Difference between revisions of "Part:BBa K1343022"
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==IMPROVEMENT REFERENCE: Guelph 2019 == | ==IMPROVEMENT REFERENCE: Guelph 2019 == | ||
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| − | A sensitive expression assay was then conducted on the functioning [[BBa_K3189015]] by, growing multiple <i>E. coli</i> [[BBa_K3189015]] transformants in a 96-well plate in varying concentrations of tetracycline labeled in Figure 1. A dark pigment was seen at 100 ng/mL tetracycline, with less colour visible at 50 ng/mL for some of the samples, and no colour visible in the absence of tetracycline. This shows that we have improved [[BBa_1343002]] by introducing [[BBa_K3189001]] upstream to act as its new promoter sequence. | + | Our improvement was adding a tet operator to the AmilCP of this sequence. A sensitive expression assay was then conducted on the functioning [[BBa_K3189015]] by, growing multiple <i>E. coli</i> [[BBa_K3189015]] transformants in a 96-well plate in varying concentrations of tetracycline labeled in Figure 1. A dark pigment was seen at 100 ng/mL tetracycline, with less colour visible at 50 ng/mL for some of the samples, and no colour visible in the absence of tetracycline. This shows that we have improved [[BBa_1343002]] by introducing [[BBa_K3189001]] upstream to act as its new promoter sequence. |
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https://2019.igem.org/wiki/images/b/be/T--Guelph--BBa_3189015a.png | https://2019.igem.org/wiki/images/b/be/T--Guelph--BBa_3189015a.png | ||
Latest revision as of 04:02, 22 October 2019
Plux->amilCP->tetR
The promoter Plux in induced by the dimer AHL-luxR. As a result, amilCP, a blue chromophoire protein, is synthesized. TetR is a repressor of the promoter Ptet.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 64
Illegal XhoI site found at 57 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
IMPROVEMENT REFERENCE: Guelph 2019
Our improvement was adding a tet operator to the AmilCP of this sequence. A sensitive expression assay was then conducted on the functioning BBa_K3189015 by, growing multiple E. coli BBa_K3189015 transformants in a 96-well plate in varying concentrations of tetracycline labeled in Figure 1. A dark pigment was seen at 100 ng/mL tetracycline, with less colour visible at 50 ng/mL for some of the samples, and no colour visible in the absence of tetracycline. This shows that we have improved BBa_1343002 by introducing BBa_K3189001 upstream to act as its new promoter sequence.
Figure 1: E. coli BL21(DE3) BBa_K3189015 transformants from 11 different colonies grown in LB+tetracycline. The three wells in the top right hand corner are negative controls (E. coli without BBa_K3189015). A dark colour of variable intensity is visible at 100 ng/mL tetracycline for all of the samples.
The construct BBa_K3189015 containing the chromoprotein amilCP (BBa_K1343022) under the control of BBa_K3189001. When the system is induced with 100 ng/mL of tetracycline, a dark blue colour is produced (Figure 2 and Figure 3). This shows BBa_K3189001 is able to function with different reporter proteins other than just gfp.
Figure 2: BBa_K3189015 containing cells in LB broth. amilCP expression being induced using 100 ng/mL tetracycline (left) and uninduced (right).
Figure 3: Pellets of cells of BBa_K3189015 induced and uninduced with tetracycline. Pellet of cells induced with 100 ng/mL tetracycline (left) and pellet of cells uninduced (right).