Difference between revisions of "Part:BBa K5034213"
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Figure 6: Transmission electron microscopy of the Shewanella morphology of WT and modified Shewanella strains with B0034-RBS | Figure 6: Transmission electron microscopy of the Shewanella morphology of WT and modified Shewanella strains with B0034-RBS | ||
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| − | <p>Details of all experiments can be found in the <a href="https://2024.igem.wiki/nanjing-china/experiments | + | <p>Details of all experiments can be found in the <a href="https://2024.igem.wiki/nanjing-china/experiments">Experiments</a>section on the Wiki.</p> |
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Revision as of 14:30, 30 September 2024
PolyP <->Pi
Contents
Basic Description
This basic part encodes the PPK1 gene which is initially from Citrobacter freundii and we performed codon optimization on, is expressed in the pBBR1MCS-terminator plasmid. This basic part is designed to facilitate the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi). The PPK1 enzyme is known for its ability to synthesize PolyP from ATP and Pi and to degrade PolyP back to Pi, with a preference for the synthetic reaction, making it a versatile tool for managing phosphate metabolism in engineered systems. In a sentence, it can reversibly convert PolyP and Pi. This reversible process favors the generation of Poly P. For the first time, we expressed this element in a strain of *Shewanella* and conducted codon optimization based on Shewanella.
Construct features
Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.
RBS: Ribosome binding site for efficient translation. In our experiments, we used BBa-B0031, BBa-B0032 and BBa-B0034, with BBa-B0031 having the lowest strength and BBa-B0034 having the highest strength.
PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.
Terminator: Efficient transcription terminator to ensure proper mRNA processing. We used rrnB T1 terminator and T7Te terminator in our experiments.
In basic parts, only coding sequence is included in our sequence. In this part, it is PPK1 Coding Sequence.
Origin (Organism)
The PPK1 gene was sourced from Citrobacter freundii. The pBBR1MCS-terminator plasmid backbone is a standard vector used for gene expression in synthetic biology applications.
Experimental Characterization and results
Trying to enhance Shewanella’s capacity to produce electricity and polymerize phosphorus, we first introduced PPK1 into it. As the strength of the RBS decreases, the electrochemical activity of Shewanella significantly increases, as detected by the Pi content detection method, while its polyphosphate capacity decreases, as observed through half-cell experiment.
We then analyzed factors contributing to the reduced electricity generation capability of SPK1 Reflection electron microscopy and transmission electron microscope results showed that the strains with high phosphorus content had abnormal morphology, indicating that allowing bacteria to be in a high phosphorus content state in advance is not conducive to the rapid transfer of electrons and is not conducive to the ability to produce electricity.
Details of all experiments can be found in the Experimentssection on the Wiki.
References
1.Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181. Sequence and Features
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