Part:BBa_K1053008:Experience
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===Applications of BBa_K1053008===
1) E. coli TOP10 strain was co-transformed by two plasmids listed bellow,
A)
pSB1A3-taR12-DT
pSB3C5-TR(12)-HHR-GFP-DT
B)
pSB1A3-empty
pSB3C5-TR(12)-HHR-GFP-DT
C)
pSB1A3-empty
pSB3C5-empty
2) Bacterial cultures were incubated in LB medium at 140 rpm and 37℃ for 12 hour.
3) Then, cultures was diluted to an OD595 of 0.0125 and induced with or without 0.1% arabinose.
4) Cultures were incubated with shaking at 140 rpm and 37℃ for 8 hours in 1 mL of LB medium.
5) OD595 and GFP fluorescence intensity were measured.
All experiments were performed using three cultures per each sample.
The result is bellow,
A: pSB1A3-PBAD-taR12-DT and pSB3C5-PBAD-TR(12)-HHR(RBS)-GFPuv-DT
B: pSB1A3-ΔRFP and pSB3C5-PBAD-TR(12)-HHR(RBS)-GFPuv-DT
C: pSB1A3-ΔRFP and pSB3C5-ΔRFP
and show relation between fluorescence of GFP/OD595 and culture time
In the presence of taR12, GFP fluorescence value divided by OD value is higher than one in absence of taR12. This result was not expected. HHR has the ability to self-cleave, but its ability is suppressed when taRNA hybridizes to HHR. Therefore, in the presence of taRNA, self-cleavage of HHR should not happen. So then, why we get such result? One factor can be due to the exposure of RBS sequence caused by the structural change of HHR when it hybridizes to taRNA.
===User Reviews===