Part:BBa_K1804001

GFP under the control of constitutive promoter lacI

Green fluorescence protein (GFP) from Aequeora victoria was placed under the control of the constitutive promoter lacI (plac).

Usage and Biology

plac promoter is constitutively active in E. coli BL21

The plac promoter is constitutively active in our strain of E. coli BL21. After construction of the pSB1C3-plac-gfp vector, the activity of the plac promoter was measured by quantification of fluorescence intensity. BL21 was used as the negative control and BL21 transformed with pAC-plac-gfp [1] as the positive control. As seen in Figures 1 and 2, GFP is expressed in both strains of BL21 containing the plac-gfp vectors but not in the negative control. When fluorescence data was quantified in arbitrary units (A.U.), the intensity of plac-gfp in pSB1C3 was lower relative to that of plac-gfp in the pAC vector (Figure 2). Nonetheless, our results indicated that plac is indeed able to drive gene expression under normal growth conditions. Supplementary Table 1 shows the descriptive statistics of the fluorescence intensities determined.

Figure 1: Representative images of bacterial cultures were taken using a confocal laser scanning microscope at 488 nm, in duplicates A. BL21 pAC-plac-gfp, positive control. B. BL21 pSB1C3-plac-gfp. C. BL21, negative control.

Figure 2: GFP is expressed in BL21 transformed with pAC-plac-gfp and pSB1C3-plac-gfp. Data is represented in Mean +/- Standard Deviation of GFP fluorescence intensity (A.U.) for the positive control of BL21 pAC-plac-gfp, BL21 pSB1C3-plac-gfp and the negative control BL21. The experiment was carried out in duplicates (n=15).

Please refer to our Materials and Methods page [http://2015.igem.org/Team:SPSingapore/Anaerobic_Promoter] for details on fluorescence intensity quantification. Our experimental procedures can be found at our Notebook page [http://2015.igem.org/Team:SPSingapore/Notebook].

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 734