Plasmid

Part:BBa_K2908671

Designed by: Dong Yufan   Group: iGEM19_CSU_CHINA   (2019-10-16)


pLN431-s(GATA3)p-GAD-miR101-BS

The CSU_CHINA iGEM team 2019 designed a fusion protein consisting of GAD and miR101-BS for sequence-specific expression of GAD.Therefore,we used our p(GATA3)p(BBa_K2908000) as a specific promoter. At the same time we used our miR101-BD(BBa_K2908668) to bind different concentrations of miR101 and fuse it to 3' end so that can regulate the transactivation domain of GAD.In this way we can ensure the specificity and independence of this protein.

T--CSU_CHINA--2908671.jpg

Physical map of this composite part.

Construction of this part
√ 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”.
As for this part, 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.

T--CSU_CHINA--module1plasmid.png

Construction method of the pocket plasmid

T--CSU_CHINA--lacz.jpg

Inserted sequence of pocket sequence lacZ

We then designed the BsmBI restriction digest location on the 5’ and 3’ top of the inserted sequence, and the final structure had been made.

Usage and Biology
We tested the specific function of synthetic promoter and specific miR-BD in TNBC cell line, and the following figure shows the results.

T--CSU_CHINA--Figure4small.jpg

Figure: (A) The luciferase assay compared the efficiency of Module1 between the TNBC cell line MDA-MB-231 and normal breast cell line HBL-100.
(B) The fluorescence image shows that in MDA-MB-231 the efficiency is significantly high than the HBL-100.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 99
    Illegal NheI site found at 584
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 360
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 279


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.<i>

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