Part:BBa_K1440066:Design
Part2_pTRE promoter with cFP and ribozyme
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 20
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 20
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 20
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
It is a component of our whole project,can work individually.
Brief
This is a component of our whole project, cannot perform complex logic calculation individually. The whole project we carried out is to construct a circuit capable of performing multiple parallel logic calculation. There are 3 reporter gene in the complete project, response to 3 permutations of two input signals -- Tet & TM. We designed two different models based on two distinct enzymatic activity of Cre recombinase, namely sequence excision activity and sequence inversion activity. We mainly focus on constructing “excision-model” , through divide this model into three tandem parts and constructed them separately. This is the third part, contains a constitutive expressed reporter and a Tet-inducible shRNA targeting this reporter gene.
Excision Model
TM is a drug that can activate CreER fusion recombinase which can recognize loxP site and modify DNA sequence directly. When two loxP sites point the same direction, Cre recombinase will execute excision activity by removing the sequence between two loxP. Based on that, we designed the excision model as shown below.
This model response to 3 different input states of 2 input drugs, Tet & TM. In this model, CFP, RFP, YFP will express and only express in high-[Tet]-low-[TM], low-[Tet]-high-[TM], high-[Tet]-high-[TM] conditions correspondingly.
Role of this Part
This part is the third part of the whole project, ranging from RFP reporter to the end. Reporter gene is constitutively expressed by CMV promoter. We put this reporter and CMV promoter in an anti-sense direction in order to connect it to the “part 2” biobrick we registered. The shRNA targeting this reporter is placed behind a Tet-inducible promoter. This promoter is derived from Pol III promoter H1T0, modified by adding 2 tetO regulatory unit. When Tet (or Dox) is added, TetR will drop off from its binding site tetO, only then will this Pol III promoter express its downstream shRNA. shRNA will undergo microRNA genesis and end up a siRNA knocking down its targeting gene.
Source
BBa_K774007 (RFP) BBa_K678012 (SV40pA) BBa_K747096 (CMV promoter)
References
3.Benson, J.D. et.al. (2009). “Single-vector inducible lentiviral RNAi system for oncology target validation”. Cell Cycle. 8: 498-504.