Revision as of 01:40, 12 October 2023

Ptac-nadE-nadD-nadM-rrnBT1-T7TE

Sequence and Features

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.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.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 encoding nicotinamide/nicotinic acid mononucleotide adenylyltransferase from Escherichia 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.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.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.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.