Difference between revisions of "Part:BBa K5034223"
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===Basic Description=== | ===Basic Description=== | ||
| − | This composite part includes the PPK2 gene from Pseudomonas aeruginosa and the NADK gene from Mycobacterium tuberculosis H37Rv, both we performed codon optimization on and are expressed in the | + | This composite part includes the PPK2 gene from Pseudomonas aeruginosa and the NADK gene from Mycobacterium tuberculosis H37Rv, both we performed codon optimization on and are expressed in the pBBR1MCS-terminator plasmid together. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), while the NADK enzyme converts PolyP to NADP. Importing them separately was successful, thus we intend to |
proceed with continued optimisation by their combination. | proceed with continued optimisation by their combination. | ||
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| + | ===Sequence and Features=== | ||
| + | <partinfo>BBa_K5034223 SequenceAndFeatures</partinfo> | ||
| + | |||
===Construct features=== | ===Construct features=== | ||
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. | ||
| + | |||
RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment. | RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment. | ||
| + | |||
PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 enzyme. | PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 enzyme. | ||
| + | |||
NADK Coding Sequence: Encodes the NAD kinase enzyme. | NADK Coding Sequence: Encodes the NAD kinase enzyme. | ||
| − | Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use | + | |
| + | Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment. | ||
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===Origin (Organism)=== | ===Origin (Organism)=== | ||
| − | PPK2 Gene: Pseudomonas aeruginosa PAO1 strain. | + | |
| − | NADK Gene: Mycobacterium tuberculosis H37Rv strain. | + | PPK2 Gene: <i>Pseudomonas aeruginosa</i> PAO1 strain. |
| + | |||
| + | NADK Gene: <i>Mycobacterium tuberculosis</i> H37Rv strain. | ||
| + | |||
| + | Plasmid Backbone: pBBR1MCS plasmid, a standard vector used for gene expression in synthetic biology. 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=== | ===Experimental Characterization and results=== | ||
Students from dry lab group using mathematical modelling to introduce the two enzymes and found an enhancement in the polyphosphate and electroproduction capabilities of Shewanella. | Students from dry lab group using mathematical modelling to introduce the two enzymes and found an enhancement in the polyphosphate and electroproduction capabilities of Shewanella. | ||
| + | |||
Using half-cell reaction(electrochemistry) to measure the electricity production ability and conducting molybdate assays to determine Pi concentration, we also found a notable enhancement in the polyphosphate, ATP and electroproduction capabilities. | Using half-cell reaction(electrochemistry) to measure the electricity production ability and conducting molybdate assays to determine Pi concentration, we also found a notable enhancement in the polyphosphate, ATP and electroproduction capabilities. | ||
The results of the half-cell experiments indicated an elevated electron transfer activity, with currents of 137.4 ± 16.34 µA/cm² for the SPPK2 strain, 134.56 ± 17.01 µA/cm² for the SNADK strain, and 164.2 ± 17.64 | The results of the half-cell experiments indicated an elevated electron transfer activity, with currents of 137.4 ± 16.34 µA/cm² for the SPPK2 strain, 134.56 ± 17.01 µA/cm² for the SNADK strain, and 164.2 ± 17.64 | ||
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===References=== | ===References=== | ||
1.Mori S, Yamasaki M, Maruyama Y, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K. Crystallographic studies of Mycobacterium tuberculosis polyphosphate/ATP-NAD kinase complexed with NAD. J Biosci Bioeng. 2004;98(5):391-3. | 1.Mori S, Yamasaki M, Maruyama Y, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K. Crystallographic studies of Mycobacterium tuberculosis polyphosphate/ATP-NAD kinase complexed with NAD. J Biosci Bioeng. 2004;98(5):391-3. | ||
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2. Zhang, H., Ishige, K., & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26), 16678-16683. | 2. Zhang, H., Ishige, K., & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26), 16678-16683. | ||
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| − | + | 3. Neville N, Roberge N, Jia Z. Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence. Int J Mol Sci. 2022 Jan 8;23(2):670. | |
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Revision as of 11:43, 30 September 2024
Pi <-> Poly P Poly P -> NADP
Contents
Basic Description
This composite part includes the PPK2 gene from Pseudomonas aeruginosa and the NADK gene from Mycobacterium tuberculosis H37Rv, both we performed codon optimization on and are expressed in the pBBR1MCS-terminator plasmid together. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), while the NADK enzyme converts PolyP to NADP. Importing them separately was successful, thus we intend to proceed with continued optimisation by their combination.
Sequence and 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]
Construct features
Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment.
PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 enzyme.
NADK Coding Sequence: Encodes the NAD kinase enzyme.
Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.
Figure 1: Basic construction of PPK2-NADK plasmid
Figure 3: Construction of PPK2-NADK plasmid
Figure 2: Bacterial PCR indicating that PPK2-NADK plasmid can replicate in Shewanella
Origin (Organism)
PPK2 Gene: Pseudomonas aeruginosa PAO1 strain.
NADK Gene: Mycobacterium tuberculosis H37Rv strain.
Plasmid Backbone: pBBR1MCS plasmid, a standard vector used for gene expression in synthetic biology. 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
Students from dry lab group using mathematical modelling to introduce the two enzymes and found an enhancement in the polyphosphate and electroproduction capabilities of Shewanella.
Using half-cell reaction(electrochemistry) to measure the electricity production ability and conducting molybdate assays to determine Pi concentration, we also found a notable enhancement in the polyphosphate, ATP and electroproduction capabilities. The results of the half-cell experiments indicated an elevated electron transfer activity, with currents of 137.4 ± 16.34 µA/cm² for the SPPK2 strain, 134.56 ± 17.01 µA/cm² for the SNADK strain, and 164.2 ± 17.64 µA/cm² for the SPPK2-NADK strain.
Figure 3: statistical data on phosphorus accumulation capacity of Shewanella with the introduction of PPK2-NADK
Figure 4: statistical data on electricity production capacity of Shewanella with the introduction of PPK2-NADK
Figure 5: statistical data on ATP level of Shewanella with the introduction of PPK2-NADK
Potential Applications
In bioelectrochemical Systems, utilizing PolyP and NADP in microbial fuel cells for further improved electron transfer and energy production.
References
1.Mori S, Yamasaki M, Maruyama Y, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K. Crystallographic studies of Mycobacterium tuberculosis polyphosphate/ATP-NAD kinase complexed with NAD. J Biosci Bioeng. 2004;98(5):391-3.
2. Zhang, H., Ishige, K., & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26), 16678-16683.
3. Neville N, Roberge N, Jia Z. Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence. Int J Mol Sci. 2022 Jan 8;23(2):670.