Part:BBa_K5300044

PnuoA-Cas12k-double Terminator-PglnK-sgRNA-GFP-tracRNA-PnifH-gfp

To verify the function of the regulatory module, we attempted to construct a plasmid with PnuoA-Cas12k-Double terminator added in front of the regulatory module.

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 4445
  Illegal AgeI site found at 5
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3850
  

Plasmid construction and validation

We first amplified the glnK promoter and nifH promoter from the genome of Sinorhizobium fredii CCBAU45436, amplified gfp and designed sgRNAs from pRJPaph-bjGFP plasmid, and ligated them using overlap PCR. To verify the effect of the regulation module alone, we connected it with the nuoA promoter-Cas12k and the linearized pBBR1MCS-2 (Figure 2-2-1). The recombinant plasmid was transformed into E. coli (DH5α).

Fig. 2-2-1 The model of validation of regulation circuit.

Triparental mating and validation

The verified correct colonies were inoculated into kanamycin-resistant LB liquid medium under shaking condition. Then we conducted triparental mating using the correct colony we obtained above, Helper and Sinorhizobium fredii CCBAU45436. Then colony PCR was performed on colonies grown on TY/NA/Kan solid medium incubated at 28°C for 24 h using the universal primer M13F/R.

Fluorescence intensity verification

We utilized its fluorescence expression intensity to verify whether the regulation module functioned properly. We set up four sets of experiment with high nitrogen and high oxygen, low nitrogen and high oxygen, high nitrogen and low oxygen, as well as low nitrogen and low oxygen for verification. Subsequently, we used M9 medium without nitrogen source, then added different concentrations of ammonium chloride to form nitrogen concentration gradient. We added petroleum jelly after boiling the medium to isolate medium against atmosphere, creating anaerobic conditions. The OD600 value of the bacterial solution was determined after a period of shaking and incubation, and the fluorescence intensity of the bacterial solution was determined using a fluorescence spectrophotometer (Figure 2-4-1 and Figure 2-4-2).

Fig. 2-4-1 Ratio of relative fluorescence intensity to OD value of bacterial solution. (a) Different oxygen conditions at low nitrogen (0 g/L ammonium chloride). (b) Different oxygen conditions at high nitrogen (0.02 g/L ammonium chloride). (c) Different conditions of nitrogen concentration during hypoxia. (d) Different conditions of nitrogen concentration during hyperoxia. Student's t-test, ns: no significant difference; *, p-value < 0.05; **, p-value<0.01; ***, p-value < 0.001.

Fig. 2-4-2 Ratio of relative fluorescence intensity to OD600 value of bacterial solution.Student's t-test, ns: no significant difference; *, p-value < 0.05; **, p-value<0.01; ***, p-value < 0.001.

We were able to clearly see that high oxygen conditions always inhibit the expression of fluorescent proteins, whether under high or low nitrogen conditions. Low nitrogen conditions also inhibit the expression of fluorescent proteins, whether under high or low oxygen conditions. This is consistent with our assumptions. Under high oxygen conditions, the nifH promoter is repressed, which reduces the expression of downstream genes, thereby the fluorescence intensity is reduced. Under low nitrogen conditions, the glnK promoter is promoted, and the expression of sgRNA is up-regulated, directing the Cas12k protein to repress the expression of fluorescent proteins. However, both low-nitrogen and high-oxygen conditions induced high expression, which is hypothesized to be the result of uninhibited constitutive expression of the promoter, as well as the possibility of leaky expression.