iGEM 2018 — Team BIT-China

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:BIT-China/Notebook

* PROJECT
  + [Background](https://2018.igem.org/Team%3ABIT-China/Background)
  + [Description](https://2018.igem.org/Team%3ABIT-China/Description)
  + [Idea & Design](https://2018.igem.org/Team%3ABIT-China/Design)
* EXPERIMENTS
  + [Regulator](https://2018.igem.org/Team%3ABIT-China/ExperimentsRegulator)
  + [Feedback](https://2018.igem.org/Team%3ABIT-China/ExperimentsFeedback)
  + [Output](https://2018.igem.org/Team%3ABIT-China/ExperimentsOutput)
  + [Results](https://2018.igem.org/Team%3ABIT-China/Results)
* MODELING
  + [Overview](https://2018.igem.org/Team%3ABIT-China/Model)
  + [Fluorescent Probe Model](https://2018.igem.org/Team%3ABIT-China/FluorescentProbesModel)
  + [H2O2
    Decomposition Model](https://2018.igem.org/Team%3ABIT-China/H2O2DecompositionModel)
  + [roGFP2-Orp1
    Michaelis equation Model](https://2018.igem.org/Team%3ABIT-China/roGFP2-Orp1MichaelisEquationModel)
* HUMAN PRACTICES
  + [Overview](https://2018.igem.org/Team%3ABIT-China/HPOverview)
  + [Integrated Human Practices](https://2018.igem.org/Team%3ABIT-China/Human_Practices)
  + [Education & Public Engagement](https://2018.igem.org/Team%3ABIT-China/Public_Engagement)
  + [Collaborations](https://2018.igem.org/Team%3ABIT-China/Collaborations)
* NOTEBOOK
  + [Lab Book](https://2018.igem.org/Team%3ABIT-China/Notebook)
  + [Methodology / Protocols](https://2018.igem.org/Team%3ABIT-China/Protocols)
  + [Material & Equipment](https://2018.igem.org/Team%3ABIT-China/Equipment)
  + [Measurement / InterLab](https://2018.igem.org/Team%3ABIT-China/InterLab)
  + [Safety](https://2018.igem.org/Team%3ABIT-China/Safety)
* ACHIEVEMENTS
  + [Judging Form](https://2018.igem.org/Team%3ABIT-China/JudgingForm)
  + [Parts](https://2018.igem.org/Team%3ABIT-China/Parts)
  + [Improve](https://2018.igem.org/Team%3ABIT-China/Improve)
  + [Applied Design](https://2018.igem.org/Team%3ABIT-China/Applied_Design)
  + [Demonstrate](https://2018.igem.org/Team%3ABIT-China/Demonstrate)
* TEAM
  + [Members](https://2018.igem.org/Team%3ABIT-China/Team)
  + [Attributions](https://2018.igem.org/Team%3ABIT-China/Attributions)
  + [Gallery](https://2018.igem.org/Team%3ABIT-China/Gallery)

[![](https://static.igem.org/mediawiki/2018/4/46/T--BIT-China--iGEM2018-A_img.png)](https://2018.igem.org/Team%3ABIT-China)

![](https://static.igem.org/mediawiki/2018/8/84/T--BIT-China--iGEM2018-top_mask.svg)
LAB BOOK
![](https://static.igem.org/mediawiki/2018/4/46/T--BIT-China--iGEM2018-lower_mask.svg)

JUNE

6.7-7.4: We got *ndi1* gene fragment by using PCR and constructed ndi1-pESC-Amp-Leu
plasmid
successfully and designed and finished some validation experiment.

JULY

### Regulator

7.3-7.8: Test: cultured yeast transformed into *ndi1* plasmid(*yca1* gene knockout)
and test its
ROS level
after 24h and 48h by Measuring the fluorescence strength.

7.9-7.12: Obtain the gene of *yno1* from the yeast genome and Overlap-extension PCR to
connect
*yno1* with pESC,then we transformed DNA fragment into TOP10 pESC-yno1-Amp-Leu plasmid was
extrated from escherichia coli.

7.13-7.16: We successfully got ESC4365 from TOP10 by Colony PCR and cultured TOP10 transformed
into ECS4365 and *yno1*.

7.17-7.20: Overlap-extension PCR to connect *yno1* with pESC,*ndi1*,and we got
pESC-ndi1,pESC-yno1,pESC-ESC4365 successfully,then transformed them into TOP10 .

7.20-7.24:Test:cultured yeast transformed into *ndi1*,ECS4365,*yno1* plasmid(*yca1*
gene knockout)in
YPD plate with 1% glucose and different concentration of galactose(0%,1%,2%) and test its ROS
after 24h and 48h by Measuring the fluorescence strength.

### Feedback

7.1-7.14 Construction of several plasmids with 9 different promoters and *egfp*.

7.15-7.22 Transformation of four plasmids (promoters are GSH1p, GSH2p, TRX2p and FLR1p).
The fluorescence intensity of all six transformed yeasts was determined.

7.23-7.29 Construction of four other plasmid (promoters are GLR1p, TRR1p, TSA1p and MSY1p).

### Output

7.5: Finished the codon optimization of roGFP2 gene sequences for yeast.

7.5-7.25:Sent the sequence of roGFP2 to the company for synthesis.

6.25-7.3:Obtained the gene of *orp1* from the yeast genome.

7.4-7.7:Finished the point mutation of *orp1*(C82S).

7.25-7.28:Synthesis the part: roGFP2-orp1 completely through OE-PCR.

7.25-8.6:Screened the promoter to find the appropriate strength of the promoter to turn on the
expression of the fusion protein gene.

6.25-7.3:Obtained the gene of promoter from the yeast genome.

AUGUST

### Regulator

7.28-8.3:Designed the primers and Overlap-extension PCR to get
f1000-ura-r1000(*ndi1*),f1000-ura-r1000(*yno1*) successfully.

8.3-8.16:Use colony PCR to verify if yeast endogenous gene *ndi1*/*yno1* has been
knocked out and
tested the codon optimization.

8.19-8.29:knock out the gene *yca1*.

### Feedback

7.30-8.5 Construction of one plasmids (promoter is gal1p).Yeast transformation of three
plasmids (promoters are gal1p, trr1p, tsa1p, glr1p and trx2p). Design primers for
standardization.

8.6-8.12 Verification of promoter strength (pre-experimental). Yeast transformation of plasmid
with promoter flr1p.

8.12-8.20 Pre-experiment of promoter strength verification for H2O2
gradient concentration.

8.23-8.26 Pre-experiment of promoter strength verification.

8.27-9.2 Design and build *pRS423-TEF1p-dcas9-yno1/ndi1—sgRNA*.

### Output

7.3-8.10:Synthesis the promoter and roGFP2-orp1 completely through OE-PCR.

8.12-8.15:Linked the promoter+roGFP2-orp1 to the plasmid pESC-Trp containing the terminator
cyc1 by restriction enzyme digestion.

8.15-8.20:Finished the expression of roGFP2-orp1.

SEPTEMBER

### Regulator

9.3-9.10: Use colony PCR to verify if yeast endogenous gene *yca1* has been knocked out.

9.13-9.18:Standardization:pGAL1-yno1-Tcyc1,pESC-ndi1,1000bp homologous+ura(Δ*yno1*).

9.21-9.30: Standardization:pGAL1-yno1-Tcyc1,1000bp homologous+ura(Δ*yca1*).

### Feedback

9.3-9.9 dcas9 promoter replacement

9.10-9.16 Redesign of the dcas9 plasmid with different promoters (promoters are
*TRX2p/GLR1p/TRR1p/SOD2p*)

9.17-9.23 Qpcr verified whether *pESC-Leu-Yno1/Ndi1* strain was overexpressed

9.24-9.30 Transfer *pRS423-dcas9-sgRNA-ndi1/yno1-GLR1p/TRX2p/SOD2p/TRR1p* into CENPK
CENPK-overexpressing *yno1/*ndi1** knockout *YCA1*.

### Output

8.20-9.5:Finished the function verification of the roGFP2-orp1.

8.20-8.28Finished the selection of promoter strength.

8.20-9.10:Tested the codon optimization.

OCTOBER

9.10-10.5:Finished parts standardization of part: promoter, part: roGFP2-orp1, part:
promoter+roGFP2-orp1+cyc1

10.1-10.7 Transfer *pRS423-dcas9-GLR1p/TRX2p/SOD2p/TRR1p-ndi1-sgRNA* plasmid into
CENPK*-yno1-yca1-TEF2p/EN*.

10.1-10.6:Standardization:pESC-ndi1,pESC-yno1.

PDF -- Notebook Regulate

PDF -- Notebook Feedback

PDF -- Notebook Output


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:BIT-China/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.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### 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/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:BIT-China/Results

* PROJECT
  + [Background](https://2018.igem.org/Team%3ABIT-China/Background)
  + [Description](https://2018.igem.org/Team%3ABIT-China/Description)
  + [Idea & Design](https://2018.igem.org/Team%3ABIT-China/Design)
* EXPERIMENTS
  + [Regulator](https://2018.igem.org/Team%3ABIT-China/ExperimentsRegulator)
  + [Feedback](https://2018.igem.org/Team%3ABIT-China/ExperimentsFeedback)
  + [Output](https://2018.igem.org/Team%3ABIT-China/ExperimentsOutput)
  + [Results](https://2018.igem.org/Team%3ABIT-China/Results)
* MODELING
  + [Overview](https://2018.igem.org/Team%3ABIT-China/Model)
  + [Fluorescent Probe Model](https://2018.igem.org/Team%3ABIT-China/FluorescentProbesModel)
  + [H2O2
    Decomposition Model](https://2018.igem.org/Team%3ABIT-China/H2O2DecompositionModel)
  + [roGFP2-Orp1
    Michaelis equation Model](https://2018.igem.org/Team%3ABIT-China/roGFP2-Orp1MichaelisEquationModel)
* HUMAN PRACTICES
  + [Overview](https://2018.igem.org/Team%3ABIT-China/HPOverview)
  + [Integrated Human Practices](https://2018.igem.org/Team%3ABIT-China/Human_Practices)
  + [Education & Public Engagement](https://2018.igem.org/Team%3ABIT-China/Public_Engagement)
  + [Collaborations](https://2018.igem.org/Team%3ABIT-China/Collaborations)
* NOTEBOOK
  + [Lab Book](https://2018.igem.org/Team%3ABIT-China/Notebook)
  + [Methodology / Protocols](https://2018.igem.org/Team%3ABIT-China/Protocols)
  + [Material & Equipment](https://2018.igem.org/Team%3ABIT-China/Equipment)
  + [Measurement / InterLab](https://2018.igem.org/Team%3ABIT-China/InterLab)
  + [Safety](https://2018.igem.org/Team%3ABIT-China/Safety)
* ACHIEVEMENTS
  + [Judging Form](https://2018.igem.org/Team%3ABIT-China/JudgingForm)
  + [Parts](https://2018.igem.org/Team%3ABIT-China/Parts)
  + [Improve](https://2018.igem.org/Team%3ABIT-China/Improve)
  + [Applied Design](https://2018.igem.org/Team%3ABIT-China/Applied_Design)
  + [Demonstrate](https://2018.igem.org/Team%3ABIT-China/Demonstrate)
* TEAM
  + [Members](https://2018.igem.org/Team%3ABIT-China/Team)
  + [Attributions](https://2018.igem.org/Team%3ABIT-China/Attributions)
  + [Gallery](https://2018.igem.org/Team%3ABIT-China/Gallery)

[![](https://static.igem.org/mediawiki/2018/4/46/T--BIT-China--iGEM2018-A_img.png)](https://2018.igem.org/Team%3ABIT-China)

RESULTS

Final Test

Verify the function of monitoring antioxidants in
real time

Our engineered yeast should finally have the function of monitoring antioxidants in real time.
To demonstrate this, we integrated the two subsystems together into ∆*yca1* yeast and had series
of tests on it, in which the increasing of ROS in yeast can be effectively captured by our
roGFP2-Orp1 protein.

In order to regulate endogenous ROS, we replaced yeast endogenous YNO1 promotor with a
galactose-inducible promotor, GAL1-GAL10. We then constructed the plasmid
pESC-TEF1p-roGFP2-Orp1-CYC1t, a shuttle vector marked with trp1. Cellular redox status were
monitored by measuring the fluorescence ratio at 488 nm (reduced state) and 405 nm (oxidized
state). Cells were cultured in SD medium supplemented with 1% galactose at 30℃, during which
GAL1-GAL10 promotor was induced when galactose added. It suggests that overexpression of *yno1*
gene will increase cells ROS level and decline the fluorescence ratio at 488 nm and 405 nm,
which means there are more ROS in our modified strain.

As the results showing, our output subsystem can reflect the accumulation of endogenous ROS
caused by regulator subsystem.

![](https://static.igem.org/mediawiki/2018/4/4d/T--BIT-China--ResultFig1.png)

Fig.1 Intracellular ROS accumulation.

The red curve indicates the fluorescence ratio at 488 nm (reduced state) and 405 nm (oxidized
state) of engineered yeast which overexpressed Yno1 and constructed the plasmid
pESC-TEF1p-roGFP2-Orp1-CYC1t. And the blue curve indicates the fluorescence ratio at 488 nm
(reduced state) and 405 nm (oxidized state) of engineered yeast which only overexpressed Yno1.
The red curve decline over time but not the blue one, which means roGFP2-Orp1 protein is able
to show the accumulation of ROS in our yeast.

Since we have proved the feasibility that the antioxidative ability of antioxidant can be
measured by fluorescence probes. We can easily set up the relationship between our system and
real antioxidative ability of antioxidant.

Detect antioxidants by engineering strain

After the whole system construction, we need to test the function of our system and then
evaluate the effect of antioxidants through our system.

To detect the antioxidant capacity of antioxidants, we have selected three antioxidants,
Vitamin C, Quercetin and Catechol, as our samples, and cultured engineered strain
(*Δyca1-yno1-roGFP2-Orp1*) until the period with high ROS accumulation (24-36 h). Then the
antioxidants were added into yeast culture and incubated for 30 minutes. The control group was
treated with aseptic water and ethanol. The fluorescence intensity ratio (I488/I408 or
I405/I488) of the reduction peak (488nm) and oxidation peak (405nm) was measured by Microplate
Reader, and the antioxidant activity of antioxidants was characterized by that ratio change
rate. And the emission wavelength is 510nm. The results are showed in Fig. 2.

![](https://static.igem.org/mediawiki/2018/8/80/T--BIT-China--ResultFig2.png)

Fig.2 Detect the Antioxidant Capability of Antioxidants (I405/I488)
Low
fluorescence ratio (I488/405) means low intracellular ROS level.

![](https://static.igem.org/mediawiki/2018/8/82/T--BIT-China--ResultFig3.png)

Fig.3 Detect the Antioxidant Capability of Antioxidants (I488/I405)
High
fluorescence ratio (I488/405) means high antioxidant capacity.

As Fig.2~3 shown, in 30 mins, group Catechol and group Quercetin both show a decreasing in
I405/I488 ratio (Fig.2), which means that Quercetin and Catechol have antioxidant activity.
Besides, the Quercetin treated group shows a faster decreasing in I405/I488 ratio than Catechol
under the same condition of time. According to our modeling, this indicates that the Quercetin
have more antioxidant activity than Catechol, which is consistent with CAA assay result. (Wolfe
K L, Liu R H et al.)[1]

But Vitamin C is less effective and had no obvious antioxidant effect in this assay. We thought
this result may had two reasons. First, the Vitamin C shows a low direct-antioxidant activity,
especially at low concentration (10 µM) compared to another two. Or the Vitamin C main
principle of anti-oxidation is its indirect-antioxidant activity, like activation of cell's
natural anti-oxidative enzymes, which is hard to reflect in fluorescence ratio level just in 30
mins.

In a word, our system gave a similar result to CAA assay in three selected antioxidant tests.
This proves our system can detect the antioxidant in living cell. But we need more antioxidant
test to verify our system's function and limitation, especially indirect-antioxidative
antioxidant, for finding "Who can get an A?"

Summary

![](https://static.igem.org/mediawiki/2018/9/98/T--BIT-China--ResultSummary1.png)

![](https://static.igem.org/mediawiki/2018/2/23/T--BIT-China--ResultSummary2.png)

![](https://static.igem.org/mediawiki/2018/8/88/T--BIT-China--ResultSummary3.png)

![](https://static.igem.org/mediawiki/2018/3/37/T--BIT-China--ResultSummary4.png)

![](https://static.igem.org/mediawiki/2018/c/ca/T--BIT-China--iGEM2018-sponsor-1.png)
![](https://static.igem.org/mediawiki/2018/0/07/T--BIT-China--iGEM2018-sponsor-2.png)

Contact

Institute of
Biotransformation and synthetic biosystem School of Chemistry and Chemical Engineering.

Beijing Institute of Technology

100081, Beijing

Email:lichun@bit.edu.cn

Copyright © 2018 BIT-China

Wolfe K L, Liu R H. Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and
dietary supplements[J]. Journal of agricultural and food chemistry, 2007, 55(22): 8896-8907.

![](https://static.igem.org/mediawiki/2018/e/ec/T--BIT-China--iGEM2018-exp-inp-BG2.png)