iGEM 2020 — Team BNU-China

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# Team:BNU-China/Notebook

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# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:BNU-China/Engineering

* abcdef
* [TEAM](https://2020.igem.org/Team%3ABNU-China/Team)

  [Members](https://2020.igem.org/Team%3ABNU-China/Team)
  [Attributions](https://2020.igem.org/Team%3ABNU-China/Attributions)
* [PARTS](https://2020.igem.org/Team%3ABNU-China/Parts)

  [Parts](https://2020.igem.org/Team%3ABNU-China/Parts)
  [Contribution](https://2020.igem.org/Team%3ABNU-China/Contribution)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3ABNU-China/Human_Practices)

  [Human Practices](https://2020.igem.org/Team%3ABNU-China/Human_Practices)
  [Science Communication](https://2020.igem.org/Team%3ABNU-China/Education)
  [Collaborations](https://2020.igem.org/Team%3ABNU-China/Collaborations)
  [Partnership](https://2020.igem.org/Team%3ABNU-China/Partnership)
* [SAFETY](https://2020.igem.org/Team%3ABNU-China/Safety)
* [MODEL](https://2020.igem.org/Team%3ABNU-China/Model)

  [Overview](https://2020.igem.org/Team%3ABNU-China/Model)
  [Cas9 Folding Rate](https://2020.igem.org/Team%3ABNU-China/Model1)
  [Predictions of Diversity of Barcode](https://2020.igem.org/Team%3ABNU-China/Model2)
  [Selection of Degradation Tags](https://2020.igem.org/Team%3ABNU-China/Model3)
  [Tracing Generations](https://2020.igem.org/Team%3ABNU-China/Model4)
  [Number of hgRNA Required](https://2020.igem.org/Team%3ABNU-China/Model5)
* [PROJECT](https://2020.igem.org/Team%3ABNU-China/Description)

  [Description](https://2020.igem.org/Team%3ABNU-China/Description)
  [Design](https://2020.igem.org/Team%3ABNU-China/Design)
  [Experiment](https://2020.igem.org/Team%3ABNU-China/Experiments)
  [Engineering](https://2020.igem.org/Team%3ABNU-China/Engineering)
  [Implementation](https://2020.igem.org/Team%3ABNU-China/Implementation)
* [HOME](https://2020.igem.org/Team%3ABNU-China)

team

**Overview**

    This year, we built the ABC (Automatic Barcode Creator) system and set a fundamental paradigm in yeast. This system can automatically label each cell with different barcodes and trace their relationships by single cell RNA sequencing. Our engineering can be divided into three modules: label each cell automatically and prolong the tracing generations, increase the variants of barcodes, and read barcodes together with transcriptomic information. Delightfully, we have validated these modules successfully by carrying out experiments, modeling and literature review.

![](https://static.igem.org/mediawiki/2020/a/ac/T--BNU-China--dnaProject.png)

# Label each cell automatically and prolong the tracing generations

   In order to achieve the automatic addition of barcodes and prolong the tracing generations, we used *CLB2* promoter to express Cas9 periodically instead of constitutive expression. We constructed the recombinant plasmid containing *CLB2* promoter [(BBa\_K3506007)](http://parts.igem.org/Part%3ABBa_K3506007) and Cas9 gene [(BBa\_K2130013)](http://parts.igem.org/Part%3ABBa_K2130013).

![](https://static.igem.org/mediawiki/2020/9/9b/T--BNU-China--engpic0000.png)

Figure 1. Gel electrophoresis results of colony PCR. Lane 1: Marker; Lane 2-5: PCR product of the upstream of integration site (1069bp); Lane 6-9: PCR production of the downstream of integration site (2862bp).

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:BNU-China/Experiments

* abcdef
* [TEAM](https://2020.igem.org/Team%3ABNU-China/Team)

  [Members](https://2020.igem.org/Team%3ABNU-China/Team)
  [Attributions](https://2020.igem.org/Team%3ABNU-China/Attributions)
* [PARTS](https://2020.igem.org/Team%3ABNU-China/Parts)

  [Parts](https://2020.igem.org/Team%3ABNU-China/Parts)
  [Contribution](https://2020.igem.org/Team%3ABNU-China/Contribution)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3ABNU-China/Human_Practices)

  [Human Practices](https://2020.igem.org/Team%3ABNU-China/Human_Practices)
  [Science Communication](https://2020.igem.org/Team%3ABNU-China/Education)
  [Collaborations](https://2020.igem.org/Team%3ABNU-China/Collaborations)
  [Partnership](https://2020.igem.org/Team%3ABNU-China/Partnership)
* [SAFETY](https://2020.igem.org/Team%3ABNU-China/Safety)
* [MODEL](https://2020.igem.org/Team%3ABNU-China/Model)

  [Overview](https://2020.igem.org/Team%3ABNU-China/Model)
  [Cas9 Folding Rate](https://2020.igem.org/Team%3ABNU-China/Model1)
  [Predictions of Diversity of Barcode](https://2020.igem.org/Team%3ABNU-China/Model2)
  [Selection of Degradation Tags](https://2020.igem.org/Team%3ABNU-China/Model3)
  [Tracing Generations](https://2020.igem.org/Team%3ABNU-China/Model4)
  [Number of hgRNA Required](https://2020.igem.org/Team%3ABNU-China/Model5)
* [PROJECT](https://2020.igem.org/Team%3ABNU-China/Description)

  [Description](https://2020.igem.org/Team%3ABNU-China/Description)
  [Design](https://2020.igem.org/Team%3ABNU-China/Design)
  [Experiment](https://2020.igem.org/Team%3ABNU-China/Experiments)
  [Engineering](https://2020.igem.org/Team%3ABNU-China/Engineering)
  [Implementation](https://2020.igem.org/Team%3ABNU-China/Implementation)
* [HOME](https://2020.igem.org/Team%3ABNU-China)

team

![](https://static.igem.org/mediawiki/2020/a/ac/T--BNU-China--dnaProject.png)

# Build a periodic expression module of Cas9

   To verify that our design enables Cas9 to be expressed periodically, we built a module for the periodic expression of Cas9, and the experiments were carried out as follows.

   1. Construct the recombinant plasmid pRH003-URA3-*CLB2* promoter-Cas9 by in-fusion cloning. Then transform it into *E. coli* DH5α. Extract plasmids from transformants. The recombinant plasmids were confirmed by colony PCR and sequencing, then transformed into *S. cerevisiae* BY4741. Screen for transformants by SD-ura culture medium.

   2.Synchronize the cell cycle of *S. cerevisiae* population to G1 phase.

   3.Release yeast cells from synchronization. Remove a time-zero fraction. Collect fractions of culture every 10 min for 150 min. Extract total protein for Western Blot. Strain without being transformed was used as a negative control. GAPDH was chosen as an internal reference.

   4.Obtain and analyze data. Draw the image of Cas9 protein levels over time.

# Degradation of Cas9 at a proper rate

   In order to verify the function of Clb2 (N124aa) as a degradation tag, we constructed the plasmids for an experimental and a control group. We hoped to characterize the protein degradation by determining the fluorescence intensity curve.

   1.The two plasmids were constructed with pYES2, containing *GAL1* galactose inducible promoter and *URA3* marker gene. The vector in the experimental group contains *CLB2* (N124aa)- Linker-GFP, while the control group only contains GFP. The successfully constructed vectors were transformed into *E.coli* DH5α strain.

   2.After the plasmids were extracted and linearized, they were transformed into *Saccharomyces cerevisiae* BY4741. We used the fluorescence microscope and differential interference microscope to verify the transformants.

   3.Both experimental and control groups were cultured in SC medium at 30 ℃ until OD600 value reached about 3.000, and then the expression was induced by galactose with a final concentration of 2%. After induction, the fluorescence intensity was measured by flow cytometry at half an hour interval.

   4.Cells were then inoculated into the medium containing glucose instead of galactose. In addition to being a carbon source, glucose also inhibits the expression of GFP by inhibiting the *GAL1* promoter. Within 7 hours after inoculation, the fluorescence intensity was measured by flow cytometry at the same interval of half an hour.

   5.We used SPSS26.0 to process the data and determined the relationship between fluorescence intensity and time, as well as the protein concentration and time. Finally, we calculated the degradation rate of protein modified by Clb2 N124aa through modeling.

# Obtaining the lineage information out of the transcriptomic information

   In order to read the lineage information out of the transcriptomic information, we design two double promoter modules. And we conducted the experiment to test the function of the cassette. To make results visible, we use gRNA that targets *ADE2* gene. *Cryptococcus neoformans* will turn red if gRNA works. The experiments were carried as follows.

   1.Construct recombinant plasmids. Get pGAP from the genome of *Cryptococcus neoformans*. Insert it upstream of pU6 on PRH003 plasmid. Use the plasmid without pGAP as the control group.

   2.Transform the product (2.5μL) into DH5α competent cells (50μL), grow cells on agar plates (containing Ampicillin). Incubate plates at 37°C overnight. Colonies were screened by colony PCR and then grown at 37℃, 200rpm. Plasmids were extracted and sent for sequencing.

   3.Use Kpn1 to linearize the plasmids and transform them into *Cryptococcus neoformans* by electroporation.

   4.The *C. neoformans* was spread on YNBA selection medium, and transformants grew after being cultured in an incubator kept at 30℃ for 4 days. Then the culture was transferred to a refrigerator at 4℃.

   5.Red colonies were selected and inoculated into YPD medium, then placed in an incubator kept at 30℃ for 4 days. Finally, it was kept in at 4℃ refrigerator.

   6.For both the experimental and the control groups, we first extracted the total mRNA of these red colonies by Trizol. Then the mRNA was reverse transcribed using oligo dT as the primer.

   7.To test whether gRNA can be transcribed, we performed PCR on the reverse transcription products by two specific primers. Then sequence the PCR product.

   We exchanged pGAL7 with pGAP and repeated steps 1-5. Then we selected red colonies and induced the expression by galactose. After expression for 30mins. We repeated steps 6-7.

Contact

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Address:19th,Xinjiekouwai St, Haidian District, Beijing, P.R.China
Postcode:100875
Email：igem2020bnu\_china@163.com

© 2020. All Right Reserved. Designed by BNU-China

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# Team:BNU-China/Results

* abcdef
* [TEAM](https://2020.igem.org/Team%3ABNU-China/Team)

  [Members](https://2020.igem.org/Team%3ABNU-China/Team)
  [Attributions](https://2020.igem.org/Team%3ABNU-China/Attributions)
* [PARTS](https://2020.igem.org/Team%3ABNU-China/Parts)

  [Parts](https://2020.igem.org/Team%3ABNU-China/Parts)
  [Contribution](https://2020.igem.org/Team%3ABNU-China/Contribution)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3ABNU-China/Human_Practices)

  [Human Practices](https://2020.igem.org/Team%3ABNU-China/Human_Practices)
  [Science Communication](https://2020.igem.org/Team%3ABNU-China/Education)
  [Collaborations](https://2020.igem.org/Team%3ABNU-China/Collaborations)
  [Partnership](https://2020.igem.org/Team%3ABNU-China/Partnership)
* [SAFETY](https://2020.igem.org/Team%3ABNU-China/Safety)
* [MODEL](https://2020.igem.org/Team%3ABNU-China/Model)

  [Overview](https://2020.igem.org/Team%3ABNU-China/Model)
  [Cas9 Folding Rate](https://2020.igem.org/Team%3ABNU-China/Model1)
  [Predictions of Diversity of Barcode](https://2020.igem.org/Team%3ABNU-China/Model2)
  [Selection of Degradation Tags](https://2020.igem.org/Team%3ABNU-China/Model3)
  [Tracing Generations](https://2020.igem.org/Team%3ABNU-China/Model4)
  [Number of hgRNA Required](https://2020.igem.org/Team%3ABNU-China/Model5)
* [PROJECT](https://2020.igem.org/Team%3ABNU-China/Description)

  [Description](https://2020.igem.org/Team%3ABNU-China/Description)
  [Design](https://2020.igem.org/Team%3ABNU-China/Design)
  [Experiment](https://2020.igem.org/Team%3ABNU-China/Experiments)
  [Engineering](https://2020.igem.org/Team%3ABNU-China/Engineering)
  [Implementation](https://2020.igem.org/Team%3ABNU-China/Implementation)
* [HOME](https://2020.igem.org/Team%3ABNU-China)

team

Science communication is of great significance to the development of science and the public's understanding of science. BNU-China have made a lot of efforts this year, trying to grasp the public's views on synthetic biology and our project as well as convey our values in order to better serve the community.

![](https://static.igem.org/mediawiki/2020/a/ac/T--BNU-China--dnaProject.png)

# Build a periodic expression module of Cas9

   We constructed a recombinant plasmid containing CLB2 promoter, Cas9 gene, and ura3 selection marker in E. coli, and introduced it into the genome of Saccharomyces cerevisiae BY4741 by homologous recombination.
  We designed two pairs of primers on both the upstream and downstream of the plasmid inserted into the genome to verify whether we have successfully recombined. The results of colony PCR showed that we successfully constructed our pathway in the genome of Saccharomyces cerevisiae BY4741（Figure 1）.

# Our Inspiration

  An article published in cell on June 17, 2020[2] established an inducible lineage tracing mouse model based on CRISPR/Cas9 (as shown in the figure below). However, the induction of cas9 still needs the addition of exogenous substances and has not yet reached automation.

# Our Solution

  Inspired, we build an inducible expression module of Cas9 in association with cell division to label each cell automatically. What’s more, to solve the problem about limited number of barcodes，we used homing guide RNA (hgRNA) to replace the small guide RNA (sgRNA) so that the diversity of barcodes can be increased greatly. Additionally, in order to make the barcode sequence read at RNA level, we designed a double promoter module, so that we can obtain the lineage information together with transcriptomic information.

# The Desired Effect

  When cells start to divide at the end of G2 phase, Cas9 is expressed under the control of Cyclin B2 promoter[3,4] and bound with hgRNA, which guides Cas9 to cut barcodes. Then at G1 phase in the next cell cycle, cells perform Non-homologous end joining(NHEJ) and introduce random mutations, generating new barcodes. The degradation tag at the N-terminal of Cas9 makes Cas9 degrade quickly[5]. After cell division, Cas9 disappears and no longer cuts the barcode until the next round of cell division.

# References

[1] Spanjaard, B., Hu, B., Mitic, N., Olivares-Chauvet, P., Janjuha, S., Ninov, N., & Junker, J. P. (2018). Simultaneous lineage tracing and cell-type identification using CRISPR-Cas9-induced genetic scars. Nature biotechnology, 36(5), 469–473. https://doi.org/10.1038/nbt.4124

[2] Bowling, S., Sritharan, D., Osorio, F. G., Nguyen, M., Cheung, P., Rodriguez-Fraticelli, A., Patel, S., Yuan, W. C., Fujiwara, Y., Li, B. E., Orkin, S. H., Hormoz, S., & Camargo, F. D. (2020). An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells. Cell, 181(6), 1410–1422.e27. https://doi.org/10.1016/j.cell.2020.04.048

[3] Eluère, R., Offner, N., Varlet, I., Motteux, O., Signon, L., Picard, A., Bailly, E., & Simon, M. N. (2007). Compartmentalization of the functions and regulation of the mitotic cyclin Clb2 in S. cerevisiae. Journal of cell science, 120(Pt 4), 702–711. https://doi.org/10.1242/jcs.03380

[4] Wäsch, R., & Cross, F. R. (2002). APC-dependent proteolysis of the mitotic cyclin Clb2 is essential for mitotic exit. Nature, 418(6897), 556–562. https://doi.org/10.1038/nature00856

[5] Hendrickson, C., Meyn, M. A., 3rd, Morabito, L., & Holloway, S. L. (2001). The KEN box regulates Clb2 proteolysis in G1 and at the metaphase-to-anaphase transition. Current biology : CB, 11(22), 1781–1787. https://doi.org/10.1016/s0960-9822(01)00564-4