Project

Part:BBa_K3972004

Designed by: Ingrid Kolen, Daniek Hoorn, Werner Doensen   Group: iGEM21_TU-Eindhoven   (2021-09-20)
Revision as of 11:58, 15 October 2021 by Ingrid Kolen (Talk | contribs) (→‎Characterization)


Tetrathionate sensor with transferred TtrR and pTtrB185-269 activated ARG1

This composite part is a combination of the parts BBa_K3972001 and BBa_K3972002, with additional functional parts BBa_R0040, BBa_K3332034 and BBa_K2507019. The ARG1 proteins are controlled by the pTtrB185-269 promoter. This promoter is activated by TtrR in the presence of TtrS and tetrathionate. In the presence of tetrathionate, TtrS is activated and in its turn TtrS activates TtrR. TtrR binds to the pTtrB185-269 promoter and induces expression of the ARG1 gas vesicle proteins

T—TU Eindhoven--designA1.png

Figure 1. The mechanism of the composite part tetrathionate sensor with transferred TtrR and pTtrB activated ARG1.


Usage and Biology

This part is optimized for expression of ARG1 in E. coli cells. This part was successfully transformed after multiple attempts in XL10-Gold and Top10 cells. The cotransformation together with TtrS did also result in colonies on plates with BL21 (DE3), Top10 and XL10-Gold cells. Unfortunately, there was no time left for testing this part with ultrasound equipment.


Characterization

Expression
This part is optimized for expression of ARG1 in E. coli cells. This part was successfully transformed after multiple attempts in XL10-Gold and Top10 cells. The cotransformation together with TtrS did also result in colonies on plates with BL21 (DE3), Top10 and XL10-Gold cells. Unfortunately, there was no time left for testing this part with ultrasound equipment.


T--TU-Eindhoven--combiB ARG.jpeg

Figure 2. Agar plate with transfected ARG1 with the insert in XL10-Gold cells.


T—TU Eindhoven--combiB.png

Figure 3. Agar plate with no cotransfected ARG1, TtrR and TtrS in BL21 (DE3).

Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1658
    Illegal EcoRI site found at 4811
    Illegal XbaI site found at 1409
    Illegal PstI site found at 404
    Illegal PstI site found at 4668
    Illegal PstI site found at 4773
    Illegal PstI site found at 5151
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1658
    Illegal EcoRI site found at 4811
    Illegal NheI site found at 1543
    Illegal PstI site found at 404
    Illegal PstI site found at 4668
    Illegal PstI site found at 4773
    Illegal PstI site found at 5151
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1658
    Illegal EcoRI site found at 4811
    Illegal BglII site found at 2162
    Illegal BamHI site found at 5567
    Illegal XhoI site found at 5255
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1658
    Illegal EcoRI site found at 4811
    Illegal XbaI site found at 1409
    Illegal PstI site found at 404
    Illegal PstI site found at 4668
    Illegal PstI site found at 4773
    Illegal PstI site found at 5151
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1658
    Illegal EcoRI site found at 4811
    Illegal XbaI site found at 1409
    Illegal PstI site found at 404
    Illegal PstI site found at 4668
    Illegal PstI site found at 4773
    Illegal PstI site found at 5151
    Illegal NgoMIV site found at 2613
    Illegal NgoMIV site found at 3015
    Illegal AgeI site found at 334
    Illegal AgeI site found at 5534
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None