Difference between revisions of "Part:BBa K4595017"
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It is a complex component composed of tac promoter, target genes nadE, nadD and nadM. | It is a complex component composed of tac promoter, target genes nadE, nadD and nadM. | ||
This component is responsible for introducing a new de novo synthetic NAD+ pathway into | This component is responsible for introducing a new de novo synthetic NAD+ pathway into | ||
− | engineered bacteria S.oneidensis MR-1 and enhancing the existing common synthetic | + | engineered bacteria <i> S.o oneidensis</i> MR-1 and enhancing the existing common synthetic |
pathway, thereby increasing the intracellular NAD+ content, increasing the intracellular NADH | pathway, thereby increasing the intracellular NAD+ content, increasing the intracellular NADH | ||
level, promoting electron transfer, and thus improving the electrical generation capacity of | level, promoting electron transfer, and thus improving the electrical generation capacity of | ||
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Ptac promoter is a functional hybrid promoter commonly used in bacteria, derived from trp and lac promoters. Ptac consists of partial sequences of two promoters, which have higher binding affinity and expression activity than both of them, and can be used for the expression of exogenous genes, and is also regulated by the regulatory factors of both [1]. There is a lacIq coding region in front of the Ptac promoter sequence, which can inhibit the normal activation of the promoter in the natural state in the system, and the induction of inducers (such as IPTG) is required to remove the inhibition and thus initiate the downstream pathway expression. We got a sequence of it through corporate synthesis. | Ptac promoter is a functional hybrid promoter commonly used in bacteria, derived from trp and lac promoters. Ptac consists of partial sequences of two promoters, which have higher binding affinity and expression activity than both of them, and can be used for the expression of exogenous genes, and is also regulated by the regulatory factors of both [1]. There is a lacIq coding region in front of the Ptac promoter sequence, which can inhibit the normal activation of the promoter in the natural state in the system, and the induction of inducers (such as IPTG) is required to remove the inhibition and thus initiate the downstream pathway expression. We got a sequence of it through corporate synthesis. | ||
<h1>nadE</h1> | <h1>nadE</h1> | ||
− | nadE is a gene encoding NH(3)-dependent NAD(+) synthetase from Escherichia coli (strainK12) . This enzyme can catalyze the nicotinic acid adenine dinucleotide (NaAD) to form NAD | + | nadE is a gene encoding NH(3)-dependent NAD(+) synthetase from <i> Escherichia coli</i> (strainK12) . This enzyme can catalyze the nicotinic acid adenine dinucleotide (NaAD) to form NAD |
by consuming ATP and using ammonia as nitrogen source. This protein catalyzes the | by consuming ATP and using ammonia as nitrogen source. This protein catalyzes the | ||
− | common pathway from NAMN to NAD+ and is found naturally in S.oneidensis MR-1. By | + | common pathway from NAMN to NAD+ and is found naturally in <i> S.o oneidensis</i> MR-1. By |
introducing exogenous nadE, we can efficiently express NH(3)-dependent NAD(+) synthetase | introducing exogenous nadE, we can efficiently express NH(3)-dependent NAD(+) synthetase | ||
to promote the efficient expression of this pathway and improve the synthesis efficiency of | to promote the efficient expression of this pathway and improve the synthesis efficiency of | ||
NAD+. | NAD+. | ||
<h1>nadD</h1> | <h1>nadD</h1> | ||
− | nadD is a gene | + | nadD is a gene eEscherichia colncoding nicotinamide/nicotinic acid mononucleotide adenylyltransferase |
− | from | + | from i (strain K12). This enzyme can catalyze reversible adenylation of nicotinic |
acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) by consuming | acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) by consuming | ||
ATP, where NaAD is a reaction precursor to catalyze the synthesis of NAD+. This protein | ATP, where NaAD is a reaction precursor to catalyze the synthesis of NAD+. This protein | ||
catalyzes the common pathway from NAMN to NAD+ and is found naturally in | catalyzes the common pathway from NAMN to NAD+ and is found naturally in | ||
− | S.oneidensis MR-1. We introduced exogenous nadD to efficiently express | + | <i> S.o oneidensis</i> MR-1. We introduced exogenous nadD to efficiently express |
nicotinamide/nicotinic acid mononucleotide adenylyltransferase and promote the efficient | nicotinamide/nicotinic acid mononucleotide adenylyltransferase and promote the efficient | ||
expression of this pathway. | expression of this pathway. | ||
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catalyzes the formation of NAD by β -nicotinamide mononucleotideNMN (NMN) and | catalyzes the formation of NAD by β -nicotinamide mononucleotideNMN (NMN) and | ||
deaminated NAD by nicotinic acid mononucleotide (NaMN). By introducing this gene into | deaminated NAD by nicotinic acid mononucleotide (NaMN). By introducing this gene into | ||
− | engineered bacteria S.oneidensis MR-1 and efficiently expressing nicotinamide | + | engineered bacteria <i> S.o oneidensis</i> MR-1 and efficiently expressing nicotinamide |
mononucleotide adenylyltransferase , we added a new reaction pathway for the synthesis of | mononucleotide adenylyltransferase , we added a new reaction pathway for the synthesis of | ||
− | NAD+ from NMN and NaMN in engineered bacteria S.oneidensis MR-1, shortening the | + | NAD+ from NMN and NaMN in engineered bacteria <i> S.o oneidensis</i> MR-1, shortening the |
reaction route from these two preforms to NAD+. The reaction efficiency was accelerated and | reaction route from these two preforms to NAD+. The reaction efficiency was accelerated and | ||
the accumulation of NAD+ was promoted. | the accumulation of NAD+ was promoted. | ||
<h1>Molecular cloning</h1> | <h1>Molecular cloning</h1> | ||
− | In order to construct the desired plasmids, we employed the <i>E.coli</i> TOP 10 amplification method. Firstly, we performed PCR amplification using specific primers for each plasmid, which results in the generation of linearized fragments harboring the target sequences in a high copy number. These fragments were then connected into complete plasmids using enzyme-cutting and enzyme-linking procedures. After transfer to Escherichia coli, colony PCR was used to confirm successful construction of the plasmid. Subsequently, the plasmids were further amplified to obtain sufficient quantities for further experiments. Finally, the complete plasmids were introduced into <i>E.coli</i> wm3064 and their successful integration was verified through colony PCR analysis. <i>E.coli</i> wm3064 was a good intermediate vector for conjugative transfer. We used it to conjugative transfer the target plasmid into <i>S.oneidensis</i> MR-1, which was verified by colony pcr. | + | In order to construct the desired plasmids, we employed the <i>E.coli</i> TOP 10 amplification method. Firstly, we performed PCR amplification using specific primers for each plasmid, which results in the generation of linearized fragments harboring the target sequences in a high copy number. These fragments were then connected into complete plasmids using enzyme-cutting and enzyme-linking procedures. After transfer to <i> Escherichia coli</i>, colony PCR was used to confirm successful construction of the plasmid. Subsequently, the plasmids were further amplified to obtain sufficient quantities for further experiments. Finally, the complete plasmids were introduced into <i>E.coli</i> wm3064 and their successful integration was verified through colony PCR analysis. <i>E.coli</i> wm3064 was a good intermediate vector for conjugative transfer. We used it to conjugative transfer the target plasmid into <i>S.oneidensis</i> MR-1, which was verified by colony pcr. |
<html> | <html> |
Revision as of 02:43, 12 October 2023
Ptac-nadE-nadD-nadM-rrnBT1-T7TE
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]
Description
It is a complex component composed of tac promoter, target genes nadE, nadD and nadM. This component is responsible for introducing a new de novo synthetic NAD+ pathway into engineered bacteria S.o oneidensis MR-1 and enhancing the existing common synthetic pathway, thereby increasing the intracellular NAD+ content, increasing the intracellular NADH level, promoting electron transfer, and thus improving the electrical generation capacity of engineered bacteria.
Usage and Biology
Ptac
Ptac promoter is a functional hybrid promoter commonly used in bacteria, derived from trp and lac promoters. Ptac consists of partial sequences of two promoters, which have higher binding affinity and expression activity than both of them, and can be used for the expression of exogenous genes, and is also regulated by the regulatory factors of both [1]. There is a lacIq coding region in front of the Ptac promoter sequence, which can inhibit the normal activation of the promoter in the natural state in the system, and the induction of inducers (such as IPTG) is required to remove the inhibition and thus initiate the downstream pathway expression. We got a sequence of it through corporate synthesis.
nadE
nadE is a gene encoding NH(3)-dependent NAD(+) synthetase from Escherichia coli (strainK12) . This enzyme can catalyze the nicotinic acid adenine dinucleotide (NaAD) to form NAD by consuming ATP and using ammonia as nitrogen source. This protein catalyzes the common pathway from NAMN to NAD+ and is found naturally in S.o oneidensis MR-1. By introducing exogenous nadE, we can efficiently express NH(3)-dependent NAD(+) synthetase to promote the efficient expression of this pathway and improve the synthesis efficiency of NAD+.
nadD
nadD is a gene eEscherichia colncoding nicotinamide/nicotinic acid mononucleotide adenylyltransferase from i (strain K12). This enzyme can catalyze reversible adenylation of nicotinic acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) by consuming ATP, where NaAD is a reaction precursor to catalyze the synthesis of NAD+. This protein catalyzes the common pathway from NAMN to NAD+ and is found naturally in S.o oneidensis MR-1. We introduced exogenous nadD to efficiently express nicotinamide/nicotinic acid mononucleotide adenylyltransferase and promote the efficient expression of this pathway.
nadM
nadM is a gene encoding nicotinamide/nicotinic acid mononucleotide adenylyltransferase from F. tularensis . This enzyme is a double-substrate specific enzyme that catalyzes the formation of NAD by β -nicotinamide mononucleotideNMN (NMN) and deaminated NAD by nicotinic acid mononucleotide (NaMN). By introducing this gene into engineered bacteria S.o oneidensis MR-1 and efficiently expressing nicotinamide mononucleotide adenylyltransferase , we added a new reaction pathway for the synthesis of NAD+ from NMN and NaMN in engineered bacteria S.o oneidensis MR-1, shortening the reaction route from these two preforms to NAD+. The reaction efficiency was accelerated and the accumulation of NAD+ was promoted.
Molecular cloning
In order to construct the desired plasmids, we employed the E.coli TOP 10 amplification method. Firstly, we performed PCR amplification using specific primers for each plasmid, which results in the generation of linearized fragments harboring the target sequences in a high copy number. These fragments were then connected into complete plasmids using enzyme-cutting and enzyme-linking procedures. After transfer to Escherichia coli, colony PCR was used to confirm successful construction of the plasmid. Subsequently, the plasmids were further amplified to obtain sufficient quantities for further experiments. Finally, the complete plasmids were introduced into E.coli wm3064 and their successful integration was verified through colony PCR analysis. E.coli wm3064 was a good intermediate vector for conjugative transfer. We used it to conjugative transfer the target plasmid into S.oneidensis MR-1, which was verified by colony pcr.