Part:BBa_K4644022

PZE-glnAP2-mcherry

        BUCT constructed the pZe-glnAP2-mCherry plasmid to overexpress the promoter under different external nitrogen source concentrations, observing the expression level of the fluorescent protein to determine the responsiveness of the promoter.

Biology

        In the system where Escherichia coli is used for the synthesis and mutual conversion of glutamate and glutamine, GOGAT-GS is a crucial component. Within this system, our interest was drawn to the promoter of the glutamine synthetase-encoding gene, glnA. As part of the Escherichia coli Nitrogen Regulation (NTR) system, this promoter can be activated when there is a lack of external nitrogen sources. It does so by phosphorylation through its transcription factor, ntrB, inducing the further formation of a hexameric structure with ntrC, which acts as a sigma factor for glnA, thereby enhancing the strength of this promoter.

        After gaining insights into such a component and its characteristics, BUCT conducted a review of relevant literature and found no reports on the specific binding domains for glutamine and ammonium nitrogen regarding this component. Consequently, we planned to design experiments for the characterization of the binding domains of this native component.

Fig 1. GOGAT-GS pathway.

Characterization of detection threshold

        Currently, in the characterization of promoter strength, the use of fluorescent reporter proteins is a highly effective method. Therefore, BUCT constructed the pZe-glnAP2-mCherry plasmid to overexpress the promoter under different external nitrogen source concentrations, observing the expression level of the fluorescent protein to determine the responsiveness of the promoter.

        In the characterization of the native promoter, we aimed to investigate whether this component could respond to different nitrogen source concentrations. In this induction phase, we used its direct precursor, glutamine, for induction.

        After electroporating the overexpression plasmid and performing selection, individual colonies were picked and cultured in a 96-well plate with shaking in LB medium for 12 hours. Then, the cultures were transferred to an enzyme-linked immunosorbent assay (ELISA) plate. In nitrogen-depleted medium, different concentrations of glutamine were added for induction, and continuous induction was carried out for 24 hours.

        The results are as follows:

Fig 2. Expression intensity changes of glnAp2 original promoter induced by glutamine.

Fig 3. Expression intensity changes of glnAp2 original promoter induced by ammonium chloride.

        It can be observed that when the glutamine concentration reaches 800 mg/L, there is a significant decrease in promoter strength. This phenomenon is undoubtedly encouraging, as it indicates that with the increase in glutamine concentration, once the "nitrogen deficiency" state is relieved, the promoter's strength shows a noticeable decline. This is in line with expectations and confirms that the overexpressed promoter is functioning properly, providing a premise for our modifications.

        However, we still found that if the original promoter was used, its expression intensity could not be significantly changed under the actual application concentration, so it was necessary to modify it.

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]