Difference between revisions of "Part:BBa K5034212"
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It can reversibly convert Poly p and AMP to ADP. For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella.We tested the effects of the introduction of this element on electricity production and phosphorus metabolism | It can reversibly convert Poly p and AMP to ADP. For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella.We tested the effects of the introduction of this element on electricity production and phosphorus metabolism | ||
| − | + | ===Basic Description=== | |
This basic part encodes the PAP gene which is initially from Acinetobacter johnsonii and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the reversible conversion of inorganic polyphosphate (PolyP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP). The PAP enzyme plays a crucial role in phosphate and energy metabolism. PAP and PPK2 have strong | This basic part encodes the PAP gene which is initially from Acinetobacter johnsonii and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the reversible conversion of inorganic polyphosphate (PolyP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP). The PAP enzyme plays a crucial role in phosphate and energy metabolism. PAP and PPK2 have strong | ||
poly(P) utilization activity, and the activity of PAP is 20-fold higher than that in PPK1, even though the Km values for utilization of all three enzymes are almost the same. This implies that PAP and PPK2 work mainly for poly(P) utilization in the cell. However, PAP has the same level of poly(P) synthetic activity (Km and Vmax) as PPK1, and this suggests that PAP also works for poly(P) synthesis at a significant level. | poly(P) utilization activity, and the activity of PAP is 20-fold higher than that in PPK1, even though the Km values for utilization of all three enzymes are almost the same. This implies that PAP and PPK2 work mainly for poly(P) utilization in the cell. However, PAP has the same level of poly(P) synthetic activity (Km and Vmax) as PPK1, and this suggests that PAP also works for poly(P) synthesis at a significant level. | ||
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Figure 1: Basic function of PAP | Figure 1: Basic function of PAP | ||
| − | + | ===Construct features(only coding sequence included in basic part)=== | |
Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. | Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. | ||
PAP Coding Sequence: Encodes the polyphosphate:AMP phosphotransferase enzyme. | PAP Coding Sequence: Encodes the polyphosphate:AMP phosphotransferase enzyme. | ||
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| − | + | ===Origin (Organism)== | |
The PAP gene was sourced from Acinetobacter johnsonii. | The PAP gene was sourced from Acinetobacter johnsonii. | ||
| − | + | ===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. | 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. | Electricity production: Using half-cell reaction(electrochemistry) to measure the electricity production ability. | ||
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Figure 5: ATP level in Shewanella with the introduction of different hydrolases | Figure 5: ATP level in Shewanella with the introduction of different hydrolases | ||
| − | + | ===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. | 1.Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181. | ||
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Revision as of 06:53, 29 September 2024
PolyP + AMP --> ADP
Contents
It can reversibly convert Poly p and AMP to ADP. For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella.We tested the effects of the introduction of this element on electricity production and phosphorus metabolism
Basic Description
This basic part encodes the PAP gene which is initially from Acinetobacter johnsonii and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the reversible conversion of inorganic polyphosphate (PolyP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP). The PAP enzyme plays a crucial role in phosphate and energy metabolism. PAP and PPK2 have strong poly(P) utilization activity, and the activity of PAP is 20-fold higher than that in PPK1, even though the Km values for utilization of all three enzymes are almost the same. This implies that PAP and PPK2 work mainly for poly(P) utilization in the cell. However, PAP has the same level of poly(P) synthetic activity (Km and Vmax) as PPK1, and this suggests that PAP also works for poly(P) synthesis at a significant level.
Figure 1: Basic function of PAP
Construct features(only coding sequence included in basic part)
Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. PAP Coding Sequence: Encodes the polyphosphate:AMP phosphotransferase 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)
=Origin (Organism)
The PAP gene was sourced from Acinetobacter johnsonii.
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 that PAP do not have a good capacity to polymerize phosphorus (but better than PPN1 and PPX).
Figure 3: statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases
Figure 4: statistical data on the phosphorus accumulation capacity of Shewanella with PAP
Figure 5: ATP level in Shewanella with the introduction of different hydrolases
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]