Reporter

Part:BBa_K3189015

Designed by: Jehoshua Sharma, Nathanael Wilms, Connor Gianetto-Hill   Group: iGEM19_Guelph   (2019-10-16)


Tet Controlled amilCP

Tet Controlled amilCP

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1758
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 146
    Illegal XhoI site found at 139
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 80


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.
T--Guelph--BBa_3189015a.png
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.

216px-T--Guelph--pTA-tetnotet.jpg
Figure 2: BBa_K3189015 containing cells in LB broth. amilCP expression being induced using 100 ng/mL tetracycline (left) and uninduced (right).

207px-T--Guelph--pTAtetnotetspun.jpg
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).

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