Part:BBa_K1840007
Device: TT-zwf-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 promotor zwf is influenced by it. The zwf promotor (BBa_K1840002) is contained in the genome of Pseudomonas putida and is situated in front of the enzyme zwf (glucose 6-phosphate dehydrogenase). The repressor HexR can bind the zwf promotor sequence and thus prevent the transcription of the zwf gene. In case the glucose derivative 2-Keto-3-deoxy-6-phosphogluconate is present, it binds to the promotor and leads to a subsequent conformational change. Hence, the repressor can no longer bind and the gene(s) 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.
1. Fluorescence after incubation in M9 minimal media and 5%, 10% or 20% glucose
Three biological replicates of zwf 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 zwf device display a significant increase in mCherry expression with increasing concentration of glucose in the media. The increase is approximately linear.
In comparison with other devices that we investigated in out project, the zwf shows a median fluorescence at 5% and 10% glucose in the media, and a relatively high fluorescence at 20% glucose in the media. Note: the other devices shown are BBa_K1840000, BBa_K1840001, and BBa_K1840003.
2. Fluorescence after incubation in LB media and 5%, 10% or 20% glucose
Three biological replicates of zwf transformed cells have been inoculated in LB media with 5%, 10% or 20% of glucose. After 12 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.
As it was when growing in M9 media with glucose, the expression of mCherry increases with increasing glucose concentration in the media. However, the absolute fluorescence values are much smaller. This is expected since other carbohydrate sources are available in LB media.
3. Fluorescence of zwf over time
Cells containing the zwf device were inoculated in M9 media with 1%, 5%, 10% or 20% glucose. After 6 hours of 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) every 30 minutes for 6 hours.
The figure shows the fluorescence standardize to OD (600nm).
As it should be for a glucose sensor that works in the same way all the time, the fluorescence of zwf does not change significantly in the samples that were incubated in 1% and 5% glucose, respectively. However, the fluorescence of the cells in 10% glucose decrease slightly, whereas the cells incubated in 20% increase. Hence, it should be considered in which range the glucose detection system is supposed to work.
Note: This experiment has only been performed with one biological replicate and the result needs to be further investigated.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 130
- 1000COMPATIBLE WITH RFC[1000]
//promoter
//terminator/double
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