Project

Part:BBa_K3972003

Designed by: Ingrid Kolen, Daniek Hoorn, Werner Doensen   Group: iGEM21_TU-Eindhoven   (2021-09-20)
Revision as of 11:08, 15 October 2021 by Ingrid Kolen (Talk | contribs)


Tetrathionate sensor with activated pTtrB185-269-ARG1

This composite part is a combination of the parts BBa_K2507019 and BBa_K3972002. The ARG1 gene is controlled by the PttrB185-269 promoter. This promoter is activated by TtrR in the presence of TtrS and tetrathionate.TtrS will be activated when tetrathionate binds and in its turn, TtrS activates TtrR. TtrR binds to the PttrB185-269 promoter and induces expression of the ARG1 gas vesicle proteins.

File:T—TU Eindhoven-- .png Figure 1. The composite part.


Usage and Biology

This composite part can be used to combine the two component TtrS/R system with the ARG1 gas vesicles as output. For visualization of this part, an ultrasound can be used to detect the gas vesicles, as described in BBa_K3972002.

Characterization

Expression

This part is optimized for expression of ARG1 in E. coli cells. Unfortunately, this part was not successfully transformed into BL21 (DE3) cells. While this part was successfully transformed in XL10-Gold cells, the co-transformation with the TtrR and TtrS plasmids in BL21(DE3) did not work. After multiple attempts with BL21(DE3) cells, there were still no colonies on the agar plates. The most probable explanation for the failed transformation is because two of the plasmids contain origins of replications from the same family. This will lead to unstable replication of the bacteria by the competition between the two plasmids. [1]


T--TU-Eindhoven--combiA plate.jpeg Figure 2. Agar plate with transfected ARG1 with the insert in XL10-Gold cells.


File:T—TU Eindhoven-- .png Figure 3. Agar plate with no cotransfected ARG1, TtrR and TtrS in BL21 (DE3).

References

[1] Morgan, K. (2014). Plasmid 101: Origin of Replication. Retrieved from: https://blog.addgene.org/plasmid-101-origin-of-replication


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 942
    Illegal EcoRI site found at 4095
    Illegal XbaI site found at 693
    Illegal PstI site found at 3952
    Illegal PstI site found at 4057
    Illegal PstI site found at 4435
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 942
    Illegal EcoRI site found at 4095
    Illegal NheI site found at 827
    Illegal PstI site found at 3952
    Illegal PstI site found at 4057
    Illegal PstI site found at 4435
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 942
    Illegal EcoRI site found at 4095
    Illegal BglII site found at 1446
    Illegal BamHI site found at 4851
    Illegal XhoI site found at 4539
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 942
    Illegal EcoRI site found at 4095
    Illegal XbaI site found at 693
    Illegal PstI site found at 3952
    Illegal PstI site found at 4057
    Illegal PstI site found at 4435
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 942
    Illegal EcoRI site found at 4095
    Illegal XbaI site found at 693
    Illegal PstI site found at 3952
    Illegal PstI site found at 4057
    Illegal PstI site found at 4435
    Illegal NgoMIV site found at 1897
    Illegal NgoMIV site found at 2299
    Illegal AgeI site found at 4818
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
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