<figcaption>Fig.6 The onservative structural domain prediction of NadM from NCBI.</figcaption>
<figcaption>Fig.6 The onservative structural domain prediction of NadM from NCBI.</figcaption>
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<h1>Molecular cloning</h1>
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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.
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<h1>Part characterisation</h1>
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<h3>Molecular cloning</h3>
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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.
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<figcaption>Fig.1 Colony PCR result of Ptac-nadE-nadD-nadM-rrnBT1-T7TE transformed <i>E.coli</i> TOP 10<br><br>The band of Ptac-nadE-nadD-nadM-rrnBT1-T7TE from colony PCR is about 2800bp, identical to the theoretical length of 2820bp estimated by the designed primer location which could demonstrate that this target plasmid had successfully transformed into <i>E.coli</i> TOP 10.</figcaption>
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<figcaption>Fig.7 Colony PCR result of Ptac-nadE-nadD-nadM-rrnBT1-T7TE transformed <i>E.coli</i> TOP 10<br><br>The band of Ptac-nadE-nadD-nadM-rrnBT1-T7TE from colony PCR is about 2800bp, identical to the theoretical length of 2820bp estimated by the designed primer location which could demonstrate that this target plasmid had successfully transformed into <i>E.coli</i> TOP 10.</figcaption>
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<h3>NADH production
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<h5>Introduction</h5>
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To verify normal gene expression in <i>S. oneidensis</i> MR-1, we measured the NADH/NAD+ content of wild type and engineered strains ycel-pncB and nadD-nadE-nadM, respectively.
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<h5>Methods </h5>
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We use NAD+/NADH Assay Kit with WST-8 from Beyotime to detect the concentration of NAD(H/+) in <i>S. oneidensis</i> MR-1. We inoculated 1 mL<i>S. oneidensis</i> MR-1 cultured overnight in 100 mL LB medium at 30℃ and 200 rpm. When OD<sub>600</sub> get to 0.6-0.8,add 0.5 mM IPTG to induce the protein expression and culture 24 hour.Remove 4mL of the bacterial solution and measure NAD(H/+) concentration according to the kit instructions.
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<br>Material and Device: NAD+/NADH Assay Kit with WST-8 from Beyotime,Tecan Infinite® 200 PRO.
<figcaption>Fig.8 The NAD(H/+) concentration of <i>S. oneidensis</i> MR-1, <i>S. oneidensis</i> MR-1(ycel-pncB), <i>S. oneidensis</i> MR-1(nadE-nadD-nadM).</figcaption>
</figure>
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We Compared to the wild type, the total amount of NAD(H/+) in <i>S. oneidensis</i> MR-1(nadD-nadE-nadM) increased by 27.34%. This indicates that, <i>S. oneidensis</i> MR-1(nadD-nadE-nadM) facilitates more efficient electron transfer.
Revision as of 08:34, 12 October 2023
Ptac-nadE-nadD-nadM-rrnBT1-T7TE
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]
Description
It is a complex component composed of tac promoter, nadE, nadD and nadM. RBS have added at the start of nadD, nadE 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
Introduction
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
Introduction
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 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(+) synthetaseto promote the efficient expression of this pathway and improve the synthesis efficiency of
NAD+.
Protein structure prediction and analysis
Fig.1 The protein structure prediction of NadE.
Protein structure prediction, as a research method, has extensive applications and significant importance. Firstly, this method can help scientists explore the relationship between protein structure and function, and further understand the important role of proteins in life processes. Specifically, the structural features of a protein affect its biological activity and interactions, and the two are directly related. Therefore, through protein structure prediction, we can predict the structure of proteins, determine their biological functions, predict the interaction modes between different components, and more accurately explain experimental phenomena, providing a reliable basis for experimental research and development. We try to predict the structure of NadE, its conservative structural domain and active site.
Fig.2 The onservative structural domain prediction of NadE from NCBI.
nadD
Introduction
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.
Protein structure prediction and analysis
Fig.3 The protein structure prediction of NadD.
We try to predict the structure of NadD, its conservative structural domain and active site.
Fig.4 The onservative structural domain prediction of NadD from NCBI.
nadM
Introduction
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.
Protein structure prediction and analysis
Fig.5 The protein structure prediction of NadM.
We try to predict the structure of NadM, its conservative structural domain and active site.
Fig.6 The onservative structural domain prediction of NadM from NCBI.
Part characterisation
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.
Fig.7 Colony PCR result of Ptac-nadE-nadD-nadM-rrnBT1-T7TE transformed E.coli TOP 10
The band of Ptac-nadE-nadD-nadM-rrnBT1-T7TE from colony PCR is about 2800bp, identical to the theoretical length of 2820bp estimated by the designed primer location which could demonstrate that this target plasmid had successfully transformed into E.coli TOP 10.
NADH production
Introduction
To verify normal gene expression in S. oneidensis MR-1, we measured the NADH/NAD+ content of wild type and engineered strains ycel-pncB and nadD-nadE-nadM, respectively.
Methods
We use NAD+/NADH Assay Kit with WST-8 from Beyotime to detect the concentration of NAD(H/+) in S. oneidensis MR-1. We inoculated 1 mLS. oneidensis MR-1 cultured overnight in 100 mL LB medium at 30℃ and 200 rpm. When OD600 get to 0.6-0.8,add 0.5 mM IPTG to induce the protein expression and culture 24 hour.Remove 4mL of the bacterial solution and measure NAD(H/+) concentration according to the kit instructions.
Material and Device: NAD+/NADH Assay Kit with WST-8 from Beyotime,Tecan Infinite® 200 PRO.
Results
Fig.8 The NAD(H/+) concentration of S. oneidensis MR-1, S. oneidensis MR-1(ycel-pncB), S. oneidensis MR-1(nadE-nadD-nadM).
We Compared to the wild type, the total amount of NAD(H/+) in S. oneidensis MR-1(nadD-nadE-nadM) increased by 27.34%. This indicates that, S. oneidensis MR-1(nadD-nadE-nadM) facilitates more efficient electron transfer.
