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Team:Tsinghua-A/Wetlab/Part2
From 2012.igem.org
Tsinghua-A::Wetlab::Eukaryotic cells
Construction in eukaryotic cells
Life process in eukaryotic cells is much more complicate, especially when it is related to signal transportation. We choose microRNA to transport cues and Bxb1 to realize the flipping process. Using microRNA can not only simplify the design of the system, making sure the transportation is precise and efficient, but also ensure the independency with other life processes due to the orthogonality of the microRNA and other signals. MicroRNA also has great scalability. Compared to Cre-loxP recombination system, Bxb1 recombination system requires different enzymes to flip back and forth, which makes the whole system more controllable. As shown in the picture, Bxb1 sites and microRNAs are located in the intron of the reporter. If the sequence has flipped, the two exons of Cyan are segregate from each other, and Cyan would not translate correctly. So we can detect whether the sequence has flipped from the expression of Cyan. From the circuit, we can get such truth table and corresponding logical relationship.
Before Reverse
| RSL | Dox | miR-21 | miR-FF4 | Cyan | mKate | EYFP |
|---|---|---|---|---|---|---|
| 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| 1 | 0 | 1 | 0 | 1 | 0 | 1 |
| 0 | 1 | 0 | 1 | 0 | 1 | 1 |
| 1 | 1 | 1 | 1 | 1 | 1 | 0 |
After Reverse
| RSL | Dox | miR-21 | miR-FF4 | Cyan | mKate | EYFP |
|---|---|---|---|---|---|---|
| 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| 1 | 0 | 1 | 1 | 0 | 0 | 0 |
| 0 | 1 | 1 | 1 | 0 | 1 | 0 |
| 1 | 1 | 1 | 1 | 0 | 1 | 0 |
