iGEM 2021 — Team Tianjin

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# Team:Tianjin/Notebook

Team:Tianjin/Notebook-2021.igem.org

* [Home](https://2021.igem.org/Team%3ATianjin)
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* Project

  [Description](https://2021.igem.org/Team%3ATianjin/Description)
  [Design](https://2021.igem.org/Team%3ATianjin/Design)
  [Engineering](https://2021.igem.org/Team%3ATianjin/Engineering)
  [Measurement](https://2021.igem.org/Team%3ATianjin/Measurement)
  [Results](https://2021.igem.org/Team%3ATianjin/Results)
  [Implementation](https://2021.igem.org/Team%3ATianjin/Implementation)
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* Lab

  [Notebook](https://2021.igem.org/Team%3ATianjin/Notebook)
  [Protocol](https://2021.igem.org/Team%3ATianjin/Protocol)
  [Safety](https://2021.igem.org/Team%3ATianjin/Safety)
* Human Practices

  [HP Overview](https://2021.igem.org/Team%3ATianjin/Human_Practices)
  [Integrated HP](https://2021.igem.org/Team%3ATianjin/Human_Practices)
  [Edu&Communication](https://2021.igem.org/Team%3ATianjin/Education)

![](https://static.igem.org/mediawiki/2021/a/ab/T--Tianjin--jiaomuduan.png)

1. [Experiment started](#0)
2. [Construction of CREATE cells](#0)

7. [Test CREATE cells](#0)

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

# Preliminary Preparations

(1) Prepare YPD solid medium
(2) Obtain the original cells, including 4741 (4741A), 4742 (4741α), and SY14.After obtaining the YPD medium, the solid plate is streaked.
(3) For bacteria to be stored in glycerol, new plates should be drawn regularly (1- 2 weeks) to maintain the vigor of the bacteria.
(4) Collection of various consumables, cleaning and recycling of test tubes, glass beads, large pipette tips, conical flasks, and reagent bottles.
(5) Laboratory cleaning and finishing work, necessary reagents and common mother liquor (such as: deionized water, amino acid mother liquor, sodium hydroxide, etc.) equipped with pipette tips, toothpicks, reagents, culture medium, centrifuge tube sterilization.

# Construction of fGFP *MAY 10 ~ AUGUST 30* The main content of this experiment is to continuously update the original to construct the GFP plasmid, so as to realize the iteration of GFP. We first use GFP with an unknown promoter to construct a plasmid and transfer it into the cell to make it often expressed. However, because the plasmid is difficult to stably exist and is ethanol-inducible, the effect is poor in subsequent induction experiments, so we abandoned this plasmid. So we replaced the promoter and used ADH1 to control its transcription to get GFP plasmid 2.0. In subsequent experiments, we found that GFP has a very stable expression intensity under the condition of normal expression, and it can still maintain a high fluorescence brightness after the cell loses the chromosome, which will affect the follow-up observation of whether the yeast cell has completed the chromosome excision. We chose to construct the fGFP plasmid. The fGFP plasmid can ensure that fGFP is normally expressed in chromosome yeast cells, while chromosome-free cells do not have fGFP and no longer produce fluorescence. Because the half-life of fGFP is short, after sorting, the brightness of the control group's gfp will be stable, while the experimental group It will get darker and darker until the fGFP is completely degraded and there is no bright light. It is more convenient for the characterization of experiments. The following will introduce the experimental process based on the three GFP plasmids. * [x] Construct a Ura-tagged GFP frequently expressed fragment and transfer it to SY14 May 10 Yeast transformation, transfer Cas9 into SY14 cells to obtain SY14-Cas9 cells Obtain 6 GFP fragments promoter P, left homology arm L, right homology arm R, terminator T, GFP main body G fragment, and Ura fragment from PRS416; May 13 Overlap connects the following fragments of GFP: Group A: P and T segments; Group B: U and R segments; Group C: L and P fragments; Group D: G and T segments; Group E: L-P-G-T fragment; And full-length fragments; PCR verifies whether the above fragments are connected. Result: P-G-T; U-R; G-T; L-P-G-T connected correctly. Culture SY14-Cas9 cells. Yeast transformation: Control group: transfer Leu plasmid into SY14-Cas cells; Experimental group: Delta6 plasmid was transferred into SY14-Cas cells. May 14 to May 29 Overlap connect B and E fragments and verify by PCR; Overlap connect L-G-U-R fragments and verify; Correct fragments are glued and stored; Verify whether Leu plasmid is transferred to SY14-Cas cells by PCR and agarose gel electrophoresis; It was verified by PCR and agarose gel electrophoresis that the Delta6 plasmid was transferred into SY14-Cas cells. May 29 Get full-length GFP. Transform the ligated full-length GFP plasmid into yeast SY14-Cas cells. May 30 to June 4 Overlap repeats the connection work of each fragment. Connect the repeatedly connected fragments and transfer them into yeast SY14-Cas cells. Verify whether the GFP plasmid has successfully entered the cell by PCR and agarose gel electrophoresis. June 4 It proved that the plasmid transferred into the cell may not exist stably, and the induction operation was not good, so it was decided to abandon the construction of the experimental plasmid. * [x] Construct the GFP2.0 frequently expressed fragment of ADH1 promoter and transfer it into SY14-Cas yeast June 5 Replace the promoter in GFP1.0 to construct plasmid GFP2.0 Connection (the following are all 2.0 fragments) Group A: P and T segments; Group B: U and R segments; Group C: L and P fragments; Group D: G and T segments; Group E: L-P-G-T fragment; Overlap group E and group B and verify. June 6 Get the full-length GFP2.0 fragment after verification, the result is correct, and the gel will be collected Transform the obtained GFP2.0 plasmid into yeast SY-14 Overlap repeatedly connects each group of fragments. June 7 to June 29 Overlap repeatedly connects each group of fragments Overlap repeatedly connects group E and group B, group D and group B to obtain the full-length plasmid GFP2.0 Transform each full-length fragment GFP2.0 into SY14-Cas yeast and perform PCR verification July 5 It was verified that the full-length GFP2.0 plasmid transferred into yeast SY14-Cas on June 29. The L, P, R, T, and G fragments were successfully ligated to the plasmid, but the Ura tag was not ligated, and subsequent ligation of the fragments and transformation were required. July 8 Overlap connect the full length GFP2.0 fragment, F-U-R fragment, U-R fragment and verify. T-U-R and U-R are successfully connected. July 8 to July 14 Link the obtained T-U-R fragment and U-R fragment with other fragments July 15 Verify GFP-Ura, the experiment is correct, it proves that the Ura tag is successfully inserted into the plasmid, and the complete plasmid GFP2.0 is obtained July 17 to July 19 Transfer the obtained GFP2.0 into yeast cell SY14-Cas Transform the obtained GFP-Ura into yeast cells SY14-Cas The verification proved that GFP2.0 was successfully transferred into yeast cell SY14-Cas, and GFP-Ura was successfully transferred into yeast cell SY14-Cas. Store the above two kinds of cells at low temperature. This step is all completed. * [x] Construct a fragment of fGFP that can be induced to rapidly degrade and transfer it to 4742 cells August 23 Obtain Escherichia coli containing fGFP fragments from the laboratory and store them in solid medium. Extract the fGFP plasmid. The fGFP fragment was transferred into yeast cells containing empty plasmids. The fGFP fragment was transformed into yeast cells containing Delta-g. The fGFP fragment was transformed into yeast cells containing 7flip-Cre. August 24 to August 30 The presence of fGFP fragments in yeast cells was verified by PCR and agarose gel electrophoresis. And ligate the ligated fGFP-Cre with the empty plasmid. Obtain FGFP-empty-Cre cells.

# Construction of carotene plasmid *MAY 17 ~ SEPTEMBER 23* May 17 Extract the carotene plasmid and transfer it into SY14 cells. May 19 to May 27 Verify whether carotene is transferred into cells by PCR and agarose gel electrophoresis. May 31 Repeat the transfer of the carotene plasmid into SY14 cells. June 5 Verify whether the carotene plasmid is successfully transferred into SY14 cells. June 30 Repeat the transfer of the carotene plasmid into SY14 cells. July 1 Extract the carotene plasmid in Escherichia coli. July 2 to July 4 Since the transfer of the carotene plasmid into yeast was not good, in order to preserve the carotene plasmid, the method of electrotransformation of Escherichia coli was adopted. The plasmid was first transferred to Escherichia coli for storage to obtain Escherichia coli 001 and E. Method, transfer the obtained plasmid into CREATE empty yeast cells. July 8 The plasmid in E. coli 002 was extracted and transferred to yeast. July 12 Yeast did not produce carotene, and PCR did not verify the presence of a carotene plasmid in yeast. July 21 to July 22 Transform tetR into E. coli 002, and verify by PCR and agarose gel electrophoresis. No good experimental results were obtained. July 26 to July 27 Transform tetR into E. coli 002, and verify by PCR and agarose gel electrophoresis. The assembled carotene plasmid is obtained. July 29-August 1 The assembled carotene plasmid was transferred into GFP-Ura yeast cells and verified by PCR and agarose gel electrophoresis. August 2 to August 9 The carotene plasmid and Delta-g plasmid were transferred into 4741 cells and passed verification. After testing the Delta-g plasmid to knock out the chromosomes of yeast cells, the carotene gene can still be expressed normally. Transfer the yeast containing Delta-g and carotene 4741 into the medium containing gal and the medium without gal, as the experimental group and the control group, let gal induce the expression of Delta-g and cut the chromosome. After 23 hours of incubation, the OD of the experimental group and the control group were measured and diluted by the same multiple (104, 103, 102) to plate the culture. Record the growth status of the cells, and record the number of colonies and the color of the colonies to verify that the carotene gene can still be expressed after the chromosome is absent. The results verified that the carotene gene did not appear in the cells, and the Delta-g band was normal. Due to the escape of the Delta-g plasmid, the carotene-Delta-g plasmid system was terminated. September 4 to September 23 The plasmid for extracting carotene is electrotransformed into Escherichia coli. The carotene plasmid was verified by PCR and agarose gel electrophoresis. After extracting the carotene plasmid, it was transferred to the chromosome-free yeast.

# Construction of loxp-cre flip plasmid *AUGUST 15 ~ AUGUST 22* August 15 to August 19 Construct loxp-Cre flip plasmid. August 20 to August 22 Transform the loxp-Cre plasmid into the yeast cell 4742 containing the 7flip plasmid to obtain the 7flip-Cre cell; The loxp-Cre plasmid was transformed into yeast cells 4742 and 4741 containing fGFP plasmid to obtain fGFP-Cre cells.

# Construction of cutting plasmid

*MAY 28 ~ SEPTEMBER 9*

We first constructed the Delta-g plasmid and verified the cutting efficiency, hoping that it can complete the construction of chromosome-free cells. However, in follow-up experiments, we found that the Delta-g plasmid is prone to escape, the Delta-g plasmid in the cell is very unstable, and gal itself is a carbohydrate substance, which is easily metabolized by the cell as an inducer, so we replaced it. The plasmid adopts 7flip-Cre and tetR-Cas9, which use estrogen as the inducer, which are more stable and difficult to escape, to complete the construction of chromosome-free cells.

* [x]

  ## Construct Delta-g plasmid and transfer it into SY14

  May 28 to July 13
  Construction of the cutting plasmid Delta-g:
  Copy the fragment from the original vector PRS42H plasmid (from the laboratory) by PCR and agarose gel electrophoresis;
  Copy the Cas9 fragment from the Cas plasmid by PCR and agarose gel electrophoresis;
  Cooperate with the company to synthesize sgRNA sequence;
  Copy the gal promoter from laboratory cells by PCR and agarose gel electrophoresis.
  July 14
  The above-mentioned fragments were transferred into yeast cells SY14 and connected in yeast cells.
  The Delta-g plasmid is obtained.
  July 18
  Transfer Delta-g to yeast 4741;
  Transfer Cas9 to yeast 4741.
  July 20
  The Delta-g plasmid was transformed into yeast cells containing GFP2.0.
  July 22 to July 31May 28 to July 13
  Transform the Delta-g plasmid into yeast cells SY-14 containing GFP2.0;
  Transform the Delta-g plasmid into yeast cells SY-14 containing GFP-Ura;
  Repeat the above operation and verify with PCR, transfer the obtained correct cells at regular intervals to ensure the viability of the cells.
  August 1 to August 9
  Due to the poor viability of SY-14 cells, we chose to replace 4741 yeast cells in subsequent experiments.
  Transform Delta-g plasmid and GFP2.0 into yeast cell 4741;
  Transform Delta-g plasmid and GFP-Ura into yeast cells 4741.
  Verify the cutting efficiency of Delta-g plasmid.
  The SY-14 yeast and the 4741 yeast containing Delta-g were respectively transferred to the medium containing gal and the medium without gal as the experimental group and the control group. The gal induced the expression of Delta-g and cut the chromosome. After 23 hours of incubation, the OD of the experimental group and the control group were measured and diluted by the same multiple (104, 103, 102) to plate the culture.
  We observed that the cut cells all lost GFP, which can prove to a certain extent that GFP was cut down. However, in the follow-up experiments, as the culture time increased, the OD of the gal-induced experimental group gradually approached or even exceeded the non-gal-induced control group, so we speculated that the cell chromosomes were not knocked out, but the escape of the plasmid occurred, so This plasmid cannot complete the subsequent construction of chromosome-free cells.
  Therefore, the Delta-g project was terminated.
* [x]

  ## Construct 7flip-Cre plasmid and transfer it into 4742

  August 20
  Obtain the 7flip-Cre plasmid from the laboratory.
  August 23 to August 30
  The fGFP was transformed into yeast cells 4741 containing the 7flip-Cre plasmid.
  The cleavage plasmid fGFP-7flip-Cre required by chromosome-free yeast cells was constructed.
* [x]

  ## Construct the tetR-Cas9 plasmid and transfer it into 4742

  September 9
  The tetR-Cas9 plasmid was obtained from the laboratory.
  Connect with Delta to obtain Delta-tetR-Cas9 plasmid.

# Construct an empty plasmid

*AUGUST 20 ~ AUGUST 30*

August 20 to August 23
An empty plasmid was constructed as a control group.
The fragments were copied from the Delta-g plasmid by PCR and agarose gel electrophoresis for gel collection.
The fragments were transformed into yeast cells 4741, and they were connected in the yeast cells.
Part of the fragments were ligated using Overlap and then transferred into yeast cells 4741.
Verify that it is fully connected by PCR and agarose gel electrophoresis.
The empty plasmid is extracted from the yeast cells and electrotransformed into E. coli to complete the storage of the plasmid.
August 24 to August 30
Transfer the extracted empty plasmid into yeast cells containing fGFP-Cre to obtain fGFP-empty-Cre cells;
Each fragment was transformed into yeast cells containing fGFP-Cre, and they were connected in yeast cells.
Connect some fragments using Overlap.
Constructed a fGFP-empty-Cre plasmid that can realize a chromosome-free yeast cell control group.

# Preparation of chromosome-free cells Through the above experiments, we can see that we have constructed a control group of achromosomal yeast cells after the fGFP-empty-Cre plasmid is transferred into yeast cells 4742. We constructed an experimental group of chromosome-free yeast cells after the fGFP-7flip-Cre plasmid was transferred into yeast cells 4742-that is, all chromosomes were removed to complete the preparation of cell-free chromosomes. The experimental group and the control group were cultured in SC-L-H-U liquid medium for 8 to 10 hours at the same time, replaced with Sg-L-H-U medium and induced by adding estrogen, and then tested 48 hours later.

# Form an efficiency display *SEPTEMBER 7 ~ SEPTEMBER 24* Stream sorting: After the induction, the cells that do not contain green fluorescence are sorted out by flow cytometry to obtain chromosome-free cells.

# Characterization of achromosome-free cells *SEPTEMBER ~ OCTOBER** [x] 96-well plate growth experiment October 1-7 During flow sorting, the chromosome-free cells/normal control cells are directly divided into the appropriate medium, 1- 5 per well (unknown). In theory, the growth status of the control group is normal, and the experimental group can hardly grow. * [x] Demolition September 11 to October The sorted achromosome cells and normal cells were separated by a cell splitter to separate dozens of single cells and cultured. Theoretically speaking, the growth status of the control group was normal, and the experimental group was basically unable to grow. * [x] CFU September 11 to October 1 Repeat the chromosome-free cell preparation experiment, and measure the OD at 48 hours, 60 hours, and 72 hours after induction, and then dilute and plate them. The dilution factor is 104, 105, 106. Count the number of colonies on the solid medium 2 to 3 days after each coating, and record the experimental data to complete the comparison. * [x] fGFP indicates no chromosome formation The fGFP in the control group is normally expressed and located on the genome, while the chromosome-free cells in the experimental group do not have fGFP and cannot produce fluorescence. Due to the short half-life of fGFP, after sorting, the green fluorescence intensity is measured by taking pictures, and the green fluorescence in the control group The brightness is stable, and the experimental group gradually darkens with the extension of the culture time until the fGFP is completely degraded and there is no bright light. * [x] Nucleic acid staining After elapsed cell sorting, the cells are stained with nuclear-specific binding dyes. The nucleus of the control group of chromosomal cells does not disappear and a bright light spot appears, while the cells of the non-chromosome experimental group show diffuse and weak fluorescence. By taking photos/videos, it can be verified that there is no chromosome formation.

# Verification of fGFP rapid degradation Take the yeast 4742 containing fGFP as the experimental group, and the wild-type yeast 4742 as the control group for inoculation and culture, change the induction medium sg for 48 hours, change back to the normal medium SC, and continuously measure the fluorescence intensity of the two. Subtract the two fluorescence intensity to get the difference, which is the fluorescence signal value. If it is observed that the half-life of its fluorescence signal value is less than the half-life of gfp in the literature, it can indicate that fGFP is rapidly degraded.

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### About Us

#### School of Chemical Engineering and Technology, Tianjin University, 135 Yaguan Road, Jinnan District, Tianjin

* E-mail：igem.tianjin@gmail.com
* Tel:+86-022-27403389
* WeChat:iGEM天津
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# Team:Tianjin/Engineering

Team:Tianjin/header-2021.igem.org

* [Home](https://2021.igem.org/Team%3ATianjin)
* Team

  [Team Members](https://2021.igem.org/Team%3ATianjin/Team_Member)
  [Attributions](https://2021.igem.org/Team%3ATianjin/Attributions)
  [Partnership](https://2021.igem.org/Team%3ATianjin/Partnership)
  [Collaborations](https://2021.igem.org/Team%3ATianjin/Collaborations)
* Project

  [Description](https://2021.igem.org/Team%3ATianjin/Description)
  [Design](https://2021.igem.org/Team%3ATianjin/Design)
  [Engineering](https://2021.igem.org/Team%3ATianjin/Engineering)
  [Measurement](https://2021.igem.org/Team%3ATianjin/Measurement)
  [Results](https://2021.igem.org/Team%3ATianjin/Results)
  [Implementation](https://2021.igem.org/Team%3ATianjin/Implementation)
* Parts

  [Parts Overview](https://2021.igem.org/Team%3ATianjin/Parts)
  [Improved Part](https://2021.igem.org/Team%3ATianjin/Improved_Parts)
  [Contribution](https://2021.igem.org/Team%3ATianjin/Contribution)
* [Model](https://2021.igem.org/Team%3ATianjin/Model)
* Lab

  [Notebook](https://2021.igem.org/Team%3ATianjin/Notebook)
  [Protocol](https://2021.igem.org/Team%3ATianjin/Protocol)
  [Safety](https://2021.igem.org/Team%3ATianjin/Safety)
* Human Practices

  [HP Overview](https://2021.igem.org/Team%3ATianjin/Human_Practices)
  [Integrated HP](https://2021.igem.org/Team%3ATianjin/Integrated_HP)
  [Edu&Communication](https://2021.igem.org/Team%3ATianjin/Education)

Team:Tianjin/Collaboration-2021.igem.org

![#](https://static.igem.org/mediawiki/2021/a/ab/T--Tianjin--jiaomuduan.png)

* [Overview](#Overview)
* [Iteration 1](#Iteration 1)
* [Iteration 2](#Iteration 2)
* [Iteration 3](#Iteration 3)

## Overview

The "Design-Build-Test-Learn" loop (DBTL) is a very important engineering framework. In the process of realizing the project, we also integrate this idea into our behavior, and constantly adjust the project, optimize the design and explore the future direction through feedback and iteration. Here is a record of our iterative process this year, showing the development of our project.

## Pre-invastigation

---

At this stage, we mainly did brainstorming to choose the general direction and theme of our project.We consulted some articles on cell-free systems, and put forward the idea of constructing chromosome-free eukaryotic cell— CREATE.

## Iteration 1

## Design

---

① The delta site was selected as the cleavage target by consulting the literature.
② The original cutting plasmid was designed.

[you can click link to the design page for details](https://2021.igem.org/Team%3ATianjin/Design#m1)

③ The initial characterization method was designed: GFP gene was integrated into yeast chromosome.
④ A mathematical model was designed to describe the change of the number of cells with respect to time.

## Build

---

① Construction of the first cleavage plasmid Δ g.
② GFP was integrated into chromosome.
③ Based on Logistic model, some known yeast-related constants (generation time, etc.) were used to predict the number of cells in advance.

## Test

---

① 4742 yeast was cleaved with the initial cleavage plasmid Δ g.
② Comparing the parameters predicted by the model with those calculated by the experimental data, there is a big deviation in the data when it is close to the environmental capacity.

## Learn

---

① We found that cas9 using pGal promoter may have leak expression problem, which makes it difficult for us to obtain successfully transformed yeast colonies from the culture medium after transforming cutting elements into yeast cells. (The transformed yeast is difficult to grow because the plasmid will be partially cut even if it is not induced after entering the cell.)
② Logistic model has better fitting effect on environmental capacity and in logarithmic growth period, but it needs to be further optimized for specific growth environment.

## Iteration 2

## Design

---

① In order to strictly control the expression of cas9 and further improve the chromosome cleavage efficiency, we designed version 2.0 of the cleavage element: 7flip + cre cleavage system .

[you can click link to the design page for details](https://2021.igem.org/Team%3ATianjin/Design#m3)

② Other methods to characterize the formation of CREATE were designed.

[you can click link to the design page for details](https://2021.igem.org/Team%3ATianjin/Design#m4)

③ After consulting the literature, we consider that the growth rate of cells can be described by the change of organic matter content in cell culture medium.
④ Refer to the previous IGEM team's modeling and related papers, and predict the degradation reaction by differential equation.

## Build

---

① Construction of a new cutting element .
② Refer to Monod model for continuous culture.
③ It is assumed that the degradation of proteins accords with the first-order kinetics.

## Test

---

① Characterization of new cutting elements.
② Two different cutting elements are compared. For example, the growth ability of yeast after transferring different cutting elements was tested. We also made further comparisons through flow cytometry .

[you can click link to the measurement page for details](https://2021.igem.org/Team%3ATianjin/Measurement#m6)

③ The specific growth rate was expressed by measuring the content of reducing sugar in yeast culture medium and compared with the experiment.

[you can click link to the measurement page for details](https://2021.igem.org/Team%3ATianjin/Measurement#m6)

④ According to the comparison of the predicted results of exponential decline and the fluorescence intensity measured experimentally, the correlation coefficient was calculated.

## Learn

---

① Through experimental verification, we can show that the newly constructed cutting element is more effective than the original cutting element.

[you can click link to the result page for details](https://2021.igem.org/Team%3ATianjin/Results#m7)

② By designing a double induction system, we have carried out more strict regulations on cas9.
③ When characterizing the new cutting element, we found that the half-life of the original GFP was too long to distinguish the CREATE from the normal cells in a short time
④ Monod model is good for describing the exponential growth period of yeast cells, but it can only be used to predict the number of cells, but can not predict other useful information;
⑤ Monod model, which predicts cell growth through nutrient change of cell culture medium, provides a new possible method for measuring cell number.
The predicted half-life is only relatively close, not accurate.

## Iteration 3

## Design

---

① By consulting the literature, we know that Saccharomyces cerevisiae SY14 has only one chromosome, and we imagine that this yeast with only one chromosome may form chromosome-free cells more easily, which will improve the formation rate of CREATE.
② A GFP with a shorter half-life is designed.
③ Design an experiment using fluorescence microscope.

[you can click link to the design page for details](https://2021.igem.org/Team%3ATianjin/design#m9)

④ An experiment was designed to verify whether the enzymes of chromosomeless cells are still active.

[you can click link to the result page for details](https://2021.igem.org/Team%3ATianjin/Design)

⑤ It is known from literature that the estimation of chromosome-free cell formation rate by the method of discrete but discontinuous equation is closer to the real situation.
⑥ The reaction count of protein degradation reaction needs to be measured experimentally. By trying to fit the curve, the reaction count can be fitted.

## Build

---

① We found the yeast strain SY14 and transferred it to the new version of the cutting element.
② We constructed fGFP that degrades rapidly and integrated this segment into the chromosome.
③ Transformation of mcherry into the engineering bacteria.
④ Based on Lotka-Volterra population competition model, an evolution rule description set by cellular automata is constructed.
⑤ The curve of fluorescence intensity changing with time was fitted, and the reaction count was calculated by physical and chemical related formulas.

## Test

---

① We used flow cytometry to compare the rate of chromosome-free cell formation between SY14 and conventional strain 4742.
② Test whether the half-life of fGFP is shortened.

[you can click link to the improvement part page for details](https://2021.igem.org/Team%3ATianjin/Improved_Parts)

③ Use a fluorescence microscope to observe the disappearance of fluorescence signals.

[you can click link to the measurement page for details](https://2021.igem.org/Team%3ATianjin/Measurement#m13)

④ We compared the chromosome-free ratio predicted by fitting curve with the experimental data of flow cytometry sorting.

[you can click link for details](https://2021.igem.org/Team%3ATianjin/Model)

⑤ We model the initial fluorescence intensity, extrapolate it reasonably and compare it with the follow-up data, and the prediction effect is good.

## Learn

---

① We were surprised to find that SY14 with only one chromosome was less efficient than 4742 with 16 chromosomes.

[you can click link to the result page for details](https://2021.igem.org/Team%3ATianjin/Result)

② We visually observed the disappearance process of nucleic acid dye and fGFP using fluorescence microscope.

[you can click link to the result page for details](https://2021.igem.org/Team%3ATianjin/Results#m17)

③ Cellular automata model is very helpful in describing cell growth, especially in co-culture system.
④ The degradation time of fGFP was greatly shortened; Although protein degradation reaction is not a simple first-order reaction, it can be used to estimate the half-life through the first-order reaction.

Groovin Bootstrap Template - Index

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### About Us

#### School of Chemical Engineering and Technology, Tianjin University, 135 Yaguan Road, Jinnan District, Tianjin

* E-mail: igem.tianjin@gmail.com
* Tel: +86-022-27403389
* WeChat: iGEM天津
* [Bilibili: iGEM\_Tianjin](https://space.bilibili.com/2045055005?spm_id_from=333.788.b_765f7570696e666f.1)

[![](https://static.igem.org/mediawiki/2021/3/39/T--Tianjin--logo02.png)](http://tsingke.com.cn)
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# Team:Tianjin/Experiments

# 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

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Tianjin/Results

Team:Tianjin/header-2021.igem.org

* [Home](https://2021.igem.org/Team%3ATianjin)
* Team

  [Team Members](https://2021.igem.org/Team%3ATianjin/Team_Member)
  [Attributions](https://2021.igem.org/Team%3ATianjin/Attributions)
  [Partnership](https://2021.igem.org/Team%3ATianjin/Partnership)
  [Collaborations](https://2021.igem.org/Team%3ATianjin/Collaborations)
* Project

  [Description](https://2021.igem.org/Team%3ATianjin/Description)
  [Design](https://2021.igem.org/Team%3ATianjin/Design)
  [Engineering](https://2021.igem.org/Team%3ATianjin/Engineering)
  [Measurement](https://2021.igem.org/Team%3ATianjin/Measurement)
  [Results](https://2021.igem.org/Team%3ATianjin/Results)
  [Implementation](https://2021.igem.org/Team%3ATianjin/Implementation)
* Parts

  [Parts Overview](https://2021.igem.org/Team%3ATianjin/Parts)
  [Improved Part](https://2021.igem.org/Team%3ATianjin/Improved_Parts)
  [Contribution](https://2021.igem.org/Team%3ATianjin/Contribution)
* [Model](https://2021.igem.org/Team%3ATianjin/Model)
* Lab

  [Notebook](https://2021.igem.org/Team%3ATianjin/Notebook)
  [Protocol](https://2021.igem.org/Team%3ATianjin/Protocol)
  [Safety](https://2021.igem.org/Team%3ATianjin/Safety)
* Human Practices

  [HP Overview](https://2021.igem.org/Team%3ATianjin/Human_Practices)
  [Integrated HP](https://2021.igem.org/Team%3ATianjin/Integrated_HP)
  [Edu&Communication](https://2021.igem.org/Team%3ATianjin/Education)

Team:Tianjin/Collaboration-2021.igem.org

![#](https://static.igem.org/mediawiki/2021/a/ab/T--Tianjin--jiaomuduan.png)

* [Overview](#Overview)
* [Measurement of nfGFP half-life](#Measurement of nfGFP half-life)
* [Characterization of the formation of CREATE](#Characterization of CREATE formation)
* [Exploration of the features of CREATE](#Exploration of the features of CREATE)

## Overview

In the Design section, we briefly introduced the experimental purpose and design ideas. In the Measurement section，we rigorously explain how we concretely realize these designs, and show the rationality of our process and the reliability of the results. In this section, we will correspond to it, summarize our final experimental data and results and make meaningful analysis to obtain new knowledge, re-evaluate the known and unknown factors in our experimental design. Finally feedback to the project to make it futher improved.
In this section，you can learn :
（1） We verify that the improved par--nfGFP has a shorter half-life.
（2） Through nucleic acid dyes, we observe the gradual disappearance of DNA in cells, thus verifying the formation of CREATE.
（3） By observing the rapid disappearance of nfGFP fluorescence signal in cells, it reflects the loss of gene expression ability of CREATE chromosome genome, thus confirming the formation of CREATE.
（4） By observing the growth status and colony number of the induced cells and normal cells on the solid medium, the results show a huge differences in the growth and reproduction capacity of the two groups of cells, which confirms the disappearance of chromosomes.
（5） Through flow cytometry analysis, we confirm the formation of CREATE by many parameters and signals and get the proportion of CREATE in the sample.
Due to the limitation of time, we have not complete all the experiments on exploring the features of CREATE, but the relevant experimental design has been determined.

[You can click here to see the design](https://2021.igem.org/Team%3ATianjin/Design#the%20study%20of%20the%20properties%20of%20chromosome-free%20cells)

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

## Measurement of nfGFP half-life

We used changes in the nfGFP fluorescence signal to characterize chromosome degradation. In the beginning, we directly took the GFP part into practice, while later, it is observed that GFP has a relatively long half-life, which is not conducive to further measurement. So, we designed and constructed the nfGFP part with a shorter half-life to improve the feasibility.

[Click here for detailed experimental design and discussion](https://2021.igem.org/Team%3ATianjin/Measurement)

![](https://static.igem.org/mediawiki/2021/e/e3/T--Tianjin--mlll.jpg)

After cessation of induction, the nfGFP signal decreased to the level when the expression was not induced, indicating that nfGFP was rapidly degraded in the cells after the fGFP gene was no longer expressed. The data show that the half-life of nfGFP is around 7.6h, which is significantly reduced compared to the half-life time of GFP on average, confirming the validity of our design.

## Characterization of the formation of CREATE

## Observe the disappearance of the nucleic acid dye

---

Nucleic acid dyes made it possible to visualize the degradation of chromosomes. Hoechest is a membrane-permeable nucleic acid dye that binds to DNA and generates a strong fluorescent signal. We stained the cells with the dye and photographed them under a fluorescent microscope to directly verify whether the cells' DNA was lost.

[Click here for detailed experimental design and discussion](https://2021.igem.org/Team%3ATianjin/Measurement#Characterization of CREATE formation)

The following two figures show the changes in the control group (Saccharomyces cerevisiae 4742 nfGFP) after 18h of staining with Hoechst dye.

![](https://static.igem.org/mediawiki/2021/1/1f/T--Tianjin--m4.jpg)

![](https://static.igem.org/mediawiki/2021/3/31/T--Tianjin--m5.jpg)
Figure 3 Hoechst control group 18h

The control group showed an increase in the number of cells in the same field of view after 18 h, and the tiny dots in the cells with solid fluorescent signals due to Hoechst dye staining were always visible, which indicates that the control chromosomes were intact.
The following video shows the changes in the experimental group (Saccharomyces cerevisiae 4742 nfGFP with 7flip plasmid + Cre plasmid (cleavage system 2.0)) from 0 to 18h.

[](https://static.igem.org/mediawiki/2021/d/d7/T--Tianjin--Measurement2-video.mp4)
Video 1 Hoechst Experimental Group Video

In the video, it is observed that most of the fluorescence signal disappears, visualizing the process of degradation of brewer's yeast DNA, which indicates that the cleavage system takes some time to work and the efficiency of CREATE formation is not yet 100%.

## nfGFP fluorescence signal attenuation

---

We use changes in the fluorescence signal of nfGFP to characterize chromosomal degradation. The nfGFP is a short half-life GFP that we designed, constructed, and experimentally validated.

[More relevant information can be reached by clicking on the nfGFP part.](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3939111)

We expressed nfGFP with a frequently expressed promoter and integrated the part on the chromosome by homologous recombination. After that, we induced both experimental and control cells in culture. Since the chromosome genome of CREATE is disrupted by cleavage and no new nfGFP can be expressed, the fluorescence signal of CREATE will be lost soon with the rapid degradation of the original nfGFP. In contrast, normal cells in the control group can continuously express GFP, and the fluorescent signal intensity does not diminish. This difference can reflect whether the chromosome genome of the cells is degraded or not.
The following two figures show the changes in the control group (Saccharomyces cerevisiae 4742 nfGFP) in the nfGFP channel after 18h of induction.

![](https://static.igem.org/mediawiki/2021/6/60/T--Tianjin--Measurement04.png)
Figure 4 nfGFP control group 0h

![](https://static.igem.org/mediawiki/2021/f/fd/T--Tianjin--Measurement05.png)
Figure 5 nfGFP control group 18h

It can be observed that the number of cells increased, and the overall average brightness was approximately the same, which indicates that the continuous production and degradation of nfGFP fluorescent protein in the control cells reached a balance, the nfGFP part works properly, and the cell growth condition was well.
The following video shows the changes in the experimental group (Saccharomyces cerevisiae 4742 nfGFP with 7flip plasmid + Cre plasmid (cleavage system 2.0)) from 0 to 18h.

[](https://static.igem.org/mediawiki/2021/b/b7/T--Tianjin--Measurement1-video.mp4)
 Video 2 nfGFP experimental group vedio

Comparing to the control group, we observe that the nfGFP fluorescence signal turns significantly dimmer, as seen in this video. Reasons for this phenomenon we assumed are :
1. The cleavage system disrupts the chromosome, and thus cells can no longer express nfGFP, which indicates that our cleavage system works appropriately.
2. The half-life of the expressed nfGFP is short, and the degradation rate is fast, which also coincides with our data on nfGFP parts, indicating that our measurement of the half-life of nfGFP is plausible. In the meantime, since the mechanism of nfGFP rapid degradation involves organelles and enzymes, it also reflects that CREATE still has a complete cellular mechanism, organelles, and metabolic activities despite the loss of the genome.
We also took pictures of the same field of view after removing the GFP channel, showing that although CREATE could no longer express nfGFP, the cell structure remained intact.

![](https://static.igem.org/mediawiki/2021/0/0c/T--Tianjin--Measurement06.png)
Figure 6 nfGFP GFP channel

![](https://static.igem.org/mediawiki/2021/a/ac/T--Tianjin--Measurement07.png)
Figure 7 nfGFP white light channel

To summarize the above analysis, we believe that CREATE is indeed being formed. Most of the CREATEs have complete cellular structures and the ability to complete some cell metabolic activities. This critical conclusion predicts that CREATE has the primary conditions to perform cellular functions and is a prerequisite for other future applications that we can perform with CREATE.

[For our thoughts on how to apply the CREATE, you can click here](https://2021.igem.org/Team%3ATianjin/Implementation)

## Measurement of growth curve

---

We used the difference in the growth curves of the experimental (CREATE) and control (without the cleavage system) groups to show that our cleavage system worked successfully and obtained a chromosome-free eukaryotic CREATE that was completely different from normal cells.
According to other research, OD600 was positively correlated with cell concentration during the logarithmic growth period.
We used a UV spectrophotometer to measure the OD600 of the bacterial solution. By measuring the optical density of CREATE and normal cells, we obtained the growth curves of both. If it was indeed CREATE, it could not grow and reproduce, while the normal cells of the control could. We confirmed this difference and thus corroborated the formation of CREATE.

![](https://static.igem.org/mediawiki/2021/6/67/T--Tianjin--m13.jpg)
Figure 8 raw data of OD600

![](https://static.igem.org/mediawiki/2021/c/c6/T--Tianjin--m14.jpg)
Figure 9 curve figure of OD600

From the figure, the experimental group that had their chromosomes cut did not significantly increase in yeast population compared to normal yeast cells, which coincides with our hypothesis that the CREATE cannot reproduce. After cutting, the growth curve of the colony in the experimental group should be below the normal yeast.
The curve formed by the modified 7flip+cre cutting system is below the curve formed by the delta cutting system in the lower position, which shows that our improvement of the cleavage system is effective.

## Flow cytometric analysis and sorting

---

The following two figures show the results of the flow cytometric analysis, the left graph is the control group, and the right is the experimental group. According to the analysis results, our highest chromosome-free eukaryotic cell CREATE formation rate reached more than 80%.

![](https://static.igem.org/mediawiki/2021/7/76/T--Tianjin--Measurement13.png)
Figure 10 Flow cytometric control group/Figure 11 Flow cytometric experimental group

During the iteration of the project, we considered using the Saccharomyces cerevisiae SY14 strain, which has only one chromosome, as a chassis to make chromosome-free eukaryotic cells CREATE. We conjectured that SY14 might form chromosome-free cells more efficiently. We used flow cytometric analysis to compare the effect of the same cleavage system within different chassis strains simultaneously. To our surprise, the rate of chromosome-free formation was lower in strain SY14 than in strain 4742.

![](https://static.igem.org/mediawiki/2021/2/22/T--Tianjin--rasd.jpg)
Figure 12 Comparison of strains in Flow cytometric

## Point dilution plate and CFU dilution

---

we can observe the different growth status between experimental group and the control group based on the results of the point dilution plate.This indicates that the part is in working condition and indirectly support the formation of the chromosomal-free eukaryotic cell CREATE.

![](https://static.igem.org/mediawiki/2021/c/c3/T--Tianjin--rasd13.jpg)
Figure 13 Different growth status between the experimental group and the control group

According to the number of colonies grown on the solid medium between the experimental group and the control group differed significantly. This indirectly support the formation of the chromosomal-free eukaryotic cell CREATE.

![](https://static.igem.org/mediawiki/2021/2/25/T--Tianjin--rasd14.jpg)
Figure 14 Different colony counts between the experimental group and the control group
The left is the experimental group (delta G) and the right is the control group (4741).The number of colonies after induction 48/120h is in the upper/lower half of the figure.

## Exploration of the features of CREATE

To investigate whether cell morphology is affected when chromosomes are removed.

![](https://static.igem.org/mediawiki/2021/thumb/d/dc/T--Tianjin--mshc.jpg/800px-T--Tianjin--mshc.jpg)
Figure 15 Microscopy of control group

![](https://static.igem.org/mediawiki/2021/f/f1/T--Tianjin--mshe.jpg)
Figure 16 Microscopy of experimental group

Compared to the control, no significant changes in cell morphology and size were observed in the experimental group, which indicates that even if the cells lose their chromosomes, the basic features of the cells will not alter within a short period.
Another result that can support this conclusion is that in experiments observe nfGFP signal when removing the GFP channel, and we can see that the morphological structure of cells without nfGFP fluorescence in vivo (CREATE) remains intact.

[Click on this link to find a relevant image](#Figure6)

### References

[1]John R. Houser,,Eintou Ford,,Sudeshna M. Chatterjea,... & Beverly Errede.(2012).An improved short‐lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae. Yeast(12), doi:10.1002/yea.2932.
[2]Hui Xu,,Mingzhe Han,,Shiyi Zhou,... & Ying-Jin Yuan.(2020).Chromosome drives via CRISPR-Cas9 in yeast. Nature Communications(1), doi:10.1038/s41467-020-18222-0.
[3]Fan Catherine,,Davison Paul A,,Habgood Robert,... & Huang Wei E.(2020).Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology.. Proceedings of the National Academy of Sciences of the United States of America(12), doi:10.1073/pnas.1918859117.
[4](2018).Cellular Structures - Chromosomes; Investigators at Fudan University Describe Findings in Chromosomes (Creating a functional single-chromosome yeast). Science Letter(), doi:
[5]Shi Shuobo,,Liang Youyun,,Ang Ee Lui & Zhao Huimin.(2019).Delta Integration CRISPR-Cas (Di-CRISPR) in Saccharomyces cerevisiae.. Methods in molecular biology (Clifton, N.J.)(), doi:10.1007/978-1-4939-9142-6\_6.

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