Difference between revisions of "Part:BBa K3859000"

Revision as of 09:57, 15 October 2021

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.

<img src="https://2021.igem.org/File:T--GreatBay_SZ--GBSZ_barcode.png" />

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

Barcode construct

Firstly, we transferred the words GBSZ ARTAG into DNA sequence using the DNA writer website(fig.1A). 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.1B).

<img src="https://2021.igem.org/File:T--GreatBay_SZ--gene_editing.png" />

【fig.1B】the process of editing the yeast gene

Producing spores with barcode

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.1A). These yeast will be used to produce spores(fig.1C)[1]. We will do a microscope examination to check whether the spores are produced or not. The microscope examination results are shown below(fig.1D)(fig.1E).

<img src="https://2021.igem.org/File:T--GreatBay_SZ--spore_formation.png" />

【fig.1C】Overview of the stages of spore formation

<img src="https://2021.igem.org/File:T--GreatBay_SZ--part_K3859000_1D.png" />

【fig.1D】yeast and spores stained with Methylene blue, we can see the endospores inside the vegetative cell(bule transparent)

<img src="https://2021.igem.org/File:T--GreatBay_SZ--part_K3859000_1E.png" />

【fig.1E】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