Difference between revisions of "Part:BBa K5034221"
The-simple (Talk | contribs) |
The-simple (Talk | contribs) |
||
Line 3: | Line 3: | ||
<partinfo>BBa_K5034221 short</partinfo> | <partinfo>BBa_K5034221 short</partinfo> | ||
__TOC__ | __TOC__ | ||
+ | |||
===Basic Description=== | ===Basic Description=== | ||
− | This composite part includes the PPK1 gene which is initially from Citrobacter freundii and we performed codon optimization on, is expressed in the | + | This composite part includes the <i>PPK1</i> gene which is initially from <i>Citrobacter freundii</i> and we performed codon optimization on, is expressed in the pBBR1MCS-terminator plasmid with the BBa-B0032 RBS, which is a medium RBS compared to others. This composite 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. |
<html> | <html> | ||
<body> | <body> | ||
Line 15: | Line 16: | ||
===Construct features=== | ===Construct features=== | ||
− | Promoter: Constitutive promoter for continuous expression. We use | + | |
− | RBS: Ribosome binding site for efficient translation. BBa-B0032 here. | + | <partinfo>BBa_K5034221 SequenceAndFeatures</partinfo> |
+ | |||
+ | Plasmid Backbone: pBBR1MCS-terminator plasmid | ||
+ | |||
+ | Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment. | ||
+ | |||
+ | RBS: Ribosome binding site for efficient translation. We use BBa-B0032 here. | ||
+ | |||
PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme. | PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme. | ||
− | Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment. | + | |
+ | Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use rrnB T1 terminator and T7Te terminator in our experiment. | ||
+ | |||
<html> | <html> | ||
<body> | <body> | ||
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
− | <img src="https://static.igem.wiki/teams/5034/ | + | <img src="https://static.igem.wiki/teams/5034/results/new/basic-structure-of-spk2.png" style="width: 500px; height: auto;"> |
</div> | </div> | ||
</body> | </body> | ||
</html> | </html> | ||
− | Figure 2: Basic construction of PPK1 with | + | Figure 2: Basic construction of <i>PPK1</i> with BBa-B0032 RBS plasmid |
<html> | <html> | ||
<body> | <body> | ||
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
− | <img src="https://static.igem.wiki/teams/5034/engineering/ | + | <img src="https://static.igem.wiki/teams/5034/engineering/pbbr1mcs-terminator-32-ppk1.png" style="width: 500px; height: auto;"> |
</div> | </div> | ||
</body> | </body> | ||
</html> | </html> | ||
− | Figure 3: Construction of PPK1 with B0032 RBS plasmid | + | Figure 3: Construction of <i>PPK1</i> with BBa-B0032 RBS plasmid |
+ | |||
+ | |||
+ | We transformed the plasmids into wild-type <i>S. oneidensis.</i>, expressed it, and performed colony PCR. The results showed that <i>PPK1</i> was successfully introduced into <i>Shewanella</i> for replication. | ||
<html> | <html> | ||
<body> | <body> | ||
Line 42: | Line 55: | ||
</body> | </body> | ||
</html> | </html> | ||
− | Figure 4: Colony PCR indicating plasmid replication in | + | Figure 4: Colony PCR indicating plasmid replication in <i>S. oneidensis.</i> |
+ | |||
+ | |||
+ | DNA agarose gel electrophoresis results showed that we obtained the plasmid with BBa-B0032 RBS, which is approximately 2.1 kb in size. | ||
<html> | <html> | ||
<body> | <body> | ||
Line 50: | Line 66: | ||
</body> | </body> | ||
</html> | </html> | ||
− | Figure 5: Agarose gel electrophoresis indicating the target gene | + | Figure 5: Agarose gel electrophoresis indicating we got the target gene with the corresponding RBS |
+ | |||
+ | |||
+ | We performed protein extraction for SDS-PAGE. SDS-PAGE results showed that protein expression of the plasmid with BBa-B0032 RBS is the medium, corresponding to the strength of RBS. | ||
<html> | <html> | ||
<body> | <body> | ||
Line 61: | Line 80: | ||
===Origin (Organism)=== | ===Origin (Organism)=== | ||
− | The PPK1 gene was sourced from Citrobacter freundii. The | + | The <i>PPK1</i> gene was sourced from <i>Citrobacter freundii</i>. The pBBR1MCS-terminator plasmid backbone is a standard vector used for gene expression in synthetic biology applications. |
===Experimental Characterization and results=== | ===Experimental Characterization and results=== | ||
− | Alteration of protein expression intensity can regulate the metabolic networks, so we focused on RBS with varying translation strengths to facilitate the regulation of PPK1 concentration in | + | Alteration of protein expression intensity can regulate the metabolic networks, so we focused on RBS with varying translation strengths to facilitate the regulation of PPK1 concentration in <i>S. oneidensis.</i> to develop the best ability to produce electricity and polymerize phosphorus. |
− | + | ||
+ | We conducted Pi content detection to determine Pi concentration and half-cell experiment to measure the electricity production ability, we found SPK2 with RBS BBa-B0032 has the medium capacity to polymerize phosphorus and a medium electroproduction capability. | ||
+ | |||
<html> | <html> | ||
<body> | <body> | ||
Line 73: | Line 94: | ||
</body> | </body> | ||
</html> | </html> | ||
− | Figure 7: | + | Figure 7: Electricity production capacity of <i>S. oneidensis.</i> after the introduction of <i>PPK1</i> with different RBS |
<html> | <html> | ||
<body> | <body> | ||
Line 81: | Line 102: | ||
</body> | </body> | ||
</html> | </html> | ||
− | Figure 8: | + | Figure 8: Phosphorus accumulation capacity of <i>S. oneidensis.</i> after the introduction of <i>PPK1</i> with different RBS |
− | = | + | Details of all experiments can be found in the <html><body><a href="https://2024.igem.wiki/nanjing-china/experiments">Experiments section on the Wiki.</a></body></html> |
− | + | ||
− | + | ===Chassis and genetic=== | |
− | === | + | Chassis:<i>Shewanella onediensis</i> MR-1 |
− | + | The gene can be expressed and function properly in <i>S. oneidensis.</i>. | |
− | + | ||
− | < | + | |
+ | ===Potential applications=== | ||
+ | The <i>PPK1</i> gene (polyphosphate kinase 1) has potential applications in: | ||
− | + | Industrial Microbial Engineering: Enhances the production of biofuels, amino acids, or antibiotics by boosting polyphosphate synthesis in microorganisms. | |
− | === | + | |
− | + | Environmental Bioremediation: Assists in the accumulation of heavy metals or radioactive substances for pollution control. | |
− | + | ||
+ | ===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. |
Revision as of 07:16, 1 October 2024
Pi <-> Poly P
Contents
Basic Description
This composite part includes the PPK1 gene which is initially from Citrobacter freundii and we performed codon optimization on, is expressed in the pBBR1MCS-terminator plasmid with the BBa-B0032 RBS, which is a medium RBS compared to others. This composite 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.
Construct 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]
Plasmid Backbone: pBBR1MCS-terminator plasmid
Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.
RBS: Ribosome binding site for efficient translation. We use BBa-B0032 here.
PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.
Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use rrnB T1 terminator and T7Te terminator in our experiment.
We transformed the plasmids into wild-type S. oneidensis., expressed it, and performed colony PCR. The results showed that PPK1 was successfully introduced into Shewanella for replication.
DNA agarose gel electrophoresis results showed that we obtained the plasmid with BBa-B0032 RBS, which is approximately 2.1 kb in size.
We performed protein extraction for SDS-PAGE. SDS-PAGE results showed that protein expression of the plasmid with BBa-B0032 RBS is the medium, corresponding to the strength of RBS.
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
Alteration of protein expression intensity can regulate the metabolic networks, so we focused on RBS with varying translation strengths to facilitate the regulation of PPK1 concentration in S. oneidensis. to develop the best ability to produce electricity and polymerize phosphorus.
We conducted Pi content detection to determine Pi concentration and half-cell experiment to measure the electricity production ability, we found SPK2 with RBS BBa-B0032 has the medium capacity to polymerize phosphorus and a medium electroproduction capability.
Details of all experiments can be found in the
Experiments section on the Wiki.Chassis and genetic
Chassis:Shewanella onediensis MR-1
The gene can be expressed and function properly in S. oneidensis..
Potential applications
The PPK1 gene (polyphosphate kinase 1) has potential applications in:
Industrial Microbial Engineering: Enhances the production of biofuels, amino acids, or antibiotics by boosting polyphosphate synthesis in microorganisms.
Environmental Bioremediation: Assists in the accumulation of heavy metals or radioactive substances for pollution control.
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.