Part:BBa_K2379010
Adaptor with FourU RNA thermometer
This construct consists of the adaptorBBa_K2379008(BBa_K2379008) and is under the influence of the pLac promoter(BBa_R0011). In addition to the adaptor, the RNA Thermometer BBa_K115002(BBa_K115002) is used to regulate the transcription of the genes downstream. Following the adaptor is the reporter protein GFP (BBa_E0040) and the double terminator(BBa_B0014). Since it is regulated by the FourU RNA Thermometer, the circuit will get activated and GFP will be produced only at 37 degree Celsius.
Usage and Biology
ABOUT THIS PART:
The RNA adaptor(BBa_K2379010) is a part that converts translational regulation into transcriptional regulation. We used this part to regulate gene expression at 37ºC. RNA thermometer(FourU)(BBa_K115002) is a temperature sensitive element used in the adaptor that shows higher expression at 37ºC and lower expression below 37ºC. The part works as expected and when GFP is placed downstream of this gene, maximum fluorescence was observed at 37ºC.
CHARACTERISATION STUDIES FOR THIS PART :
1.The above graph indicates the fluorescence expression at three different time periods (each recorded at an interval of 60 minutes).
2.The cell cultures were grown initially at temperature 37ºC until it reached an OD600 of 0.3. Then the cultures were transferred and incubated at three different temperatures: 32ºC, 37ºC and 42ºC.
3. Normalised fluorescence was calculated by dividing observed fluorescence by cell density at OD600, background fluoresence was minimized using a negative control.
4. Triplets were used for characterisation and the error bars indicate the Standard deviation of their mean values.
CONCLUSION:
Similar results were obtained for this part containing RNA thermometer with adaptor as obtained for RNA Thermometer (FourU) + GFP except for the fact that in this case RNA thermometer(FourU) is able to control and regulate the transcription of downstream genes with the help of adaptor element which was not possible earlier. We can infer that the adaptor is working on the following logical basis: The adaptor is working because it controlled the expression of GFP through the RNA thermometer, which is only possible had the RNA thermometer regulated thermosensitive expression at the transcriptional level and not the translational level as GFP has its own constitutive RBS. Had the adaptor not worked, even at 32 degrees celcius the construct would have given high levels of expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1068
| None |