Part:BBa_K3859000

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).

【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

Characterization by iGEM22_Worldshaper-HZ

• Group: iGEM22_Worldshaper-HZ
• Author: Haoyu Xu
• Summary:Characterization of BBa_K3859000 working efficiently as a guide sequence in gRNA for the CRISPR-Cas12a system.

We tested the efficiency of BBa_K3859000 designed by iGEM_21_GreatBay_SZ as the guide sequence to guide gRNA to recognize dsDNA compared to sequence utilizing GAPDH designed by us. Here is a figure to illustrate the difference between 2 sequences.

Figure 1 The difference in two sequences of our original template and BBa_K3859000

Experiment process

We first constructed two gRNA sequence listed above. The Cas12a handle region is the same in two sequences and the only difference is between the guide sequences. One contains GAPDH sequence and one contains BBa_K3859000. Thus, the aim of this experiment is to compare the effect of GAPDH and BBa_K2859000 serving as guide sequences.

Table 1 Specific sequences of the two gRNAs

Then we inserted this two igRNA sequence into pet-28a plasmids. After seeing the successful gel electrophoresis results, we send the plasmid for gene sequencing and checks for the gene sequencing results.

Figure 2 Verification of BBa_K4409013 by gene sequencing (The dark blue region shows the region that is uniform between parts we design, and the region between TCTAGA and CTCGAG are the specific base we want)

Figure 3 Verification of BBa_K4409014 by gene sequencing (The dark blue region shows the region that is uniform between parts we design, and the region between TCTAGA and CTCGAG are the specific base we want--being marked out by black box)

Then the comparison between two gRNA sequences is achieved by adding dsDNA, igRNA, RNA trigger (circRNA), buffer, fluorescent probe and Cas12 enzyme into a TXTL system and comparing the fluorescence under UV light.

Experiment results

Figure 4 The fluorescent results of sample tubes using different guide sequences of igRNAs. From left to right: 1) Our sequence utilizing GAPDH; 2) not for this test; 3) BBa_K3859000 as the guide sequence; 4) negative control group

Using ImageJ, we can compare the relative brightness of each group, and make the following charts.

Chart 5 Relative Brightness of fluorescence produced (Error bars are printed based on standard deviation)

The percentage difference of fluorescence produced is also calculated.

Chart 6 Percentage difference of fluorescence [Calculated by the following formula: (Brightness-negative control brightness)/negative control brightness] (Error bars are printed based on standard deviation)

Using ImageJ, we found that BBa_K3859000 produces the brighter fluorescence than BBa_K4409013 with no significant difference in brightness level, but both are brighter than negative control groups.

Conclusion

Both sequences of GAPDH and BBa_K3859000 are workable since they are significantly brighter than negative control group. However, the BBa_K3859000 might be slightly more suited to serve as guide sequence since it produces stronger fluorescence.

References

Jiao, Yang & Zhu, Baocun & Chen, Ji-Hua & Duan, Xiaohong. (2015). Fluorescent Sensing of Fluoride in Cellular System. Theranostics. 5.173-187. 10.7150/thno.9860.

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]