Part:BBa_K1806005
T7+ ibPB RNA Thermometer + RFP
The part is designed to produce RFP in higher temperatures. The RNA Thermometer acts as a heat-shock RBS.
Cloning
The already linear part was ligated to the pSB1C3 vector, and then transformated to be verified with Colony PCR. The RFP in the iGEM Registry contained Hind3, unlike the RFP in this part. For this reason, the part was cut with the EcoR1 and Hind3 restriction enzymes. The gel runs of the part were examined. The part was tagged as G-Block 7 and sample 7-6 yielded the accurate base pair length.
Functional Assay
The bacteria that were transformated with G-Block 7 were cultured in 5 ml and incubated for 13 hours. After this incubation, the cultures were incubated at respective temperatures for 4 hours and were given 50uM of iPTG twice with 4 hours periouds. There were a total 5 different temperatures that the cultures were incubated in: 4, 25, 37, 42 and 50 C. The proteins in the cultures were then isolated and the protein concentrations were measured. Data on RFP concentration were acquired at 584 nm of emission and 607 nm of excitation. The acquired values were divided to the total amount of protein to acquire the following ratio.
| Medium Temperature | iPTG Presence +/- | Total Amount of Protein | RFP Fluorometric Measurement | RFP/Total |
|---|---|---|---|---|
| 4 | + | 14.09 | 22.32 | 1.584 |
| 4 | - | 11.349 | 10.36 | 0.912 |
| 25 | + | 17.87 | 14.35 | 0.802 |
| 25 | - | 11.054 | 4,313 | 0,390 |
| 37 | + | 16.808 | 40.2 | 2.391 |
| 37 | - | 15.216 | 17.36 | 1.140 |
| 41 | + | 7.637 | 44.83 | 5.870 |
| 41 | - | 5.557 | 11.13 | 2.002 |
| 50 | + | 5.463 | 60.06 | 10.993 |
| 50 | - | 7.997 | 20.95 | 2.619 |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 720
Illegal AgeI site found at 832 - 1000COMPATIBLE WITH RFC[1000]
| n/a | T7+ ibPB RNA Thermometer + RFP |