Part:BBa_K1333317:Experience

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Applications of BBa_K1333317

We use 50000μM L-arabinose, the highest concentration, to test inducible mRFP expression with RNAT-fourU element under araBAD promoter.The results are as follows.

Figure 1. A) The groups of bacteria containing 4 plasmid: Negative control, Pbad-RBS-GFP, Pbad-FourU-RFP(induced by 0μM L-arabinose) and Pbad-FourU-RFP(induced by 50000μM L-arabinose) after centrifugation. B) After cultured for 24h, the expression of RFP protein regulated by FourU element at 37°C is dramatically higher than the one at 30°C; C) After cultured for 24h, there is no significant difference of the expression of GFP protein regulated by normal RBS at 37°C and 30°C.

Figure1 shows that temperature almost has no impact on the expression level of reporter protein regulated by normal RBS, however, to the experimental group regulated by the FourU element, the expression quantity of reporter protein cultured at 30°C induced by 50000μM L-arabinose is dramatically lower than the group cultured at 37°C, and there is no detectable fluorescence intensity of the reporter protein without any L-arabinose. The results show that the araBAD promoter has litte leaked expression and works well with the FourU element.

Then we test the response of the inducible plasmid to the temperature changes from 30°C to 37°C. We cultured both the positive control and experimental bacteria at 30°C overnight and changed them to 37°C, and then test the fluoresce intensity of reporter protein after 1h, 2h and 3h. The result are as follows.

Figure 2. A) Fluorescence intensity changes of RFP protein regulated by the FourU element after shifted from 30°C to 37°C for 5h. B) Fluorescence intensity changes of GFP protein regulated by the normal RBS after shifted from 30°C to 37°C for 5h.

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