Part:BBa_K3189005
Tet-Regulated VioC
This composite part is a minimal package of DNA that contains all the essential components necessary to have VioC from the violacein pathway under the control of TetO. When tetracycline is present it allows for the transcription of VioC.
E. coli cells already containing BBa_K3189013 were transformed with BBa_K3189005 were grown on LB+Ampicillin at 37°C for 24 hours at which point they were moved to 4°C for 48 hours. Figure 1a is a raw picture of the plate with faint colours able to be seen. To more clearly distinguish the pigments being produced, Figure 1b has had the saturation increased, brightness decreased, and contrast increased. This image more clearly shows the pigmentation of the colonies ranging from green to pink. It can also be seen that the colour of the colonies is dependent on cell density with the more isolated colonies being green and the more closely associated ones being pink. Although all colonies are not pink, this image still shows a working system that has another influence from cell density affecting expression.
Figure 1: Colonies transformed with BBa_K3189013 and BBa_K3189005 plated on LB+Ampicillin showing a gradient of pigment colour correlated to colony density. (A) Shows the raw image of the transformation plate and (B) shows the same image with the contrast and saturation increased along with a decrease in brightness to highlight the gradient of pigment on the plate. The colour gradient bar in the center emphasizes the pigment change thought out the pate and the colony density bar shows the high to low density transition on the plate.
From cells that contained BBa_K3189013 and BBa_K3189005, colonies were resuspended in PBS and spotted onto LB agar containing 100 μg/mL ampicillin, 100 μg/mL tryptophan, 1mM IPTG, and 50 ng/mL tetracycline. After 1 week at room temperature, colonies produced a dark, almost black pigment seen in Figure 2, that was impossible to discern with the naked eye. What is interesting to note is that the colonies produced both green and pink halos around them. The colour enhanced colonies in Figure 2c show green halos pointed towards the outside of the plate when the part of the colony facing the inside produced a pink halo. The colony in the center of the plate however, only produced a pink halo in every direction. Since the interior of the plate is more populated than the outside, this lends more evidence to the density-dependent pigmentation seen in previous tests documented on our wiki.
Figure 2: Colonies containing BBa_K3189013 and BBa_K3189005 plated on LB agar containing 100 μg/mL ampicillin, 100 μg/mL tryptophan, 1mM IPTG, and 50 ng/mL tetracycline. (A) Shows the raw image of the transformation plate and (B) shows the same image with the contrast and saturation increased. (C) Shows the pigment front in relation to the other colonies on the plates with the lower density sides appearing green and the higher density sides appearing pink.
These results presented here show a working biobrick that is capable of producing a pink pigment in the presence of tetracycline. It is also clear from these results that some other mechanism is influencing the production of VioC. Further characterization of the influence of cell density is needed.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1520
Illegal NheI site found at 2250 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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