iGEM 2020 — Team Peking

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# Team:Peking/Notebook

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![](https://static.igem.org/mediawiki/2020/7/78/T--Peking--lab.png)

Lab

---

* [Notebook](https://2020.igem.org/Team%3APeking/Notebook)
* [Result](https://2020.igem.org/Team%3APeking/Results)
* [Lab Attribution](https://2020.igem.org/Team%3APeking/Lab-Attribution)
* [Safety](https://2020.igem.org/Team%3APeking/Safety)

![](https://static.igem.org/mediawiki/2020/f/f7/T--Peking--achievement.png)

Achievement

---

* [Contribution](https://2020.igem.org/Team%3APeking/Contribution)
* [Proof of Concept](https://2020.igem.org/Team%3APeking/Proof%20Of%20Concept)
* [Human Practices](https://2020.igem.org/Team%3APeking/Human%20Practices)
* [Education](https://2020.igem.org/Team%3APeking/Education)

![](https://static.igem.org/mediawiki/2020/a/a8/T--Peking--modeling.png)

Algorithm

---

* [Music to DNA](https://2020.igem.org/Team%3APeking/music%20to%20DNA)
* [DNA to Music](https://2020.igem.org/Team%3APeking/DNA%20to%20music)
* [Scoring Algorithm](https://2020.igem.org/Team%3APeking/Fitness%20function)
* [Engineering](https://2020.igem.org/Team%3APeking/Engineering)

![](https://static.igem.org/mediawiki/2020/f/fd/T--Peking--team.png)

Team

---

* [Team Members](https://2020.igem.org/Team%3APeking/Team)
* [Attributions](https://2020.igem.org/Team%3APeking/Attributions)
* [Acknowledgement](https://2020.igem.org/Team%3APeking/Acknowledgement)
* [Collaboration](https://2020.igem.org/Team%3APeking/Collaborations)

![](https://static.igem.org/mediawiki/2020/4/41/T--Peking--project.png)

Project

---

* [Background](https://2020.igem.org/Team%3APeking/Background)
* [Description](https://2020.igem.org/Team%3APeking/Description)
* [Design](https://2020.igem.org/Team%3APeking/Design)
* [Implementation](https://2020.igem.org/Team%3APeking/Implementation)

Notebook

# Experimental principles

Between the mutual transformation of music and DNA, the most important thing is to introduce mutations. In
addition to computer simulation, we also carried out a series of laboratory work to further demonstrate our
mutation results.

First, we thought of making CRISPR/Cas9 based mutagenesis enzymes directly work on raw information sequences.
However, we may come to a stop a few generations later when sgRNA could not recognize its target anymore. We
either end up with rarely mutated sequences or have to adjust sgRNA itself, neither of which would be a good
result. Thus, we come up with the idea to separate each sequence into two parts. One is the information
sequence which stores actual music information, and ahead of it is the anchor sequence where sgRNA binds.

The sequence of each section consists of a coding region and an anchor interval. The anchor interval is
targeted by the gRNA, and then the coding sequence is mutated by the base editor. Because the mutation
accuracy of the base editor is not 100%, it may mutate the adjacent sites of the mutation site. We chose
early base editors (less accurate and more prone to random mutations), and simultaneously introduced them
into E. coli along with our music coding sequence and separate gRNA expression plasmids. The base editors
included both ABE (adenine base editor) and CBE (cytosine base editor), so that their combination can change
both A-T and C-G base pairs. In this way, we can carry out large-scale random mutations in the coding range
of the whole music coding sequence, and then re-sequence these mutated sequences. By using our algorithm, we
can re-import the high scoring sequences into E. coli, and then repeat the cycle, and we can get the
mutation sequences with higher scores.

## Introduction of EvolvR

[click here](https://static.igem.org/mediawiki/2020/b/bf/T--Peking--introduction_of_EvolvR.pdf)

## Introduction of ABE and CBE

[click here](https://static.igem.org/mediawiki/2020/c/ce/T--Peking--introduction_of_ABE_and_CBE.pdf)

# Lab notebook

| Date | Process |
| --- | --- |
| 7.29 | Cloning of plasmid I0050, J23119, B0015 with pBAD, RBS, ddTerm part respectively / Extracting a small number of plasmids and measuring the concentration |
| 7.30 | Selection of bacteria with various components / Enzyme digestion and ligation of pBAD, ddTerm, RBS and PSB1C3 by BioBrick |
| 8.3-8.4 | Preparation and split charging of chloramphenicol / Enzyme digestion and ligation of 1C3-ddTerm, B0015, 3C5-pBAD-dcas9-J23119-sgRNA, pBAD-dcas9-J23119-sgRNA / Transforming and amplifying / Extracting a small number of plasmids |
| 8.5-8.8 | Build ABE, CBE part with 15 AA linker and 10 AA linker / Enzyme digestion and ligation of plasmids / Extracting a small number of L-pmCDA-I-UGL-I (Amp+) |
| 8.9-8.10 | Ligation of sgRNA scaffold by ePCR / PCR was used to verify ABE-dCAS9, CBE-dCAS9 and sgRNA scaffolds |
| 8.12-8.13 | Each element was cut off from PUC57, incluing UGI, CDA+UGI, pBAD / Construction of pBAD+RBS, pBAD-long+RBS / PCR was used to verify pBAD+RBS, pBAD-long+RBS, TadA, dCAS9 |
| 8.14 | Amplifying PSB1C3-pBAD-RBS, Puc57-ABE-UGI, Puc57-CBE-UGI, PSB1C3-sgRNA scaffold / The bacterial liquid was verified by PCR and sequencing |
| 8.15 | The EvolvR system was divided into five fragments with more than 1000bp, which could be connected with each other through Bsa1 and synthesized by the company |
| 9.29 | All the plasmids mentioned before were of low concentration and needed to be re extracted / Retransform PSB1C3-pBAD-RBS, Puc57-ABE-UGI, Puc57-CBE-UGI, PSB1C3-sgRNA scaffold |
| 9.30 | Most of the colonies did not grow and the original bacterial solution should be resuscitated and coated |
| 10.1 | Extracting a small number of plasmids pSB1C3-pBAD-RBS puc57-ABE-UGI puc57-CBE-UGI pSB1C3-sgRNA scaffold and measuring the concentration / Verified by PCR, Most of them have problems |
| 10.2 | Reusing another batch of preserved plasmids and transforming plasmidsp SB1C3-pBAD-RBS puc57-ABE-UGI puc57-CBE-UGI pSB1C3-sgRNA scaffold |
| 10.3-10.4 | There are some problems in PSB1C3-sgRNA scaffold and PSB1C3-sgRNA scaffold / 5 parts of EvolvR was coated and extracted / Retransforming PSB1C3-sgRNA scaffold, PSB1C3-sgRNA scaffold |
| 10.5-10.6 | PSB1C3-sgRNA scaffold and PSB1C3-sgRNA scaffold were verified successfully by PCR / Puc57-ABE-UGI puc57-CBE-UGI carriers were linearized by BioBrick but lost with plasmids in accident |
| 10.7-10.8 | Restart (see 10.2) |
| 10.11 | Verifying by PCR / There are some problems in PSB1C3-pBAD-RBS and restart transforming it. |
| 10.12-10.13 | PSB1C3-pBAD-RBS was verified successfully by PCR / Puc57-ABE-UGI, Puc57-CBE-UGI, PSB1C3-sgRNA scaffold were digested by enzymes and linearized / PBAD-RBS was attained / Amplifying PSB1C3-ddTerm and EvolvR parts |
| 10.14 | We got ABE-final and CBE-final by ligating Puc57-ABE-UGI, Puc57-CBE-UGI and ddterm, PBAD-RBS |
| 10.15 | EvolvR parts were digested from the synthesized vector and PSB1C3 was digested at BSA1 site / Ligating them |
| 10.16-10.17 | ABE-final, CBE-final, EvolvR-final / PCR confirmed that it was positive |

* [Experimental principles](#a1)
* [Lab notebook](#a2)

© 2020 Peking iGEM. All Rights Reserved.

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# Team:Peking/Engineering

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[![](https://static.igem.org/mediawiki/2020/3/3c/T--Peking--home.png)](https://2020.igem.org/Team%3APeking)  Home

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Lab

---

* [Notebook](https://2020.igem.org/Team%3APeking/Notebook)
* [Result](https://2020.igem.org/Team%3APeking/Results)
* [Lab Attribution](https://2020.igem.org/Team%3APeking/Lab-Attribution)
* [Safety](https://2020.igem.org/Team%3APeking/Safety)

![](https://static.igem.org/mediawiki/2020/f/f7/T--Peking--achievement.png)

Achievement

---

* [Contribution](https://2020.igem.org/Team%3APeking/Contribution)
* [Proof of Concept](https://2020.igem.org/Team%3APeking/Proof%20Of%20Concept)
* [Human Practices](https://2020.igem.org/Team%3APeking/Human%20Practices)
* [Education](https://2020.igem.org/Team%3APeking/Education)

![](https://static.igem.org/mediawiki/2020/a/a8/T--Peking--modeling.png)

Algorithm

---

* [Music to DNA](https://2020.igem.org/Team%3APeking/music%20to%20DNA)
* [DNA to Music](https://2020.igem.org/Team%3APeking/DNA%20to%20music)
* [Scoring Algorithm](https://2020.igem.org/Team%3APeking/Fitness%20function)
* [Engineering](https://2020.igem.org/Team%3APeking/Engineering)

![](https://static.igem.org/mediawiki/2020/f/fd/T--Peking--team.png)

Team

---

* [Team Members](https://2020.igem.org/Team%3APeking/Team)
* [Attributions](https://2020.igem.org/Team%3APeking/Attributions)
* [Acknowledgement](https://2020.igem.org/Team%3APeking/Acknowledgement)
* [Collaboration](https://2020.igem.org/Team%3APeking/Collaborations)

![](https://static.igem.org/mediawiki/2020/4/41/T--Peking--project.png)

Project

---

* [Background](https://2020.igem.org/Team%3APeking/Background)
* [Description](https://2020.igem.org/Team%3APeking/Description)
* [Design](https://2020.igem.org/Team%3APeking/Design)
* [Implementation](https://2020.igem.org/Team%3APeking/Implementation)

Engineering

Utilizing Python programs, we have successfully made this set of theoretical codons converting DNA into music a reality. DNA, a fantastic molecule bearing the beauty of life, is interpreted by the nature to form rich and diverse creatures. And we, by deciphering the DNA code to melodious music, offers another way for human beings to appreciate its beauty.

Hemoglobin is a key protein in the organism, which carries oxygen, allowing the transportation of oxygen to break through the method of direct penetration from the body surface to the tissue cells, thereby allowing organisms to gain large-scale development. At the same time, it has a homologous relationship in most vertebrates, which means that the gene sequence encoding it is not only the key code for making life so colorful, but also a page in this great book on the evolution of life.

For example, a cheetah needs to move at a high speed to capture prey in the grassland, and its hemoglobin's strong oxygen-carrying capacity is one of the essential qualities to support this high-speed movement. And if we listen to this music decoded by the gene sequence encoding the beta chain of the cheetah's hemoglobin, we will find that it is as energetic and passionate as the cheetah itself, as if it were running on the grassland.

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© 2020 Peking iGEM. All Rights Reserved.

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# Team:Peking/Experiments

![](https://static.igem.org/mediawiki/2020/b/b9/T--Peking--team-icon-green.png)

[![](https://static.igem.org/mediawiki/2020/3/3c/T--Peking--home.png)](https://2020.igem.org/Team%3APeking)  Home

![](https://static.igem.org/mediawiki/2020/7/78/T--Peking--lab.png)

Lab

---

* [Notebook](https://2020.igem.org/Team%3APeking/Notebook)
* [Result](https://2020.igem.org/Team%3APeking/Results)
* [Lab Attribution](https://2020.igem.org/Team%3APeking/Lab-Attribution)
* [Safety](https://2020.igem.org/Team%3APeking/Safety)

![](https://static.igem.org/mediawiki/2020/f/f7/T--Peking--achievement.png)

Achievement

---

* [Contribution](https://2020.igem.org/Team%3APeking/Contribution)
* [Proof of Concept](https://2020.igem.org/Team%3APeking/Proof%20Of%20Concept)
* [Human Practices](https://2020.igem.org/Team%3APeking/Human%20Practices)
* [Education](https://2020.igem.org/Team%3APeking/Education)

![](https://static.igem.org/mediawiki/2020/a/a8/T--Peking--modeling.png)

Algorithm

---

* [Music to DNA](https://2020.igem.org/Team%3APeking/music%20to%20DNA)
* [DNA to Music](https://2020.igem.org/Team%3APeking/DNA%20to%20music)
* [Scoring Algorithm](https://2020.igem.org/Team%3APeking/Fitness%20function)
* [Engineering](https://2020.igem.org/Team%3APeking/Engineering)

![](https://static.igem.org/mediawiki/2020/f/fd/T--Peking--team.png)

Team

---

* [Team Members](https://2020.igem.org/Team%3APeking/Team)
* [Attributions](https://2020.igem.org/Team%3APeking/Attributions)
* [Acknowledgement](https://2020.igem.org/Team%3APeking/Acknowledgement)
* [Collaboration](https://2020.igem.org/Team%3APeking/Collaborations)

![](https://static.igem.org/mediawiki/2020/4/41/T--Peking--project.png)

Project

---

* [Background](https://2020.igem.org/Team%3APeking/Background)
* [Description](https://2020.igem.org/Team%3APeking/Description)
* [Design](https://2020.igem.org/Team%3APeking/Design)
* [Implementation](https://2020.igem.org/Team%3APeking/Implementation)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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

# Team:Peking/Results

![](https://static.igem.org/mediawiki/2020/b/b9/T--Peking--team-icon-green.png)

[![](https://static.igem.org/mediawiki/2020/3/3c/T--Peking--home.png)](https://2020.igem.org/Team%3APeking)  Home

![](https://static.igem.org/mediawiki/2020/7/78/T--Peking--lab.png)

Lab

---

* [Notebook](https://2020.igem.org/Team%3APeking/Notebook)
* [Result](https://2020.igem.org/Team%3APeking/Results)
* [Lab Attribution](https://2020.igem.org/Team%3APeking/Lab-Attribution)
* [Safety](https://2020.igem.org/Team%3APeking/Safety)

![](https://static.igem.org/mediawiki/2020/f/f7/T--Peking--achievement.png)

Achievement

---

* [Contribution](https://2020.igem.org/Team%3APeking/Contribution)
* [Proof of Concept](https://2020.igem.org/Team%3APeking/Proof%20Of%20Concept)
* [Human Practices](https://2020.igem.org/Team%3APeking/Human%20Practices)
* [Education](https://2020.igem.org/Team%3APeking/Education)

![](https://static.igem.org/mediawiki/2020/a/a8/T--Peking--modeling.png)

Algorithm

---

* [Music to DNA](https://2020.igem.org/Team%3APeking/music%20to%20DNA)
* [DNA to Music](https://2020.igem.org/Team%3APeking/DNA%20to%20music)
* [Scoring Algorithm](https://2020.igem.org/Team%3APeking/Fitness%20function)
* [Engineering](https://2020.igem.org/Team%3APeking/Engineering)

![](https://static.igem.org/mediawiki/2020/f/fd/T--Peking--team.png)

Team

---

* [Team Members](https://2020.igem.org/Team%3APeking/Team)
* [Attributions](https://2020.igem.org/Team%3APeking/Attributions)
* [Acknowledgement](https://2020.igem.org/Team%3APeking/Acknowledgement)
* [Collaboration](https://2020.igem.org/Team%3APeking/Collaborations)

![](https://static.igem.org/mediawiki/2020/4/41/T--Peking--project.png)

Project

---

* [Background](https://2020.igem.org/Team%3APeking/Background)
* [Description](https://2020.igem.org/Team%3APeking/Description)
* [Design](https://2020.igem.org/Team%3APeking/Design)
* [Implementation](https://2020.igem.org/Team%3APeking/Implementation)

Result

David Liu’s lab created the first base editor in 2016 (Komor et al., 2016) and since then has been trying to expand their precision editing capabilities. Base editors make specific DNA base changes and consist of a catalytically impaired Cas protein (dCas or Cas nickase) fused to a DNA-modifying enzyme, in this case a deaminase. Base changes from C•G-to-T•A are mediated by cytosine base editors (CBEs) and base changes from A•T-to-G•C are mediated by adenine base editors (ABEs). How does this work? Through molecular biology teamwork. The guide RNA (gRNA) specifies the editing target site on the DNA, the Cas domain directs the modifying enzyme to the target site, and the deaminase induces the DNA base change without a DNA double-strand break. But base editors aren’t perfect. They may be slow, can only target certain sites, or make only a subset of base substitutions. (addgene blog by Susanna Bachle)

We used the existing plasmids for enzyme digestion and ligation, and ePCR was added to the BioBrick connector. After multiple rounds of splicing and assembly, we obtained the ABE and CBE we needed. The schematic diagrams are as follows:

# CBE

Until 2016, precise single base changes were only possible through exploiting the homology-directed repair (HDR) pathway which occurs in cells at low frequencies and relies on the efficient delivery of donor DNA to facilitate repair. Since the development of CRISPR-mediated base editing (BE), these types of repairs can now be done more efficiently than before. A base editor precisely changes a single base with an efficiency typically ranging from 2575%, while the success of precise change via HDR limited to 0-5%. This blog post covers a brief review of different basic BE technologies and their adaptation for plant genome editing. (addgene blog by Guest Blogger)

In 2016, two independent groups (komor et al., 2016 and Nishida et al., 2016) invented CRISPR base editor by linking cytosine deaminase with cas9 cleavage enzyme (ncas9), thus achieving accurate and efficient base rewriting in the genome. Ncas9 creates a gap in DNA by cutting only one single strand, thus greatly reducing the possibility of harmful insertion deletion. After binding with DNA, CBE deamination of target cytosine (C) into uracil (U) base. Later, the resulting U•G pairs were repaired through the cell mismatch repair mechanism to convert the original C•G pair into T•A, or reduced to the original C•G through the uracil glycosylase mediated base excision repair. The presence of UGI minimizes the second result and increases the production of required T•A base pairs.

![](https://static.igem.org/mediawiki/2020/b/b4/T--Peking--CBE-5.jpg)

(addgene blog by Guest Blogger)

![](https://static.igem.org/mediawiki/2020/2/2f/T--Peking--CBE-1.png)

![](https://static.igem.org/mediawiki/2020/e/e6/T--Peking--CBE-2.png)

![](https://static.igem.org/mediawiki/2020/6/64/T--Peking--CBE-3.png)

![](https://static.igem.org/mediawiki/2020/6/6c/T--Peking--CBE-4.png)

# ABE

![](https://static.igem.org/mediawiki/2020/f/f2/T--Peking--ABE-4.jpeg)

(Gaudelli et al., 2020.)

Gaudelli et al. have successfully developed an adenosine deaminase, which can act on DNA for adenine base editing. They first created a defective chloramphenicol resistance gene (CamR) by introducing a point mutation (H193Y). Reversal of this mutation by adenine base editor will restore antibiotic resistance. To find such a protein, they created a mutant library of E.coli tRNA adenosine deaminase (ecTadA), fused it with dcas9, and transformed it into E.coli containing the defective CamR gene. Screening of viable colonies and subsequent rounds of evolution and engineering produced a mutant TadA (TadA \*), which accepted DNA as a substrate satisfactorily.

The artificially evolved adenosine deaminase catalyzes the transformation of target "A" into "I" (inosine), which is regarded as "G" by cell polymerase. Subsequently, a primitive genome A•T base pair was transformed into a G•C base pair. Since inosine excision repair is not as active as uracil excision, ABE does not require any additional inhibitor proteins, such as UGI in CBE.

![](https://static.igem.org/mediawiki/2020/d/d8/T--Peking--ABE-1.png)

![](https://static.igem.org/mediawiki/2020/f/f7/T--Peking--ABE-2.png)

![](https://static.igem.org/mediawiki/2020/f/f7/T--Peking--ABE-3.png)

# gRNA-Scaffold

![](https://static.igem.org/mediawiki/2020/5/51/T--Peking--gRNA-Scaffold.png)

# EvolvR

## 1. Introduction of EvolvR

The capacity to diversify genetic codes advances our ability to understand and engineer biological systems. Several methods for generating mutant libraries exist, but none provide a means to continuously diversify all nucleotides within a user-defined genomic region. EvolvR, a CRISPR-Cas9 based targeted mutagenesis method developed by the Dueber Lab at Berkeley, provides a new approach for generating novel genetic variants in bacteria.

By fusing error prone polymerase Poll3M to nCas9, the nick site, introduced by sgRNA targeting and nCas9 nicking, serves as the initiate point for error prone PolI3M function. Due to its modular nature, the Dueber Lab created a few versions of EvolvR. PolI5M, generated by two additional mutations to PolI3M which increased EvolvR's mutagenesis rate to ~10-3 mutations per nucleotide per generation.

Here we use PolI5M and eSpnCas9 to target the music sequence, expecting to generate mutated music sequence and "listen to the mutation".

## 2. Construction of EvolvR

We use GoldenGate Assembly to construct EvolvR. By synthesizing eSpnCas9-linker-PolI5M 1kb per 1kb, we add GoldenGate tag and linked the parts. After that, the part was linked to standard pSB1C3 vector by T4 DNA ligase mediated ligation.

## 3. Experimental progress of EvolvR

Constructing pEvolvR

Ligating EvolvR

Cutting pSB1C3 vector

Linking EvolvR and pSB1C3 vector

Introduce pEvolvR to bacteria

Sequencing for the editing outcome

# Citation

[1] Madej T, Lanczycki CJ, Zhang D, Thiessen PA, Geer RC, Marchler-Bauer A, Bryant SH. " MMDB and VAST+: tracking structural similarities between macromolecular complexes. Nucleic Acids Res. 2014 Jan; 42(Database issue):D297-303

[2] Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature. 2017 Nov 23;551(7681):464-471. doi: 10.1038/nature24644. Epub 2017 Oct 25. Erratum in: Nature. 2018 May 2;: PMID: 29160308; PMCID: PMC5726555.

[3] Banno S, Nishida K, Arazoe T, Mitsunobu H, Kondo A. Deaminase-mediated multiplex genome editing in Escherichia coli. Nat Microbiol. 2018 Apr;3(4):423-429. doi: 10.1038/s41564-017-0102-6. Epub 2018 Feb 5. PMID: 29403014.

* [CBE](#a2)
* [ABE](#a1)
* [gRNA-Scaffold](#a3)
* [EvolvR](#a4)

© 2020 Peking iGEM. All Rights Reserved.