBBR1MCS plasmid backbone is a standard vector used for gene expression in synthetic biology applications. The plasmid backbone(BBa_K5034201) of this part is a modified version of pBBR1MCS, with a double terminator(BBa_B0015) on it.

Experimental Characterization and results

We conducted colony PCR assays to verify that the plasmids can replicate in S. oneidensis. The results showed that all plasmids can replicate normally.(Fig.5)

Figure 5: Colony PCR indicating that different plasmids can replicate in S. oneidensis

In our team’s previous research, we found that the introduction of PolyP synthase in Shewanella decrease the current significantly. So we planned to improve the situation by introducing different polyphosphate hydrolases which influence the phosphate metabolism of S. oneidensis, and this part(PPX) was one of the PolyP hydrolases. However, there is no significant improvement on electricity producing capacity after the introduction of PPX gene.(Fig.6) In addition, the phosphorus accumulation capacity also decreased compared with WT.(Fig.7)

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

We postulated that PolyP hydrolases could promote the conversion of PolyP to phosphorus-containing small molecules, such as ATP or NADPH, which could have an impact on electricity generation and phosphorus accumulation. So, the concentration of ATP in the bacteria was measured. The results showed that PPX had no significant impact on ATP concentration of engineered bacteria.(Fig.8)

Figure 8: ATP level in S. oneidensis with the introduction of different hydrolases

In the end, PPK2(BBa_K5034205) and NADK(BBa_K5034206) was selected to further improve electricity producing capacity and phosphorus accumulation capacity.

• 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.

Details of all experiments can be found at the Experiments section on the Wiki.

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.