Part:BBa_K1819008

Promoter test circuit to analyze the kill switch efficiency

BBa_K1819008 construction indicates lactose promoter (pLac) efficiency to control a TetR promoter inhibited by TetR protein. This circuit was used to verify a kill switch mechanism (See more details in Brasil-USP team wiki)

In the absence of pLac inductor (Lactose or IPTG), RFP will be constitutively expressed under TetR promoter control. If inducer is added, TetR promoter is repressed and, consequently, RFP expression ceases and GFP expression starts.

Characterization

DH5-alpha

    We first started with DH5α. For protocols, we refer to our Protocols section. We evaluated the fluorescence per cell by measuring Optical Density (OD) of our colonies and their fluorescences. We show in figures 1 and 2 our results.

Figure 1 - Optical Density (OD) measurements over time, showing that all colonies were in their log phase.

Figure 2 - Fluorescence per cell for each promoter, normalized by the fluorescence with no induction. In the x-axis, the higher the concentration label, the higher the concentration, with zero means no induction at all. Only Rhamnose showed some actual induction. All errors may be considered about 0.05 a.u.

    Except for Rhamose, fluorescence was very weak and basically at the level of our negative control (E. Coli without plasmid), confirmed by a Mann-Whitney U test. Rhamnose showed some level of activity which would be considered weak, compared to, for instance, our Interlab Results. Finally, no considerable changes were detected in the RFP measures. Since there results are not good indicatives, we performed this experiment three different times (to reduce possible experimental artifacts with assembling or other steps in our protocol). Yet we have always observed the same behavior consistently.

BL21 DE3

    We performed the same tests in BL21 strain. For this test, Arabinose was not used as we couldn't confirm its transformation in our gels. For protocols, we refer to our Protocols section. Again, our colonies were all in log phase (omitted) when we measured their fluorescence over time. Our results are in figure 3 and 4.

Figure 3 - Fluorescence per cell for pLac, normalized by the fluorescence with no induction. We see that with IPTG, GFP activity incresases and RFP activity decreases, as it should happen.

Figure 4 - Fluorescence per cell for each promoter, normalized by the fluorescence with no induction. Not only the induced activity is lower than the activity without induction - for the first 3 concentrations of Rhamnose, but also the RFP is also growing.

Discussion

    We successfully expressed and induced promoters pRha in DH5α strain and pLac in BL21 strain. Mann-Whitney U tests confirmed induction, i.e., induced fluorescence per cell was (statistically) significantly higher than our negative controls. Yet, the other promoters weren't successfully expressed or induced, and there are definitely several possible factors to explain it. Since we had no issues at all with any of our constitutive promoters (see for instance our Interlab Study, Pveg characterization in E. Coli, Linker-GFP part), our primary concern is that our protocols should be modified and optimized for induced promoters.

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 AgeI site found at 2437
  Illegal AgeI site found at 2549
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 870