Part:BBa_K3859003
ALL KNOWLEDGE BEGINS IN WONDER barcode
BBa_K3859003 is one of our barcodes which contains specific DNA sequences. This barcode has the meaning of ALL KNOWLEDGE BEGINS IN WONDER. 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 sentence ALL KNOWLEDGE BEGINS IN WONDER into DNA sequence using the DNA writer website(fig.4A). The picture below shows the DNA sequence and words translation chart (fig.2). Then we attached a cpf1-PAM sequence(TTTA)to the 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).
【fig.1】The process of transferring words sentence into DNA sequence.
【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).
【fig.3】The process of editing the yeast gene
【fig.4】Overview of the stages of spore formation
【fig.5】Yeast and spores stained with Methylene blue, we can see the endospores inside the vegetative cell(bule transparent)
【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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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