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Part:BBa_K3859000

Designed by: TianYi Huang   Group: iGEM21_GreatBay_SZ   (2021-10-01)


GBSZ ARTAG barcode

BBa_K3859000 is one of our barcodes which contains specific DNA sequences. This barcode has the meaning of GBSZ ARTAG. This word includes our team name and the products we made, which are very representative. Barcode is a unique DNA sequence which we inserted into yeast spore in order to achieve an efficient, simple and durable spore detection system.[2] We will use Cas12a to detect the barcode in spore in order to identify the authenticity of items, such as artworks.

Barcode design

Firstly, we transferred the words GBSZ ARTAG into DNA sequence using the DNA writer website(fig.1). The picture below shows the DNA sequence and words translation chart (fig.2). Then we attached a cpf1-PAM sequence(TTTA) to upstream of the barcode sequences for CRISPR Cas12a detection[3]. Finally, this short segment of DNA was inserted into yeast for spore production(fig.3).


T--GreatBay SZ--GBSZ barcode.png


【fig.1】The process of transferring words sentence into DNA sequence.

T--GreatBay SZ--DNA sequence translation.png

【fig.2】Character and DNA sequence translation chart

Spores producing

Firstly, we replace the the GFP with barcode using golden gate assembly. Then the plasmid is cut in to linear by notl digestion. At last we transfer the linear DNA into yeast, it will insert into yeast plasmid by homologous recombination(fig.3). These yeast will be used to produce spores(fig.4)[1]. We will do a microscope examination to check whether the spores are produced or not. The microscope examination results are shown below(fig.5)(fig.6).

T--GreatBay SZ--gene editing.png

【fig.3】The process of editing the yeast gene


T--GreatBay SZ--spore formation.png

【fig.4】Overview of the stages of spore formation

T--GreatBay SZ--part K3859000 1D.png

【fig.5】Yeast and spores stained with Methylene blue, we can see the endospores inside the vegetative cell(bule transparent)

T--GreatBay SZ--part K3859000 1E.png

【fig.6】Spore staining of yeast after sporulation, the red small dot represent yeast and green dot represent yeast spores.

References

1. Neiman A. M. (2005). Ascospore formation in the yeast Saccharomyces cerevisiae. Microbiology and molecular biology reviews : MMBR, 69(4), 565–584. https://doi.org/10.1128/MMBR.69.4.565-584.2005

2. Qian, J., Lu, Z. X., Mancuso, C. P., Jhuang, H. Y., Del Carmen Barajas-Ornelas, R., Boswell, S. A., Ramírez-Guadiana, F. H., Jones, V., Sonti, A., Sedlack, K., Artzi, L., Jung, G., Arammash, M., Pettit, M. E., Melfi, M., Lyon, L., Owen, S. V., Baym, M., Khalil, A. S., Silver, P. A., … Springer, M. (2020). Barcoded microbial system for high-resolution object provenance. Science (New York, N.Y.), 368(6495), 1135–1140. https://doi.org/10.1126/science.aba5584

3. Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S. E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., & Zhang, F. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3), 759–771. https://doi.org/10.1016/j.cell.2015.09.038

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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