Part:BBa_K4595017

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. We utilized this composite element to increase the intracellular NADH concentration to modulate the electroproduction capacity of S.o oneidensis MR-1 and succeeded in making the engineering bacteria S.o oneidensis MR-1 more efficient than the wild type in electroproduction. Therefore, this component can effectively increase the intracellular NADH concentration, and can be utilized to increase the intracellular NADH concentration to improve the yield of target products if NADH is required as a cofactor for the enzymes used by other iGEM teams in the future.

Fig.1 Schematic of modular design to enhance NAD+ biosynthesis and EET rate in S. oneidensis MR-1.

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

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.
The nadE gene sequence was translated using ExPasy and was deduced to encode 275 amino acids with a molecular weight of 30636.83 Da, a theoretical isoelectric point pI of 5.41, and Pfam predicted to have 1 NAD_synthase functional structural domain. CDD predicts that it has an active-site loop, and A conformational change in the major and minor loops is required for enzyme function.From Uniprot, we know NadE has 6 binding site of deamido-NAD+, 6 ATP binding sites.Such multiple binding sites contribute to the high activity of the enzyme.

Fig.2 The protein structure prediction of NadE.

Fig.3 The onservative structural domain prediction of NadE from NCBI.

nadD

Introduction

nadD is a gene eEscherichia colncoding nicotinamide/nicotinic acid mononucleotide adenylyltransferase from E.coli (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

The nadD gene sequence was translated using ExPasy and was deduced to encode 213 amino acids, with a molecular weight of 24528 Da, a theoretical isoelectric point pI of 5.52, and a Cytidylyltransferase-like functional domain predicted by Pfam. CDD predicts that it has an Active site motif (T/H)XGH.It is involved in catalysis of nucleotidyltransfer reaction which is conserved in all members of the superfamily

Fig.4 The protein structure prediction of NadD.

We try to predict the structure of NadD, its conservative structural domain and active site.

Fig.5 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

The nadM gene sequence was translated using ExPasy, and was deduced to encode 352 amino acids, with a molecular weight of 40880.77 Da, a theoretical isoelectric point pI of 6.09, and Pfam predicted to have one functional structural domain of Cell cycle protein.

Fig.6 The protein structure prediction of NadM.

We try to predict the structure of NadM, its conservative structural domain and active site.

Fig.7 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.8 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 0.1 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.9 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.

Electricity production

Introduction

We built a microbial fuel cell device to test the electricity-producing performance of S. oneidensis MR-1.

Methods

We inoculated S. oneidensis MR-1 cultured overnight in 100 mL LB medium at 30℃ and 200 rpm to OD₆₀₀ to 0.6-0.8. The cultured cells were collected by centrifuge and re-suspended in 140 mL anoditic solution. 50 μg/mL kana antibiotics and 18mM lactate were added to maintain constant culture conditions and 0.1 mM IPTG inducer to initiate gene expression. The two electrode chambers of MFC are separated by proton exchange membrane, using carbon cloth as cathode and anode electrodes, and then adding 140mL cathode solution to the cathode chamber, using 2kΩ external resistance to close the external circuit.
After construction the MFC, we use a digital multimeter to measure the output voltage of microbial fuel cell. We measured the voltage of S. oneidensis MR-1(nadD-nadE-nadM) and compared them with the voltage of S. oneidensis MR-1. Measure a set of voltages every 6 hours.

Material and Device

Dual-chamber electrochemical devices (140 ml working volume), Nafion 117 proton exchange membranes (DuPont, USA) Carbon cloth (PHYCHEMI, Taiwan, China),VC980 + digital multimeter (VICTOR, China).

Results

Fig.10 The out put voltage of S.oneidensis MR-1, S.oneidensis MR-1(ycel-pncB), S.oneidensis MR-1(nadD-nadE-nadM) when the anoditic solution is M9 buffer and 18mM lactate.

The results showed that S. oneidensis MR-1(nadD-nadE-nadM) significantly higher discharge peak and prolonged high-efficiency discharge duration compared to the wild type. The highest out put voltage was up to 150.7 mV, with a 42.32% increase in the hightest power output .. It is speculated that this could be attributed to the ability ofS. oneidensis MR-1(nadD-nadE-nadM) to accelerate intracellular NADH synthesis, resulting in a higher power output.