Difference between revisions of "Part:BBa K4595017"
Line 26: Line 26:
 
<h1>Usage and Biology</h1>
 
<h1>Usage and Biology</h1>
 
<h3>Ptac</h3>
 
<h3>Ptac</h3>
 +
<h5>Introduction</h5>
 
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.
 
<h3>nadE</h3>
 
<h3>nadE</h3>
 +
<h5>Introduction</h5>
 
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 NAD by consuming ATP and using ammonia as nitrogen source. This protein catalyzes the  
 
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 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 <i> S.o oneidensis</i> 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  
 
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(+) synthetaseto promote the efficient expression of this pathway and improve the synthesis efficiency of  
 
NAD+.
 
NAD+.
−
<h5>Protein structure prediction
+
<h5>Protein structure prediction and analysis</h5>
 
<html>
 
<html>
 
<figure><center>
 
<figure><center>
Line 37: Line 39:
 
alt=""  
 
alt=""  
 
src="https://static.igem.wiki/teams/4595/wiki/parts/nade-pro.jpg"
 
src="https://static.igem.wiki/teams/4595/wiki/parts/nade-pro.jpg"
−
width="700"
+
width="400"
 
title="">
 
title="">
 
<figcaption>Fig.1  The protein structure prediction of NadE.</figcaption>
 
<figcaption>Fig.1  The protein structure prediction of NadE.</figcaption>
Line 49: Line 51:
 
width="700"
 
width="700"
 
title="">
 
title="">
−
<figcaption>Fig.1 The protein structure prediction of NadE.</figcaption>
+
<figcaption>Fig.2 The onservative structural domain prediction of NadE from NCBI.</figcaption>
 
</figure>
 
</figure>
  
  
−
<h1>nadD</h1>
+
<h3>nadD</h3>
 +
<h5>Introduction</h5>
 
nadD is a gene eEscherichia colncoding nicotinamide/nicotinic acid mononucleotide adenylyltransferase
 
nadD is a gene eEscherichia colncoding nicotinamide/nicotinic acid mononucleotide adenylyltransferase
 
from i (strain K12). This enzyme can catalyze reversible adenylation of nicotinic  
 
from i (strain K12). This enzyme can catalyze reversible adenylation of nicotinic  
Line 62: Line 65:
 
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.
−
<h1>nadM</h1>
+
<h5>Protein structure prediction and analysis</h5>
 +
<html>
 +
<figure><center>
 +
<img
 +
alt=""
 +
src="https://static.igem.wiki/teams/4595/wiki/parts/nadd pro.jpg"
 +
width="400"
 +
title="">
 +
<figcaption>Fig.3  The protein structure prediction of NadD.</figcaption>
 +
</figure>
 +
We try to predict the structure of NadD, its conservative structural domain and active site.
 +
<html>
 +
<figure><center>
 +
<img
 +
alt=""
 +
src="https://static.igem.wiki/teams/4595/wiki/parts/nadd-cd.png"
 +
width="800"
 +
title="">
 +
<figcaption>Fig.4  The onservative structural domain prediction of NadD from NCBI.</figcaption>
 +
</figure>
 +
<h3>nadM</h3>
 +
<h5>Introduction</h5>
 
nadM is a gene encoding nicotinamide/nicotinic acid mononucleotide adenylyltransferase
 
nadM is a gene encoding nicotinamide/nicotinic acid mononucleotide adenylyltransferase
 
from F. tularensis . This enzyme is a double-substrate specific enzyme that  
 
from F. tularensis . This enzyme is a double-substrate specific enzyme that  
Line 72: Line 96:
 
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.
−
 
+
<h5>Protein structure prediction and analysis</h5>
 +
<html>
 +
<figure><center>
 +
<img
 +
alt=""
 +
src="https://static.igem.wiki/teams/4595/wiki/parts/nadm-pro.jpg"
 +
width="400"
 +
title="">
 +
<figcaption>Fig.5  The protein structure prediction of NadM.</figcaption>
 +
</figure>
 +
We try to predict the structure of NadM, its conservative structural domain and active site.
 +
<html>
 +
<figure><center>
 +
<img
 +
alt=""
 +
src="https://static.igem.wiki/teams/4595/wiki/parts/nadm-cd.png"
 +
width="800"
 +
title="">
 +
<figcaption>Fig.6  The onservative structural domain prediction of NadM from NCBI.</figcaption>
 +
</figure>
 
<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 <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.
 
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.

Revision as of 08:13, 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.

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.1 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.