Part:BBa_K1333317

I13458-I13453-FourU-RFP [[File:]]

Usage and Biology

Considering of the different transcription level provided by B2H system, we replace the constitutive promoter(BBa_J23119) in BBa_K1333309 by an inducible promoter, for using different concentration of inducer to simulate this situation. Avoiding destructing the structure of RNAT, we choose the pBAD(BBa_K1333315),induced by L-arabinose, whose operon are in the upstream of promoter.

In the 2014 distribution of biobricks, we found the parts of the promoter: arabinose C (AraC) with Promoter C (PC) (BBa_I13458) and PBad (BBa_I13453) modified by MIT in 2005, and integrated the two parts into araBAD operator (BBa_K1333315). And then, we constructed this araBAD operator before FourU element and RFP.

Figure1. The inducible mRFP expression with RNAT-fourU element under PBad.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1247
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1187
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 1873
  Illegal AgeI site found at 1985
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

Functional Parameters

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 2. 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 3. 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.