Part:BBa_K190022

Added by XHD-Wuhan-Pro-China

We first examined the efficiency of the BBa_K190022 zinc promoter. Then BBa_K190022 zinc promoter BBa_K1755001 (ribB coding sequence) was made into BBa_K4155001.

We do parallel validation for BBa_K190022 and BBa_K4155001, respectively. The experimental results show that the effect of BBa_K4155001 is significantly improved compared with BBa_K190022.

Validation of Zinc Sensitive Promoters

Figure 1

We used molecular biology tools to construct plasmids pUC19 and pETDuet-1, and useda direct DNA synthesis method to synthesize zinc responsive promoter (pzntR) and riboflavin synthesis gene (ribB).

Firstly, pzntR was linked to Green fluorescent protein(GFP), and the linked gene fragment was double digested with HindⅢ and BamHⅠ, and then inserted into plasmid pUC19. The recombinant plasmid, named pUC19-pzntR-GFP, was introduced into E.coli BL21 and expressed.

Engineered E.coli BL21 strains and wild-type E.coli BL21 strains were cultured in LB liquid medium at 37℃, and different concentrations (0, 30, 60, and 90 μM) of Zn2+ were added when OD (bacterial density value) 600 was 0.6. Three hours after the addition of Zn2+, engineered and wild-type cells induced by Zn2+ were observed under a fluorescence microscope. The 96 Well Plate Reader was used to measure GFP (excitation wavelength of GFP was 488nm, emission wavelength was 507nm).

Figure 2

The engineered bacteria containing the zinc promoter plasmid in the left panel express green fluorescence, while the wild bacteria in the right panel do not. This suggests that zinc-sensitive promoters can be primed.

Figure 3

With the increase of zinc ion concentration, the absorption intensity of wild bacteria did not change and was maintained at 10. However, with the increase of zinc ion concentration, the green fluorescence of green fluorescent protein expression has been increasing, that is, the stimulation intensity of the zinc-sensitive promoter is different.

This proves that the zinc-sensitive promoter can work.

Working System Verification

Firstly, we synthesized the riboflavin synthesis gene (ribB) by direct DNA synthesis method. The riboflavin synthesis gene for riboflavin expression promotes electron transfer from the cell to the electrode and then produces current voltage changes.

Figure 4

Firstly, pzntR was ligated with ribB, and the ligated gene fragment was double digested with BamHⅠ and EcoRⅠ, and then inserted into plasmid pETDuet-1. The recombinant plasmid, named pETDuet-1pzntR-ribB, was transferred to Escherichia coli BL21 for expression.

We set up a two-compartment MFC-operated reactor with a working volume of 240 mL in each chamber, and the electrodes were pretreated before use. Carbon felt with an area of 16cm2 is used as an anode and cathode. These electrodes are connected via titanium wires to a 1000-ω external resistor.

In the MFC operating system, the anodic medium was supplemented with different concentrations (0-500μM) of Zn2+ in an M9 liquid medium for use by the Zn2+ response regulator. The cathode solution was potassium ferricyanide (100 mM ferricyanide in 50 mM phosphate buffer, pH 7.0), and the voltage was recorded at 10-min intervals in an MFC biosensor using a data acquisition device.

Figure 5

There is a significant linear relationship between zinc ion concentration and the maximum voltage of the constructed MFC biosensor.

The maximum voltage t-test of engineered bacteria and wild bacteria at 500μM zinc ion concentration showed that P < 0.001, indicating that riboflavin synthesized by ribB riboflavin synthesis gene could significantly promote electron transfer. This proves that our system works properly.

Zinc Promoter (ZntR regulated) with own RBS

The pZntR from E.coli K.12 has a specific RBS site behind it in the genome. Here the RBS site is attachted to the promoter region. The RBS site might influence the activity of the promoter and will be tested in the same way as BBa_K190016. ZntR activates transcription when Zn(II) is bound (1).

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]

1) C.E. Outten, F.W. Outten, T.V. O’Halloran (1999) DNA Distortion Mechanism for Transcriptional Activation by ZntR, a Zn(II)-responsive MerR Homologue in Escherichia coli, The Journal of Biological Chemistry, Vol. 274, No. 53, Issue of December 31, pp. 37517–37524.