Part:BBa_K4595013

Ptac-ycel-pncB-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

This is a composite component composed of tac promoter, target gene ycel and pncB, which is responsible for introducing NAMN synthesis pathway with Na as a precursor into engineered bacteria S.o oneidensis MR-1, thereby increasing intracellular NAD+ content, improving intracellular NADH level and promoting electron transfer. The electricity generation capacity of engineered bacteria was improved.

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 . 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.

ycel

Our engineered bacterium S.o oneidensis MR-1 mainly relies on electron transfer of NADH for bioelectricity generation. We hope to increase the level of intracellular NADH by substantially increasing the level of NAD+, the precursor of NADH, and then converting NAD+ into NADH through EMP/PF/TCA pathway, so as to increase the level of NADH.Through data review, it was found that the synthesis of NAD+ in S.o oneidensis MR-1 was mainly divided into three modules, namely, the de novo pathway (from L-Asp or Trp to NAMN) with L-aspartic acid (LAsp) or TRP as the precursor; Nicotinamide (Nm) or nicotinic acid (Na) were used as precursors for rescue pathway (from Nm or Na to NAMN); The common part of these two biosynthetic pathways is often referred to as the universal biosynthetic pathway (from NAMN to NAD+). Accordingly, we designed multiple proteins to improve the efficiency of these modules. Since our engineered bacteria S.o oneidensis MR-1 lacked the protein to transport and absorb Na and could not complete the salvage pathway using Na as a precursor, we introduced ycel. ycel is a transmembrane protein encoding NiaP from Bacillus subtilis (strain 168), which has been suggested to be involved in niacin/niacinamide transport. The introduction of this protein can not only increase the content of intracellular Nm, promote the synthesis of NAMN with Nm as a precursor, but also increase the source of intracellular Na, providing a precursor for the engineered bacteria S.o oneidensis MR-1 to complete the pathway from Na to NAMN.

pncB

pncB is a gene sequence from Salmonella Typhimurium encoding nicotinate phosphoribosyltransferase (NAPRTase), which contains a 1,200 bp open reading frame encoding 400 residues of the protein. The protein can catalyze the synthesis of β-nicotinicacid-D-ribonucleotide from niacin and 5-phospho-D-ribose-1-phosphate by consuming ATP. By introducing pncB, our engineered bacteria added a new NAMN synthesis pathway using Na as a precursor, thereby further increasing the intracellular NAD+ content.

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-ycel-pncB-rrnBT1-T7TE transformed E.coli TOP 10

The band of Ptac-ycel-pncB-rrnBT1-T7TE from colony PCR is about 2600bp, identical to the theoretical length of 2624bp estimated by the designed primer location which could demonstrate that this target plasmid had successfully transformed into E.coli TOP 10.