iGEM 2022 — Team tianjin

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{% block title %}Notebook{% endblock %}
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1. [April](#0)
2. [May](#0)
3. [June](#0)
4. [July](#0)
6. [August](#0)
8. [September](#0)
10. [October](#0)

* [Prev](#0)
* [Next](#0)

# April

Establishing the subject—multi-site editing—after taking other projects and skills of our members into consideration.
Interviewed Prof. Yi Wu.

April 29
Chinese characters stored in the computer was outputted in binary, then converted into ternary, and we coded the character/base conversion program based on ternary system.

# May

Around May 12
TPrevious plan of ternary system went through a few difficulties and had problems with realization. Inspired by opinions from other universities, especially some art schools, we decided to modify songs via base editing technique.

May 29
We realized that music has become universal as a form of digital information. Therefore, the coding system focused on already digitalized music, and designed music tracks based on MIDI.

End of May
Questionnaire 1.

# June

June 8
We designed the first generational coding rules focusing on music editing, with hopes of combining Chinese characters coding in gRNA design to increase density of the characters.

June 9
Previous code's lack of density led to further thinking, we were inspired after communicating with members from other crews and went through other projects, began to plan for the second generational code.

June 11
The base sequences we acquired cannot satisfy our requirements for gRNA design, so we turned to another coding system: we generated pseudo-random numbers, converted them into ternary, each group of numbers were transformed into six bases. Next step, estimated whether the base sequence is suitable for gRNA design and, if the answer is yes, define it as the base expression of one character.
We basically shaped the second generation, largely increased degrees of freedom. Concepts such as events, branches and switches were brought in with our combination of tip tree. Code density went up but needed further improvement.

June 20
The second generational code was used to design the information plasmid DNA sequence.

June 30
Repeat the transfer of the carotene plasmid into SY14 cells.

June 24
Dry lab made first attempt to construct gRNA sequences, the original plan was to combine gRNA sequences with both textual information and musical information. However, due to the long period of designing gRNA, the textual part was postponed.We used constructed gRNA arrays to design DNA.
Coded for viewing image size; acquired microvenus.png; coded for image cropping.

June 25
Coded Chinese character search and replace program.

June 26
Coded to generate the empty pixels and created the black and white pixel units; Coded program for pixel segmentation and converting them into 0s and 1s.

June 27
Interviewed Dr. Mingzhe Han.

# July

*July 1 ~ July 15*

July 1
First and second generational decoding took off. Information plasmid design completed.

July 3
Coded the first generational encoding program and decoding program of images.

July 8
Decoding part is completed, and we started to code the music module (.MID) which can be played by the system according to the decoding rules. We used winhex software to decipher and reverse the low-level code, iterating the file to obtain the corresponding track (inputted as DNA fragments).
Coded the second generational encoding program of images.

July 9
Coded the second generational decoding program of images.

July 10
Coded the GIF generating program.

July 13
Coded the program to generate original image; coded generating program for modifying target points.

July 14
Coded the program for decoding second type of sequence.

July 15
Design of information plasmid storing Ode to Joy accomplished and sent to a biotech company for synthesis.
Coding and music track are basically completed, also generally connected with wet lab experiment.
Starting to optimize the code itself, aiming to increase coding efficiency and information density.
Due to the time cycle length and shortage of time, third generational code did not go through wet experiment verification, but it is theoretically achievable.

# July

*July 16 ~ July 31*

July 16-July 17
Participated in the meetup.
Communicated with TJU\_SLS offline.

July 18
Coded the program to generate images from 0s and 1s.
Acquired the GIF generated in second type of sequence; generated informative sequences 1 and 2.

July 20
Accomplished combination of informative sequences 1 and 2, DNA sequence synthesis was outsourced to a company.
Coding the GUI.

July 21
Information plasmid storing GIF was successfully designed and sent to a biotech company for synthesis.

July 22
Formation of third generational code was completed, we started to use software such as C, Python, eclipse (JAVA) and winhex to code the third generation. (Modifying input and output according to the underlying code, iterating file format and fine-tuning internal parameters).

July 25
We began to practice yeast galactose-induction, modified the metabolic pathways of violacein in yHX0378 to check the modification efficiency of gRNA array.
Inoculated X-16-2, X-16-4, X-22-4 to SC-L-H-U liquid medium for cell expansion.
Designed premiers for metabolic pathways of violacein to check the effect of our modification.

July 26
Transferred X-16-2, X-16-4, X-22-4 to SG-L-H-U+1/3ade liquid medium for inducing.

July 27
We coated Induced X-16-2, X-16-4, X-22-4 on SC-L-H-U+1/3ade medium.

July 29
Obtained premiers for metabolic pathways of violacein.

July 30
We performed PCR to verify the modification of metabolic pathways of violacein.

July 31-September 7
Coded the first edition of GUI for GIF generating program.

End of July
Interviewed Prof. Yingxiu Cao

# August

*Volunteer educating during summer vacation about synthetic biology and iGEM.*
*AUGUST 1 ~ AUGUST 19*

August 1- August 2
First time to have acquired results of modifying metabolic pathways of violacein.

August 4
First time to have selected successfully grown red colonies of yNuwa002 to streak plate culture on SC-L-U+1/3ade medium.
Secondary inoculated X-16-2, X-16-4, X-22-4 to SC-L-H-U liquid medium for cell expansion.
Third generational coding basically finished, we were able to code simple music tracks, as for more complex tracks, coding is still in progress due to technical problems and lack of time.

August 5
Secondary transferred X-16-2, X-16-4, X-22-4 to SG-L-H-U+1/3ade liquid medium for inducing.

August 6
The red colonies streak plate cultured once were streak plate cultured again.
Second time to have coated Induced X-16-2, X-16-4, X-22-4 on SC-L-H-U+1/3ade medium.

August 11
Lectured about DNA storage on Bilibili.com.

August 13
Second time to have selected successfully grown red colonies of X-16-2 on SC-L-U+1/3ade medium to first time streak plate culture.

August 17
Red colonies successfully grown were streak plate cultured again.

August 19
We obtained Escherichia coli with integrated GIF plasmid from GENERAL BIOL.

# August

August 20
Used streak plate technique to culture GIF plasmid integrated Escherichia coli.

August 21
Lectured for international students.

August 26
Designed premiers for GIF plasmid for synthesis.

August 27
We inoculated eight tubes of Escherichia coli with GIF plasmid integrated to LB+AMP liquid medium.
Inoculated one tube of yHX0362 primary yeast to SC-U liquid medium.

August 28
We inoculated one tube of yHX0362 secondary yeast to SC-U liquid medium.
Extracted 50μL×8 GIF plasmids.
Performed transformation of constitutive and inducible yeast.

August 29
Constitutive yeast coated on SC-L-U-H+1/3ade+hyg medium.
Inducible yeast coated on SC-L-U+1/3ade+hyg medium.

August 30
We obtained premiers for GIF plasmid.

August 31
We obtained successfully synthesized Ode to Joy plasmid from GENERAL BIOL.

End of August
Questionnaire 2
Interviewed Prof. Yunzi Luo

# September

*September 1 ~ September 16*

September 1
We acquired successfully transformed inducible GIF strain: yNuwa002.
Inoculated yNuwa002 to SC-L-U+hyg liquid medium for cell expansion.
We gathered our current possessed software and programs to form a keyboard game.

September 2
Transferred yNuwa003 to SG-L-U+1/3ade+hyg liquid medium for inducing.

September 3
Induced yNuwa003 were coated on SC-L-U+1/3ade+hyg medium.
We designed premiers for Ode to Joy plasmid and sent them for synthesis.
Basically accomplished modifying the metabolic pathways of violacein.
Communications with freshmen.

September 5
We obtained successfully synthesized premiers for Ode to Joy plasmid.

September 6
We selected successfully grown red colonies of yNuwa002 to streak plate culture on SC-L-U+1/3ade medium.

September 8-September 25
Coded the second edition of GUI for GIF generating program.
Decoding of Sequencing results.

September 11
Construction of Ode to Joy plasmid accomplished.

September 14
Extracted Ode to Joy plasmid.

September 16
We inoculated one tube of yHX0362 primary yeast to SC-U liquid medium.

# September

*September 17 ~ September 28*

September 17
We inoculated one tube of yHX0362 secondary yeast to SC-U liquid medium.
Performed transformation of constitutive and inducible yeast.

September 18
Constitutive yeast coated on SC-L-U-H+1/3ade+hyg medium.
Inducible yeast coated on SC-L-U+1/3ade+hyg medium.

September 21
We acquired successfully transformed inducible Ode to Joy strain: yNuwa003.
First batch of GIF red colonies were performed PCR and sent for sequencing.

September 22
Inoculated yNuwa003 to SC-L-U+hyg liquid medium for cell expansion.
Obtained first batch of GIF plasmid modification results.

September 22-September 27
Improved the decoding program, successfully acquired the decoding results for single-cell sequencing.

September 23
Transferred yNuwa003 to SG-L-U+1/3ade+hyg liquid medium for inducing.

September 24
Induced yNuwa003 were coated on SC-L-U+1/3ade+hyg medium.

September 27
Second batch of GIF red colonies were performed PCR and sent for sequencing.

September 28
Obtained second batch of GIF plasmid modification results.

# October

October 5
Red colonies of yNuwa003 were picked out for PCR and sent to GENEWIZ for sequencing.

October 6
Obtained modification results of Ode to Joy plasmid.

October 8
Summarizing information plasmid modification results accomplished.

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* [Overview](#Overview)
* [The Round of GIF Coding](#The Round of GIF Coding)
  + [Design](#The Round of GIF Coding Design)
  + [Build](#The Round of GIF Coding Build)
  + [Test](#The Round of GIF Coding Test)
  + [Learn](#The Round of GIF Coding Learn)
* [The Round of Music Coding](#The Round of Music Coding)
  + [Design](#The Round of Music Coding Design)
  + [Build](#The Round of Music Coding Build)
  + [Test](#The Round of Music Coding Test)
  + [Learn](#The Round of Music Coding Learn)

## Overview

---

Through iteration, three final programs are obtained in the dry experiment part. The Chinese
character base conversion program can convert 3,000 common characters into corresponding base
sequences. The image-storage program can store a 35-pixel image within 2,500 bp of DNA sequence,
while allowing six different images to be made at a time. A music program can store "Ode to Joy"
in DNA sequences of up to 2, 000bp and make a single revision to produce another song, "Auld
Lang Syne". In the wet experiment section, the most suitable chassis cells and experimental
conditions for multi-site editing were obtained.

## The Round of GIF Coding

---

#### Design

---

![](https://static.igem.wiki/teams/4362/wiki/engineering/p-1.png)

1) We planned to develop a program capable of converting 7X5 pixel maps into DNA sequences.

2) The sequence would be introduced into yeasts, and the multi-site editing technique was used to
convert the bases.

3) We planned to develop the decoding system to resolve the changes and get another pixel map.

#### Build

---

1) We developed the first generation of photo storage programs. But it took about 2,500 bp to store
such a pixel map, and we planned to store seven-pixel maps. Large pieces of DNA take too long to
synthesize. we developed a second coding system to reduce the length of DNA that needs to be
synthesized. The second encoding system created a new pixel map based on the previous image by
encoding only the pixels that need to be changed, so that the remaining six images total only about
2,500 bp, greatly reducing the length of DNA we need to synthesize.

2) we developed a first-generation decoding program for decoding the sequencing results of
individual
cells.

3) However, there are many unedited sites in a single cell. Not only do we need to get single colony
editing results, we need to synthesize all the strains tested for decoding. So we developed a
second-generation decoding program that can synthesize the sequencing results of five cells。

#### Test

---

By the image coding program, we obtained a DNA sequence of a Micro Venus pixel map . with the
guidance of dry experiment, we also conducted wet experiments via multi-site editing.

#### Learn

---

Successfully decoded the modified seven images, and verified our image coding design.

[Click here for more
information](https://2022.igem.wiki/tianjin/software)

## The Round of Music Coding

---

#### Design

---

![](https://static.igem.wiki/teams/4362/wiki/engineering/p-2.png)

1) We planned to develop a program that can convert music into DNA sequences.

2) By transforming the sequence into yeasts and using multi-site editing system, we planned to
change
the bases.

3) We planned to develop the decoding system to convert nonsense notes into a piece of reasonable
music, such as Ode to joy.

#### Build

---

1) We have developed the first music coding program capable of storing the Ode to Joy in a DNA
sequence. Professor Wu Yi, the instructor, thought that the storage density was not satisfied, and
there may be some targets misaligned in double-stranded coding.

2) In order to improve the data density, we believe that it is more advantageous to adopt a
MIDI-like encoding format.
We developed the second generational code according to the underlying logical code of MIDI.

3) Since the second generation coding system was developed too late, we chose to design plasmids
with the first generation coding system.

4) We developed a decoding program.

#### Test

---

We conducted multi-site editing wet experiments and We sequenced the plasmid.

#### Learn

---

Using the decoding program, we successfully converted the encoded sequence into the MIDI format of
Ode to Joy which could play on the computer.

[Click here for more
information](https://2022.igem.wiki/tianjin/proof-of-concept)

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* [Overview](#Overview)
* [Construction of yNuwa001](#Construction of yNuwa001)
* [Construction of pRS-hyg-joy](#Construction of pRS-hyg-joy)
* [Construction of yNuwa002 and
  yNuwa003](#Construction of yNuwa002 and yNuwa003)
* [Expression of base editor by
  inducing](#Expression of base editor by inducing)
* [Sequencing](#Sequencing)

## Overview

---

| Strains | Description | Source |
| --- | --- | --- |
| yHX0362 | *Saccharomyces cerevisiae* integrating CBE coding sequence on chromosome | Basic items owned by the experimental center |
| yHX0378 | *Saccharomyces cerevisiae* integrating violacein pathway on chromosomeⅩ | Basic items owned by the experimental center |
| S.cerevisiae strain BY4741 | Original *Saccharomyces cerevisiae* used for transformation | Basic items owned by the experimental center |
| S.cerevisiae strain BY4742 | Original *Saccharomyces cerevisiae* used for transformation | Basic items owned by the experimental center |
| Escherichia coli top10 | Universal *Escherichia coli* clone host | Basic items owned by the experimental center |
| yNuwa001 | yHX0378 containing pZJ-leu-30gRNA and pRS413-CBE | Construction of this work |
| yNuwa002 | yHX0378 containing pZJ-leu-30gRNA and pRS413-CBE | Construction of this work |
| yNuwa003 | yHX0362 containing pZJ-leu-30gRNA and pRS-hyg-joy | Construction of this work |

| Plasmids | Description | Source |
| --- | --- | --- |
| pZJ-leu-30gRNA | Carried by pCCI, contains gRNA arrays that target multiple editing sites and leu tag | Basic items owned by the experimental center |
| pRS413-CBE | Carried by pRS413, contains CBE that can convert C bases to T bases | Basic items owned by the experimental center |
| pDG1730 | E.*coli* and B.*subtilis* shuttle expression vector used for partnership | From SJTU |
| pRS-hyg | Carriers containing hygromycin tags for multi-site editing of information sequences | Basic items owned by the experimental center |
| pRS-hyg-joy | Carried by pRS-hyg, contains Ode to joy message | Construction of this work |
| pRS-hyg-GIF | carried by pRS-hyg, contains GIF message | Construction of this work |

| Primer | Sequence(5'-3') | Purpose |
| --- | --- | --- |
| FRAG-1-F | GTTCAAATCTCACGTGCAGC | Verify the modification of yHX0378 sites |
| FRAG-1-R | CTCCTCAAATTGCTACCACGAC |
| FRAG-2-F | CATTCTGGATTTCCCGCGTAT |
| FRAG-2-R | GCGTAATCCACTTCTTCGACG |
| FRAG-3-F | CCGAAAGCTCGTTTGTTCTTGA |
| FRAG-3-R | CAAGAACAATGCAATAGCGCATC |
| FRAG-4-F | GATGTCGGAGGTTGGTAATGC |
| FRAG-4-R | GGACGGCTCATTATGATGCAC |
| FRAG-5-F | GCGCATGCCTATAAGTATAGCG |
| FRAG-5-R | AAGATCCAGTCTCCAGATCTGC |
| FRAG-6-F | GTGCGATTATCGTTGGTGGC |
| FRAG-6-R | CTTCAGGTTGTCTAACTCCTTCC |
| MF-1-F | CCTCTTCGCTATTACGCCAGC | Verify the modification of pRS-hyg-GIF sites |
| MF-2-R | CTTGGACATCGACTCCTAGAAGTTCC |
| MF-3-F | CCACTCTCGATTGGACCGTG |
| MF-3-R | CACCATGATAGGTCAGCTAACAAGG |
| MF-4-F | GCGTCTGGAAGTCGAGGACAAG |
| MF-4-R | CAAAAGCTGGAGCTCCACCG |
| SM-F | CCTTAGACCAGAACGACTTGTTGGAC |
| SM-R | CCGCCACAGCACGAATTGTAT |
| HLS-F | GTGCACCATATGCGGTG | Verify the modification of pRS-hyg-joy sites |
| HLS-R | GGATCCGATAGCGGATAACAA |

## Construction of yNuwa001

---

We used rapid yeast transformation for yHX0378, this type of transformation is simple and
time-saving, therefore
we were able to free up more time for our remaining experiments.

1.The yeast colonies were incubated in 5 mL of yeast peptone dextrose (YPD) overnight at 30℃ in
the rocking
device at 220 rpm.

2.The next day, we extracted 500 µL of the culture to 5 mL of YPD.

3. When OD600 reached 0.4-0.6, 1 mL of the yeast cells were harvested and washed twice with
sterile water.

4.Then we mixed 240µL of 50% polyethylene glycol (PEG) 3350, 50 µL of ssDNA and 36 µL of 1 M
LiOAc together,
mixture served as transformation buffer. The ssDNA was preheated at 101 ℃ for 12 mins and then
placed on ice
for cooling down promptly.

5.Each kind of plasmid would be added 2 µL (pZJ-leu-30gRNA,pRS413-CBE) and about 20µL sterile
water was added to
the yeasts. The preconditioned yeast cells (about 34 µL) then were immersed into the
transformation buffer and
then mixed by being inverted up and down 7-10 times, afterwards we incubated the mixture at 42℃
for 40 min in
water bath/metal bath.

6.The solution was centrifuged at 4000rpm for 1 min, after which we discarded the supernatant.
Remained cells
were plated onto a selective media.

## Construction of pRS-hyg-joy

---

We got a plasmid from the GENERAL BIOL which contained the information of Ode to joy, however,
it is not reasonable to directly import the plasmids into Saccharomyces cerevisiae. The
following operations are performed.

1.Firstly, we obtained the Ode to joy information fragments by restriction enzyme EcoRI and
BamHI double digestions and gel extraction.

2.Meanwhile, KOD ONE PCR was performed on our original GIF plasmids and we also used gel
extraction to obtain vectors. After the vectors and fragments were assembled by seamless
cloning, we obtained the required information plasmids, pRS-hyg-joy.

3.Next, we transformed the plasmids into Escherichia coli for amplification. It also ensured the
normally progress of our subsequent experiments.

4.After the plasmids were extracted from Escherichia coli, we added the designed primers and
performed colony PCR and electrophoresis verify.

In order to make sure of the results' accuracy, we sent them to GENEWIZ for sequencing and
obtained accurate plasmids sequences. The sequencing results will determine whether the
plasmid-construction succeeded or not.

![](https://static.igem.wiki/teams/4362/wiki/experiment-1.png)

## Construction of yNuwa002 and yNuwa003

---

Although the rapid yeast transformation method is the same, it should be noted that there are
some differences in the transformation of yHX0362.

1.We first integrated the CBE fragment on yHX0362 genome, used SC-U culture for incubation.

2.Then, we designed two sets of experiments of induced yeasts and constitutive yeasts to observe
the differences between the two transformation methods. Induced yeasts contained pZJ-leu-30gRNA
and information plasmids (pRS-hyg-joy and pRS-hyg-GIF). The constitutive yeasts possessed one
more pRS413-CBE than the induced yeasts.

3.Because the plasmid has antibiotic tag, the yeasts need to be returned to its resistant growth
state by overnight incubation after a heat shock. We used YPD to help them recover and gain the
ability of growing in culture containing antibiotics (hygromycin).

4.We coated constitutive yeasts to SC-L-U-H+1/3 ADE + HYG and the other yeasts to SC-L-U+1/3 ADE
+ HYG.

## Expression of base editor by inducing

---

1.The yeast colonies were incubated in 5 mL of SC-L-U + HYG about 1 day at 30 ℃ with shaking
device at 220 rpm.

2.Take 1000µL yeast after culture, centrifuge(4000rpm×1min), clean the waste liquid, add 1mL
sterile H2O and centrifuge again (4000rpm×1min). Repeat for two to three times, and 5mL
SG-L-U+1/3 ADE + HYG (Galactose and Raffinose replaced the Glucose in SC) was put into the
shaking tubes.

3.We then extracted 200µL SG-L-U+1/3 ADE + HYG into the centrifuge tubes, suspended yeast cells
by suction, extracted 100 µL of which and transferred them back into the previous tubes, and
induced them at 30℃ for 24h.

4.After inducing, we extracted 4µL yeast solution and add 200µL sterile water, put 102 µL of the
mixture onto the appropriate selective SC media, waited for them to grow visible red colonies,
then they were picked and sent for sequencing.

## Expression of base editor by inducing

---

1.The yeast colonies were incubated in 5 mL of SC-L-U + HYG about 1 day at 30 ℃ with shaking
device at 220 rpm.

2.Take 1000µL yeast after culture, centrifuge(4000rpm×1min), clean the waste liquid, add 1mL
sterile H2O and centrifuge again (4000rpm×1min). Repeat for two to three times, and 5mL
SG-L-U+1/3 ADE + HYG (Galactose and Raffinose replaced the Glucose in SC) was put into the
shaking tubes.

3.We then extracted 200µL SG-L-U+1/3 ADE + HYG into the centrifuge tubes, suspended yeast cells
by suction, extracted 100 µL of which and transferred them back into the previous tubes, and
induced them at 30℃ for 24h.

4.After inducing, we extracted 4µL yeast solution and add 200µL sterile water, put 102 µL of the
mixture onto the appropriate selective SC media, waited for them to grow visible red colonies,
then they were picked and sent for sequencing.

## Sequencing

---

Before sequencing, we need do some preprocessing. Select induced red colonies, boil and add
primers for PCR amplification. They will then be sent to GENEWIZ for the results. The relevant
experimental details of PCR can be referred to Protocol.
 [(click to protocol)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)

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{% block title %}Results{% endblock %}
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* [Overview](#Overview)
* [Design and
  Construct Micro Nuwa Multi-site Editing System](#Design and Construct Micro Nuwa Multi-site Editing System)
* [Measurement of Micro nuwa editing
  system](#Measurement of Micro nuwa editing system)
* [Editing of picture (Micro Venus) and music (Ode to Joy) data sequences](#Editing of picture (Micro Venus) and music (Ode to Joy) data sequences)
* [Verifying editing result via
  Sanger Sequencing](#Verifying editing result via Sanger Sequencing)
  + [The sequencing result of GIF](#The sequencing result of GIF)
  + [The sequencing result of Ode to Joy](#The sequencing result of Ode to Joy)

## Overview

---

On this page, we will focus on the final results and statistical data of each experimental
part. It consists of the following main parts:

1. We have constructed a multi-site editing system, which consists of ultra-long gRNA array
and CBE. In addition, plasmids containing pictures(Micro Nuwa) and music(Ode to Joy) data
were also successfully constructed.

2. We also verified the editing efficiency of Micro nuwa through the cell phenotype at the
colony level.

3. It has proved that Micro nuwa successfully edited the data sequence of pictures (Micro
Venus) and music (Ode to Joy).

4. The editing effect of Micro Nuwa editing system is verified by sanger sequencing. Micro
Nuwa. We have re-edited 33 out of 36 sites, and up to 27 sites in a single colony were
re-edited simultaneously.

In the following subsections, we will analyze all the experimental results in detail.

## Design and Construct Micro Nuwa Multi-site Editing System

---

We designed and constructed a 30-gRNA array, and transferred it into Saccharomyces cerevisiae
yHX0362, which integrates the base editor with galactose-inducible promoter in the genome, to
construct the engineering strains of our Micro Nuwa editing system.

![](https://static.igem.wiki/teams/4362/wiki/results/01.png)

Figure 1: Ultra -long gRNA arrays.

![](https://static.igem.wiki/teams/4362/wiki/results/02.png)

Figure 2: The structure of nCDA1Δ198-BE3

In addition, we constructed the data plasmid pRS-hyg-joy. The method of constructing plasmid is
seamless cloning, and the method of verifying whether succeed is PCR and sequencing of the two
junctions. The recombinant plasmid was transformed into a strain containing the multi-site editing
system, and the strains yNuwa002 and yNuwa003 were obtained.

![](https://static.igem.wiki/teams/4362/wiki/results/3.png)

Figure 3: The connection verification of the junctions (Marker: Trans2K PlusⅡDNA Marker, Odd
numbers are the results of PCR of junction1, Even numbers are the results of PCR of junction2 )

## Measurement of Micro nuwa editing system

---

We transferred Micro Nuwa editing system into the strain yHX0378 expressing violacetin, in which
gRNA was designed to target five genes of VIO pathway. We verified the multi-site editing
ability of the Micro Nuwa editing system through the cell phenotype at the colony level and
sanger sequencing. Among the 25 editing sites, 23 sites were edited. Up to 20 sites were edited
in single colony. The average number of edited sites is 17.25.

![](https://static.igem.wiki/teams/4362/wiki/results/4.png)

Figure 4: Editing situation of VIO pathway. The edited sites are arranged in horizontal rows. The
8 colonies sequenced were arranged in columns. The sequencing results validated the editing
capability of our 30-gRNA array.

## Editing of picture (Micro Venus) and music (Ode to Joy) data sequences

---

We induced yNuwa002 and yNuwa003 with galactose at 30 ℃ for 24h. Under the condition of
OD600=0.3, SC-Leu-Ura+1/3ade+hyg nutrient deficient medium is used for screening.

![](https://static.igem.wiki/teams/4362/wiki/results/5.png)

Figure 5: Multi-site editing results. (A) Average editing efficiency 58.83%. (B) Average editing
efficiency 53.53%.

## Verifying editing result via Sanger Sequencing

---

We obtained the data sequences in pRS - hyg – joy and pRS - hyg – GIF plasmids through PCR, and
then carried out agarose gel electrophoresis on the PCR products. We split the GIF data sequence
into three fragments for PCR. The length of the sequence was verified by electrophoresis.

![](https://static.igem.wiki/teams/4362/wiki/results/6.png)

Figure 6: Electrophoresis Results of Segmented Information Fragments(Marker: Trans2K PlusⅡDNA
Marker, NO.1 to 8 are the results of PCR of fragments)

## The sequencing result of GIF

---

After obtaining the sequence from GENEWIZ, we compared the sequencing file with the original
file and analyzed the editing results. In the pRS - hyg – GIF plasmid, we designed 36 editing
sites, including: 33 sites were edited, and up to 27 sites were edited in the colony, with an
average of 13.8 sites edited.

![](https://static.igem.wiki/teams/4362/wiki/results/7.png)

Figure 7: The number of colonies with editing events at each site.

![](https://static.igem.wiki/teams/4362/wiki/results/8.png)

Figure 8: The number of editing events per colony.

The editing event on B-7, B-12 and C-5 did not occur. The C-G content of these gRNA sequences is
high, which may form a secondary structure after being released. This will affect the binding of
dCas9 and sgRNA.

![](https://static.igem.wiki/teams/4362/wiki/results/9.png)

Figure 9: All sites in yNuwa002

When the sequence is input into the decoding program, the actual GIF can be obtained, and the
[proof of concept](https://2022.igem.wiki/tianjin/proof-of-concept#Proof%20of%20Concept%20for%20Dancing%20Venus%20Data%20Storage) can be done.

## The sequencing result of Ode to Joy

---

After obtaining the sequence from GENEWIZ, we compared the sequencing file with the original
file and analyzed the editing results. In the pRS - hyg – JOY plasmid, we designed 9 editing
sites, including: 8 sites were edited, and up to 7 sites were edited in the colony, with an
average of 3.2 sites edited.

![](https://static.igem.wiki/teams/4362/wiki/results/10.png)

Figure 10: The number of colonies with editing events at each site.

![](https://static.igem.wiki/teams/4362/wiki/results/11.png)

Figure 11: The number of editing events per colony.

![](https://static.igem.wiki/teams/4362/wiki/results/12.png)

Figure 12: All sites in yNuwa003.

When the sequence is input into the decoding program, the actual piece can be obtained, and the
[proof of concept](https://2022.igem.wiki/tianjin/proof-of-concept#Proof%20of%20Concept%20for%20Ode%20To%20Joy%20Data%20Storage) can be done.

{% endblock %}