Team:CSU CHINA/Composite Part
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Composite Parts
| Section | Name | Type | Description |
| Module1 | BBa_K2908671 | Plasmid | pLN431-s(GATA3)p-GAD-miR101-BS |
|---|---|---|---|
| Module2 | BBa_K2908679 | Plasmid | s(ESR1)p-miR141spe-EGFP |
| BBa_K2908680 | Plasmid | s(ESR1)p-miR148bspe-EGFP | |
| BBa_K2908681 | Plasmid | s(ESR1)p-miR101spe-EGFP | |
| Module3 | BBa_K2908670 | Plasmid | pLN431-G8p-yCD-miR101BS |
Overview
√ Golden Gate assay to construct the plasmids
First step: Construction of pocket plasmid
Next: ‘Golden Gate’
In 2008, Engler etc. reported a kind of cloned strategy through IIS restriction enzymes , called IIS cloning, namely ‘Golden Gate’ cloning. Type IIS restriction enzyme can specifically identify the target sites on double-stranded DNA, and non-specifically cut the DNA double-stranded downstream of the target sites to produce a cohesive end at the 5 'or 3' end of the DNA double-stranded [1].
"Golden Gate" cloning method is in the same reaction system, using IIS type restriction endonuclease, cut beyond recognition site of DNA, DNA fragments containing cohesive end, at the same time by ligase connect several DNA fragments according to the established order, and spliced into excluding enzyme recognition site of DNA fragments, is like a linear puzzles are correctly spliced together, make the multiple objective DNA fragments in the order set to realize "seamless connection”.
Module1
pLN431-s(GATA3)p-GAD-miR101BD (BBa_K2908671)
We use the pLN431[2] as the back-clone of the Module1, then we cut the vector and an insert sequence (lacZp+lacZ) with MluI and NotI to form the pocket plasmid.
Pocket plasmid construction
Detailed pocket plasmid construction
Supplementary Figure: lacZ
We designed the BsmBI restriction digest location on the 5’ and 3’ top of the inserted sequence, the following figure shows the pattern of construction method.
Module1-Golden Gate
Module2
p(ESR1)p-miR141spe-EGFP (BBa_K2908672)
p(ESR1)p-miR148bspe-EGFP (BBa_K2908674)
p(ESR1)p-miR101spe-EGFP (BBa_K2908675)
The construction method is the same as module1, only except the paired sequence of each inserted fragment, which shows in the following picture.
Module2-Golden Gate
Module3
pLN431-G8p-yCD-miR101BD (BBa_K2908670)
References
[1] Waclaw Szybalski”, Sun C. Kim”, Noaman Hasan” and Anna J. Podhajskab, Class IIS restriction enzymes, a review.1991, Gene, 100: 13-26.
[2] Lior Nissim, Ming-Ru Wu, Erez Pery, Adina Binder-Nissim, Hiroshi I. Suzuki, Doron Stupp, Claudia Wehrspaun, Yuval Tabach, Phillip A. Sharp, and Timothy K. Lu, Cell, (2017),Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy.