Difference between revisions of "Part:BBa K1440000:Design"

Latest revision as of 08:44, 27 October 2014

Part2_pTRE promoter with cFP and ribozyme

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 20
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 20
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 20
1000
COMPATIBLE WITH RFC[1000]

Design Notes

It is a component of our whole project,can not 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 second part, contains two type of regulatory units --- pTRE and ribozyme.

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.

alt text

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 second part of the whole project, ranging from pTRE promoter to the second loxP site. pTRE is the promoter derived from ordinary Tet-on system, comprise of 6 tandem tetO upstream the minimal CMV core promoter. CFP will be expressed by pTRE promoter when Tet (or Dox) is added. Additional plasmid contains tTA ( tetracycline-controlled transactivator) is required for its expression. Ribozyme can perform self-cleavage when it is transcribed into mRNA. The ribozyme used here derives from 3’UTR region of mouse CLEC2 gene, serves as a regulatory unit of RFP in the third part of our project, and has no impact on expression of CFP. Notably, the ribozyme sequence here is currently in reverse direction, and a functional ribozyme should be the reverse complement of the ribozyme sequence present there.

Source

BBa_K812032 (mCFP) BBa_K678012 (SV40pA)

References

1.Scott WG. et.al. (2008). “A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA”. Nature 454(7206):899-902. 2.Hayashi, S & McMahon, AP. (2002). “Efficient Recombination in Diverse Tissues by a Tamoxifen-Inducible Form of Cre: A Tool for Temporally Regulated Gene Activation/Inactivation in the Mouse”. Dev Biol 244(2): 305-318.

@@ Line 9: / Line 9: @@
 It is a component of our whole project,can not work individually.
-Brief
+'''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 second part, contains two type of regulatory units --- pTRE and ribozyme.
-Excision Model
+'''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.
@@ Line 21: / Line 23: @@
 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
+'''Role of this Part'''
 This part is the second part of the whole project, ranging from pTRE promoter to the second loxP site.
 pTRE is the promoter derived from ordinary Tet-on system, comprise of 6 tandem tetO upstream the minimal CMV core promoter. CFP will be expressed by pTRE promoter when Tet (or Dox) is added. Additional plasmid contains tTA ( tetracycline-controlled transactivator) is required for its expression.