Part:BBa_K5300045

Composite part composed of PglnK and gfp

The glnK promoter responds to low nitrogen signals, activating downstream gene transcription under low-nitrogen conditions, and becoming inactive in high-nitrogen environments. In this system, gfp serves as the reporter gene. By quantitatively measuring gfp’s expression, we can assess the ability of glnK promoter to initiate downstream gene expression under low-nitrogen conditions.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1309
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 714
  

First, we validated the glnK promoter alone and explored its expression under different nitrogen concentrations. We constructed a “promoter + fluorescent protein” system in pBBR1MCS-2 (Figure 2-1-1) to reflect the ability of the promoter based on the expression of the fluorescent protein. Since glnK promoter is regulated by nitrogen in both rhizobium and E. coli and the growth cycle of rhizobium is longer, we chose to validate the system in E. coli. The successfully constructed plasmid was introduced into E. coli (DH5α) and colony PCR was performed for verification (Figure 2-1-2).

Fig. 2-1-1 The model of promoter validation.

Fig. 2-1-2 The results of colony PCR. Positive colonies were expected at around 1.5 kb, which all ten picked colonies from the Gibson assembly turned out to be.

Subsequently, we explored the induction conditions by using M9 medium without nitrogen source as culture conditions and adding different concentrations of ammonium chloride as an additional nitrogen source. The OD600 value of the bacterial solution was determined after overnight shock incubation, and the fluorescence intensity of the bacterial solution was measured using a fluorescence spectrophotometer (Figure 2-1-3).

Fig. 2-1-3 Ratio of relative fluorescence intensity to OD600 value of bacterial solution. (a) Column diagram. (b)Broken line graph. Student's t-test, ns: no significant difference; *, p-value < 0.05; **, p-value<0.01; ***, p-value < 0.001.

Obviously, there was a significant difference between the fluorescence expression intensity under nitrogen-free conditions and that in the presence of a rich nitrogen source, successfully verifying that the glnK promoter was repressed by high nitrogen conditions.