Project

Part:BBa_K1728019

Designed by: Yung-Li Chen, Katy Chang, Timothy Chan En Haw   Group: iGEM15_CGU_Taiwan   (2015-09-17)
Revision as of 01:25, 19 September 2015 by Timothykoala (Talk | contribs) (Usage and Biology)

SAT toehold switch RNA sensor with T7 promoter & luciferase reporter

SAT toehold switch RNA sensor with T7 promoter & luciferase reporter

Usage and Biology

Toehold switch is a secondary structure of RNA. Because of special sequence design, it can form a hairpin structure and hide ribosome binding site (RBS)in the loop. There are three basic elements of toehold switches including 30bp trigger sequence(GCACCTAACCGTTCAATAACATAGAACTCC), bacteria ribosome binding site and linker.The trigger sequence of this part is complementary to a part of Signal Transducer and Activator of Transcription (SAT) mRNA(SAT partial sequence, BBa_K1728007). Once the SAT mRNA is presented, the toehold structure will open. Therefore, the ribosome is able to bind on the RBS and translate the down stream gene. In order to highly transcribe, we add a T7 promoter sequence in front of the toehold switch sequence(SAT toehold switch RNA sensor, BBa_K1728003). This part, we use luciferase(Firefly luciferase - luciferase from Photinus pyralis, BBa_I712019)as our down stream gene and even a highly sensitivity reporter.

In Vivo Assay

CGU_Taiwan_SAT_in_vivo.png

We lysed the bacteria to get the lysate. There are two groups, one is toehold switch only; the other is toehold switch treated with trigger, which is made by cotransformation of the two constructed plasmids: BBa_K1728019 and BBa_K1728011. The two plasmids can be selected for that they were constructed with different backbones which contains different antibiotics resistance. Thus, the bacteria can survive the environment with ampicillin and chloramphenicol antibiotics added if the two plasmids are successfully cotransformed.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 922


[edit]
Categories
//chassis/prokaryote/ecoli
Parameters
None