Part:BBa_K1840008

Device: TT-gad-mCherry

This device acts as a detector for glucose (and certain derivatives of it). The double transcription terminator ensures, that only the gene downstream of the gad promotor is influenced by it. The promotor (BBa_K1840003) is contained in the genome of Pseudomonas putida and is situated in front of the enzyme gad (gluconate oxidase). The repressor PtxS can bind the gad promotor sequence and thus prevent the transcription of the gad gene. In case the glucose derivative 2-ketogluconate is present, it binds the promotor and leads to a subsequent conformational change. Hence, the repressor can no longer bind and the gene downstream can be transcribed. In our device, the downstream gene is mCherry, codon optimized for Pseudomonas (BBa_K1840004). Therefore, in the presence of glucose, mCherry is expressed. In various experiments, we could see that this device is in deed working.

Applications of BBa_K1840008

This part can be used as a glucose sensor. It expresses mCherry according to the level of glucose in the environment. Please see the following experiments and their results for characterization:

1. Fluorescence after incubation in M9 minimal media and 5%, 10% or 20% glucose

Three biological replicates of gad transformed cells have been inoculated in M9 minimal media with 5%, 10% or 20% of glucose. After 16 hours incubation, 100ul of each sample was transferred to a black 96 well plate with transparent bottom. The plate reader measured the fluorescence (excitation:584nm, emission:620nm) and the OD (600nm). Three technical replicates were measured. Following figure shows the average of all technical and biological replicates with standard deviation as error bar. Cells containing the gad-device displayed and increasing expression of mCherry with increasing glucose concentration in the environment. Even though the tendency line was calculated for a linear correlation, it might be possible that the correlation between glucose concentration and mCherry is different, for example exponentially. Further measurement with concentrations in between have to be done to confirm that theory.

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 325
  
• 1000
  COMPATIBLE WITH RFC[1000]