Part:BBa_K1053006:Experience
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===Applications of BBa_K1053006===
We examined whether TR-HHR-taRNA’s output can be repressed by corresponding taRNA.
1) The plasmids as shown below was used to transform E. coli Top10.
A)
pSB1A3-PBAD-TR(42)HHR-taR12-DT
pSB3C5-crR12-GFP
B)
pSB1A3-Pconst(H)-taR42-DT-PBAD-TR(42)HHR-taR12-DT
pSB3C5-crR12-GFP-DT
C)
pSB1A3-PBAD-taR12-DT
pSB3C5-crR12-GFP
D)
pSB1A3-ΔRFP
pSB3C5-crR12-GFP
E)
pSB1A3-ΔRFP
pSB3C5-ΔRFP
2) The transformants were pre-cultured in LB medium at 37 degrees celcius for 8 hours.
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.
The result is bellow,
Result and Discussion
In order to evaluate a function of TR(42)-HHR-taR12, we used crR12-GFP as a reporter.
taRNA is complementary to the sequence of crRNA, and by the binding of taRNA to crRNA, RBS is exposed and gene expression is activated. In this result, the GFP fluorescence value divided by OD value of B was lower than that of A. It shows that taR42 suppressed self-cleavage of TR(42)-HHR and caused less GFP expression. Therefore, we prove that the self-cleavage activity of TR(42)-HHR and amount of taR12 can be controlled by taR42.
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