Part:BBa_K1679041

pBAD and GFP

We constructed this part with pBAD (BBa_I0500) and GFP (BBa_I13504) to measure pBAD activity at different concentrations of arabinose.

Usage and Biology

Based on our experiments, the best concentration of arabinose to induce in liquid medium is 0.002% while 2% in agar medium.

Experiments

We characterized the inducible promoter (BBa_I0500) in both liquid and agar medium.

liquid

The liquid cultures were diluted to OD600 0.0015 in LB and added to the 96-well plate together with arabinose at 7 different concentrations (0%, 0.00002%, 0.0002%, 0.002%, 0.02%, 0.2%, 2%, m/V). The absorbance at 600nm and green fluorescence (excitation: 485 nm, emission: 520 nm) were measured every 30min in a plate reader, until the E. coli cells reached stationary phase (that is, the absorbance and fluorescence no longer increase: about 8 hours from OD 0.5). Both absorbance and fluorescence were background (LB only) corrected. The fluorescence was then normalized for the number of cells by dividing by the absorbance.

Fif.1 PBAD induced by different concentrations of arabinose in liquid.

solid

The tested cultures were incubated at different concentrations of arabinose (0.002%, 0.02%, 0.2%, 2% m/V). We put 150μL (OD=0.15) liquid culture into a 90mm-diameter plate contained 20mL LB agar medium. One hour later, a piece of 1cm-diameter sterile filter paper was put in the center of each plate. 15μL Ara liquid was put on the center of the paper. Eighteen hours later, the black and white photos were taken under 362nm excitation light, another ten hours later, the colorful photos were taken by a digital camera. It showed that 2%(m/V) Ara had the best result. Each concentration had three biological replicates. And you could have an object image of the fluorescence of GFP under different concentration of Ara.

Fif.2 PBAD induced by different concentrations from 0.002% to 2% on solid medium.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1880
  Illegal SapI site found at 961