Regulatory

Part:BBa_K4644009

Designed by: Han Cao   Group: iGEM23_BUCT   (2023-09-29)


glnAP2-D5

        This part consists of the nitrogen-sensitive promoter glnAp2-D5 which Obtained through random mutation. When in low-nitrogen circumstance, the promoter will be activated and start fast transcription, while high-nitrogen environment will suppress the transcription.

Biology

        In E.coli K 12, the relevant elements of its nitrogen regulatory system include glnAP2, ntrB, and ntrC. By responding to changes in the concentration of glutamine, ntrB transfers the phosphorylation state to ntrC, forming a hexamer expression product. According to relevant reports, ntrC is one of glnA σ Factor, under external nitrogen deficiency conditions, induces the expression of glutamine synthase.

        By detecting the fluorescence signal intensity of mcherry, the detection threshold is characterized under low nitrogen induction with gradient ammonia concentration. At the same time, the mutant gene library is obtained by random mutation of the promoter, and the desired promoter is continuously screened and attempted to be obtained. In the future, we will also synchronously verify the indirect corresponding limit of this promoter for ammonia under the condition of overexpressing gdhA.

usage

1.glnAP2(random)

        Using the original glnAP2 promoter sequence as a template, we performed error-prone PCR amplification using the Beyotime QuickMutation™ Gene Random Mutagenesis Kit. The resulting mutant sequence library was then homologously recombined with the mCherry fluorescent protein and the pZe plasmid backbone, creating a fusion plasmid containing the entire mutation library, referred to as pZe-glnAP2(random)-mCherry.

Fig 1. PET12-glnAP2-mcherry.

Characterization

        This plasmid was transformed into Escherichia coli Nissle 1917, which already contained the integrated 50trc-gdhA gene (details in the results section), using electroporation. Single colonies were picked and cultured in deep-well plates with LB medium. Well A1 served as the control (original). The cultures were grown at 37°C and 1000 rpm for 12 hours to create a seed culture. Subsequently, the seed culture was transferred into a 96-deep-well plate with nitrogen-free medium containing 10 g/L glucose. (The seed deep-well plate was stored at 4°C for later use.) Ammonium chloride (NH4CL) was added at the appropriate concentrations (0, 150, 300, 400 mg/L), and the cultures were incubated at 37°C and 1000 rpm for 24 hours. Fluorescence intensity was measured (excitation at 580 nm, emission at 620 nm).

        Selection criteria: Groups 0 and 150 displayed normal fluorescence, while groups 300 and 450 exhibited weak fluorescence.

        For each corresponding bacterial strain, plasmid DNA was extracted, and 5 μL was reserved, while the remainder was sent to Huada Company for sequencing.

After a period of screening, BUCT obtained a sample, referred to as "B6 B7 C6 D5",this one is D5.

Fig 2. Expression intensity changes of random mutation samples with ammonium chloride concentration.

D5 Sequence map

Fig 3. D5 Sequence map.


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]


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