Part:BBa_K862000

precA-LacZ-doubleTerminator

This is a part for the precise quantification of UV-radiation or radioactive radiation in E. coli (recA+) strains, i.e. BL21(DE3). It consits of a recA Promoter (part BBa_J22106) fused to a LacZ reporter cloned in front of a double terminator (part BBa_K173004).

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]

Characterisation

This part was characterized during the 2012 HS competition by the Heidelberg_LSL team

Fig. 1: Comparison of parts BBa_K862000 (precA_LacZ) and BBa_K862001 (psulA_LacZ) via ONPG assay of Bl21(DE3) UV-irradiated for different times. Both constructs show a strong correlation between the UV-irradiation time and the LacZ activity (as measured by the production of o-nitrophenol).

Fig. 2: X-gal assay of Bl21(DE3) transformed with BBa_K862000 (precA_LacZ), BBa_K862001 (psulA_LacZ) and BBa_K862002 (precB_LacZ) and UV-irradiated for different times. All constructs show a strong positive correlation between UV induction time and coloring of the wells. PrecA-LacZ (BBa_K862000) gives the lowest reporter background expression whereas psulA-LacZ (BBa_K862001) gives the highest overall coloring of the samples.

Fig. 3: Test of precA-GFP reporter construct (part BBa_J22106 cloned into GFP backbone BBa_E0040 ) by fluorescence microscopy. E.coli transformed with our precA-GFP reporter were either UV-irradiated for 30 min or left uninduced. measurement of GFP expression show a 10-fold (!) increase in GFP expression due to UV-irradiation compared to the control.