Part:BBa_K3995002

Pprovoin5

Pprovoin5

BBa_K3995002

Usage and Biology

This operator region has a leftward-facing repressible promoter and a rightward-facing activated promoter. AtzR remains bound to the DNA. In the natural system, the left-facing promoter is upstream of the atzR CDS, and atzR is autoregulatory; in the presence of cyanuric acid, the binding of AtzR changes to cause the right-facing promoter to become activated, allowing for the expression of the downstream atzDEF CDS. The presence of cyanuric acid "frees" AtzR from being bound to the atzDEF promoter so polymerase can now bind and transcribe following gene. AtzR remains bound to the atzR promoter region even in the presence of cyanuric acid. Note that high glnK concentrations are also needed, as glnK is a co-activator of downstream operator.

Construct design

In our group, amilGFP is a key functional factor that show the signs of fluorescence which is controlled by a Pprovoin5 promoter (Figure 1). The Pprovoin5_amilGFP is inserted in the pUC57 mini vector (Figure 2).

Figure1. Pprovoin5_amilGFP box..

Figure2. Schematic maps of Pprovoin5_amilGFP (reporter plasmid)..

Experimental approach

Construction of recombinant plasmid The plasmid pUC57_mini and gene aiilGFP were digested by enzyme and these two fragments were connected by T4 DNA. Finally we got the recombinant plasmid pUC57-amilGFP.

Figure 3. The electrophoresis results of bacteria PCR..

Lane pUC57 -Pprovoin5-GFP-1 to 3: Bacteria PCR of monoclonals of amilGFP with size of 703bp. 1 to 3 were positive monoclonals. Extract 1 to 3 plasmids for sequencing.

Proof of function

Table 1. Fluorescence intensity when the concentration of CYA equals to 30uM and the duration is 4 hours.

Figure 4. Histogram of the fluorescence intensity when the concentration of CYA equals to 30uM and the duration is 4 hours.

As seen from figure 4, comparing to the blank control, bacteria C presents an obvious higher fluorescence reaction to the cyanuric acid, the derivative from Atrazine. In such a case, it could indicate that our engineered bacteria could work for detecting cyanuric acid.

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]