iGEM 2021 — Team FAFU-CHINA

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 [Description](#Description)

Notebook

![](https://static.igem.org/mediawiki/2021/9/94/T--FAFU-CHINA--Lab--100-1.jpeg)

#### DESCRIPTION

Memorabilia of the experimental group

| DATE | EVENT |
| 2021.2.27 | Screening test, written test |
| 2021.3.6 | The interview part, the first design of relevant synthetic biology topics, lasted for one month. 2021.4.6  The first project report and personnel selection, exchange of literature review methods, theoretical research of synthetic biology. |
| 2021.4.9 | The establishment of the experimental group and the design of the second synthetic biology project can improve the previous project or choose a new project. Requirements, meet the basic principles of synthetic biology, and meet the basic design conditions such as biosafety, experimental design and economic saving. Finally, the best topic of iGEM this year will be selected under the review of teachers. |
| 2021.4.15 | At the first group meeting, members told other group members about the progress and achievements of their research, listened to their opinions and improved them. |
| 2021.4.27 | In the second group meeting, prepare the PPT, communicate and improve the topic. |
| 2021.5.6 | The teacher reviewed the subject and appointed Chen Qinyu as the leader of the experimental group, and the microbial aroma production plant as the iGEM subject in 2021. All members of the experimental group will assist in the follow-up theoretical design and specific experimental design . |
| 2021.5.12 | In the third group meeting, for the follow-up experimental design, the team members need to find suitable chemical elements and culture medium. |
| 2021.5.18 | The fourth group meeting will exchange the reading results of the paper for a week. At the same time, the experimental group will discuss the specific experimental design from next week. The team members need to put forward effective experimental specific schemes according to the basic experiments required in the early stage. |
| 2021.6.23 | The fifth group meeting, the time arrangement of summer school retention personnel, the arrangement of summer experiment, the expansion and extension of the subject, and the discussion of the final experimental method. |
| 2021.7.1 | The sixth group meeting, change of school stay time in summer vacation, design of publicity video content and temporary experimental adjustment. |
| 2021. 8.28 | The school starts, the team members return to school, and the instructor arranges laboratory training. |
| 2021.9.4 | Escherichia coli transformation experiment. Transform our B3 plasmid into the DH5α strain, and inoculate the transformed DH5α into LB plates containing chloramphenicol. |
| 2021.9.5 | Pick the Escherichia coli transformants in the resistant plate, and perform bacterial liquid PCR. Use a fluorescent stereo microscope to observe the petri dish, and combine the PCR results to select the correct transformants. |
| 2021.9.13 | The first growth curve determination is over, the growth of E. coli and yeast is good, and the co-cultivation medium is successfully designed |
| 2021.9.15 | Preparation of Saccharomyces cerevisiae (PS100) protoplasts |
| 2021.9.23 | Pick several yeast transformants from the resistant plates, inoculate them into YPD medium with related antibiotics resistance, and extract the total DNA. |
| 2021.9.27 | Pick several yeast transformants from the resistant plates and inoculate them into YPD medium with related antibiotics resistance to prepare total DNA extraction. |
| 2021.10.1 | Use the total DNA template to perform PCR to select the correct yeast transformants. |
| 2021.10.02 | Yeast liquid PCR:5ul bacterial solution was added into 20ul 0.02m NaOH solution and lysed at 99℃ for 10 minutes. The supernatant was used as template for PCR identification |
| 2021.10.02-2021.10.09 | We conducted a seven-day co-cultivation experiment and drew the corresponding growth curve. We set the E. coli culture as group A, yeast culture as group B, and the mixed culture of the two as group C, and each group had 3 bottles of bacterial liquid at the same time. nourish. The cultivation was started at 4 o'clock in the afternoon of the same day, and the corresponding amount of linalool and nerol were added after the growth of No. 3 in group A, No. 3 in group B, and No. 3 in group C reached the growth burst. After No. 1 of group A, No. 1 of group B, and No. 1 of group C reached a stable growth, the corresponding amounts of linalool and nerol were added. Measure the OD value of each bottle of bacterial liquid in real time during the whole experiment. |
| 2021.10.15 | Yeast inverters were selected and added into the corresponding resistant medium, and cultured at 30℃ and 180rpm for 3 days. 6ml of bacterial solution was added into aMicro-Reaction Vial and detected by gas chromatography. |

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# Team:FAFU-CHINA/Engineering

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 [Preface](#Preface)
 [Oscillator](#Oscillator)
 [Photoinduced Electron Transfer Systems](#Photoinduced_Electron_Transfer_Systems)
 [Stability of co-culture system](#Stability_of_co-culture_system)
 [Construction of odoriferous strain](#Construction_of_odoriferous_strain)
[Reference](#Reference)

Engineering

![](https://static.igem.org/mediawiki/2021/9/93/T--FAFU-CHINA--flower--100-7.jpeg)

#### DESCRIPTION

### Preface

We designed and evaluated our two generations of microbial aroma production systems in order to enable
microbes to produce dynamic and attractive fragrances. Through literature review, design system,
brainstorming and literature review cycle, we finally determined the operation mechanism of microbial
incense production factory.

### Oscillator

Gene oscillator can realize the dynamic expression of target gene. Using a gene oscillator to
dynamically express the core enzymes used to produce linalool and neroli, we could achieve dynamic
changes in aroma, which was our initial idea. We understand the working principle of the gene oscillator
in detail, and predict the actual effect of the oscillator through modeling.

![](https://static.igem.org/mediawiki/2021/2/23/T--FAFU-CHINA--4-1.png)

In E. coli, oscillators are not difficult to construct. However, we also noted that neroli and linalool
may be toxic to E. coli, and we were concerned that continuous production of neroli and linalol by E.
coli would accelerate the decline of E. coli communities. And the oscillator is a closed loop control
system, even if the fragrance components can be dynamically changed, it may not be in accordance with
our will to change.

### Photogenetic Systems

Our goal is to achieve open-loop regulation and automatic rhythm.
Photoperiod gave us the idea that we could use the dynamics of light intensity throughout the day as
input to the system. And we can do that by making the substrate organisms that produce linalool and
neroli respond in reverse to the light signal. Therefore, BBa\_K3732000 and BBa\_K3127998 were used in
yeast and E.coli respectively.

![](https://static.igem.org/mediawiki/2021/6/67/T--FAFU-CHINA--4-2.png)

BBa\_K3127998

Under dark conditions, YF1 phosphorylation response modulator FixJ then drives the repressor gene
expression downstream of FixK2 promoter, resulting in the repressor target gene expression. Under light
conditions, YF1 activity is greatly reduced, FixJ is dephosphorylated, the repressor gene cannot be
expressed, and the target gene (GFP) can be activated.
The plasmid carrying BBa\_K3127998 was transfected into competent E.coli DH5α, and the transformants were
screened in chloramphenicol resistant plate. The transformants were inoculated into 1.5 mL
chloramphenicol LB medium and cultured in dark for 9 hours. A small amount of bacterial solution was
dipped into the inoculum needle and strewed on the resistant plate. The transformants were cultured
under light/dark conditions respectively. Two days later, the petri dishes were examined under a
fluorescence stereoscopic microscope.

![](https://static.igem.org/mediawiki/2021/4/4f/T--FAFU-CHINA--ert.png)

Single colonies with green fluorescence were selected and inoculated into 1.5 mL LB medium containing
chloramphenicol and cultured in light/dark

![](https://static.igem.org/mediawiki/2021/b/bd/T--FAFU-CHINA--tyu.png)

BBa\_K3127998

The E. coli cultured under light condition gave out strong green fluorescence, while the E. coli
cultured under dark condition gave out very weak green fluorescence. This shows that BBa\_K3127998 can be
induced by white light, and the induction effect meets the expectation.

![](https://static.igem.org/mediawiki/2021/0/08/T--FAFU-CHINA--4-5.png)

We fused the DNA sequence of BBa\_K3732000 with the 3 'end of the DNA sequence of the red fluorescent
protein in the plasmid to study the biological function of BBa\_K3732000 in the early stage. Meanwhile,
the BBa\_K3732000 element and kanMX were connected in series in the plasmid. We intend to design primers
with upstream and downstream homologous arms of the termination codon of the target gene to clone 5
'mRFP1 to kanMX 3' for homologous recombination after the element is tested. As a result, BBa\_K3732000
and KanMX were added to the loci of the target gene(The characterization diagram of BBa\_K3732000 is
added below).

BBa\_K3732000 can couple the degradation of the target protein with the light signal. The half-life of
the target protein did not change under black conditions. CODC1 fused to the tail of the target protein
is recognized by proteasome in eukaryotes under light conditions, and the target protein degrades.

![](https://static.igem.org/mediawiki/2021/9/95/T--FAFU-CHINA--4-6.png)

### Stability of co-culture system

First, we inoculated E. coli and yeast into co-culture medium, respectively, and measured their growth
curves at 35 ℃ and 180rpm. In order to test whether (A, B, and C represent E.coli, S. cerevisiae, and E.
coli and S. cerevisiae, respectively. Co-culture, 1, 2 and 3 respectively represent the addition of
linalool and neroli in the stable phase, without linalool and neroli in the whole process, and the
addition of linalool and neroli in the logarithmic phase).

![](https://static.igem.org/mediawiki/2021/5/51/T--FAFU-CHINA--4-7.png)

![](https://static.igem.org/mediawiki/2021/a/ad/T--FAFU-CHINA--4-8.png)

![](https://static.igem.org/mediawiki/2021/1/11/T--FAFU-CHINA--4-9.png)

The results showed that E.coli and yeast (nutrient deficiency type) grew well in the co-culture medium,
and the design of the medium was successful. On the other hand, linalool and neroli at the concentration
of 1.1g/L seemed to have no obvious inhibitory effect on the growth of the strain. Instead, the microbes
seemed to grow faster when linalool and neroli were added to the culture, possibly because they
metabolized them as nutrients. In addition, we observed that the biomass of the control group was
slightly higher than that of the experimental group with linalool and neroli at the later stage of the
stationary phase, possibly because linalool and neroli could accelerate the decline of the flora. After
a week of growth curve measurement, we found that there was no significant decline in the number of
bacteria, OD value kept at about 1.5. The co-culture system was basically stable, and linalool and
nerolol at the concentration of 1.1g/L had no obvious inhibitory effect on the growth of the strain.

### Construction of odoriferous strain

With HP, we obtained IPP-enriched yeast (PS100) from the laboratory of Zhejiang University of
Technology.
The genes that can synthesize nerolol were downloaded from NCBI, codon optimization was carried out, and
the sequence was synthesized by Bioengineering.
Protoplasts (PS100) are prepared using kits, and then plasmids carrying the target genes are transferred
into (PS100). Subsequently, the inverters were screened by neomycin-resistant medium plate. Several
transformants were selected and inoculated into special medium for co-culture.

### Reference

[1] Lutz AP, Renicke C, Taxis C. Controlling Protein Activity and Degradation Using Blue Light. Methods
Mol Biol. 2016;1408:67-78.

[2] Jungbluth M, Renicke C, Taxis C (2010) Targeted protein depletion in Saccharomyces cerevisiae by
activation of a bidirectional degron. BMC Syst Biol 4:176

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# Team:FAFU-CHINA/Experiments

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 [1.Medium configuration](#Preface)
 [2.Growing overnight cultures](#Growing_overnight_cultures)
 [3.Construction](#Construction)
 [4.Verification tests](#Verification_tests)

Experiments

![](https://static.igem.org/mediawiki/2021/8/8e/T--FAFU-CHINA--Lab--100-2.jpeg)

#### DESCRIPTION

### 1.Medium configuration

**1.1 A synthetic medium**
**Materials & concentration：**

| Component A | |
| --- | --- |
| 20g/L glucose | 8g/L xylose |
| 1.00 g /L MgSO4·7H2O | 1.10 g/L KCl |
| 0.15 g/L CaCl2 ·2H2O | 1.00 g /L (NH4)2HPO4 |
| 8.75 g/L (NH4)2SO4 |  |

Sterilize at 115° C for 15minutes.

| Component B | |
| --- | --- |
| 60.3 mg/L myoinositol | 30.0 mg/L Ca pantothe-nate |
| 6.0 mg/L thiamine HCl | 1.50 mg/L pyridoxine HCl |
| 20.0 mg/L leucine | 20.0mg/L histidine |
| 20.0mg/L uracil |  |

Filter with 0.22μm needle filter.

| Component C | |
| --- | --- |
| 15.0mg/L FeCl3·6H2O | 10.6 mg/L MnCl2 |
| 9.0 mg/L ZnSO4·7H2O | 2.4 mg/L CuSO4·2H2O |

Sterilize at 121 ° C for half an hour.
**1.2 LB broth Materials & concentration：**

Tryptone：10g/L NaCl：5g/L Yeast Extract：5g/L
( solid medium：Add Agar 20g/L)
Sterilize at 121 ° C for half an hour
**1.3 YPD (Yeast Extract Peptone Dextrose Medium) Materials & concentration：**

Tryptone：20g/L Dextrose：20g/L
( solid medium：Add Agar 20g/L)
Sterilize at 121 ° C for half an hour (Glucose solutionat 115 ° C for 15minutes)
Mix when it reaches room temperature.

### 2 Growing overnight cultures

① Prepare 20ml of LB broth or YPD medium in 250ml Erlenmeyer.
② Pipette 10μL of culture into the medium.
③ 30℃ 118rpm.

### 3 Construction

**3.1 Glycerol stock preparation**

① Prepare 40% Glycerin.(Use a measuring cylinder to measure 40ml of glycerin,then dilute to 100ml
with ddH2O. Sterilize at 121°C for half an hour).
② Sterilize the Eppendorf tubes, then store them in a sterilized clean box.
③ After labeling the Eppendorf tube, pipette 1ml bacteria culture into it.Then add 1ml glycerin
into the tube. (\*This step operates in the clean bench).
④ Gently invert the tube multiple times to mix thoroughly.
⑤ Seal it with a sealing film, then store at -20°C.
**3.2 Agarose gel electrophoresis**
① To separate medium size fragments, the agarose gel is used in a concentration of 1% w/v. Weigh
0.25/0.5/1g of agarose powder in an erlenmeyer flask.
② Measure 25/50/100 mL of TBE buffer and add it to the erlenmeyer.
③ Put the mix in the microwave until the agarose is completely dissolved.
④ Add the gel nucleic stain(Fluorescein) by 0.01%.
⑤ Let the mix cool down till it can be added to the electrophoresis tray.
⑥ When agarose gel is solidified, remove the comb and put the gel into the Electrophoresis Cells

filled with TBE buffer.
⑦ Add 6X purple loading buffer mixed each DNA sample.
⑧ Load the DNA samples in the remaining wells(1.5μL for detection, all for purification).
⑨ Run the gel with the voltage 200V for 15 mins.
⑩ Visualize the DNA fragments using a UV trans illuminator.
**3.3 Polymerase Chain Reaction**

50μl PCR system

| component | volume |
| dd H2O | 35.5μl |
| 10xtag buffer(Mg2+free) | 5μl |
| 25mM MgCl2 | 3μl |
| Primer -F | 2μl |
| Primer -R | 2μl |
| dNTP MIX | 1μl |
| Template DNA | 1μl |
| Hieff Taq DNA polymerase | 0.4μl |

PCR procedure

| Temperature | 90℃ | 94℃ | 50-60℃ | 72℃ | 72℃ |
| Time | 30s-5min/10min | 30s | 30s | 60s/kb | 10min |
| Cycle | 1 | 30 | 30 | 30 | 1 |

**3.4 Infusion**

50μl PCR system

| component | volume |
| dd H2O | 1μl |
| Target gene fragment | 4μl |
| Liner plasmid | 3μl |
| Mix | 2μl |

①Mix gently and centrifuge to the bottom of the tube.
②Incubate at 37 ℃ for 1 h.
③Ice bath for 5 min.
④Store the product at -20 ℃.

**3.5 DNA purification after gel electrophoresis**

① Use a clean scalpel or blade to cut out the gel containing the target DNA fragment. The gel should be
cut out whenever possible. The gel block containing DNA was placed in a pre weighed 1.5ml centrifuge
tube and weighed. Write down the weight of the rubber block.
Note: the DNA fragments recovered from X are used for cloning, so it is necessary to avoid damage to DNA
by UV irradiation. Minimize exposure time under UV.

②adding 1:1 volume binding fluid (volume: weight) to the rubber block (for example, adding 100 binding
liquid into the gel of 100 mg): injection of agarose gel for T concentration of human I2%, adding 2:1
volume binding liquid to the rubber block.

③ Place the gel mixture at 10 min for 50-60 ℃ until the rubber block is completely dissolved. Invert the
centrifuge tube every few minutes to speed up the dissolution process. Gel must be completely dissolved.
Add a short vortex gel mixture before recovery column. Observe the color of the solution. Yellow
indicates the best pH for DNA binding. If the color of the solution is orange or purple, the color of
the mixture will turn yellow by adding 10μl 3M of sodium acetate solution.

④ Transfer the 800ul dissolved gel solution (from steps 3 and 4) to the GcneJET recovery column.
Centrifuge for 1 minute. Pour out the waste liquid and place the column in the recovery header.
Note: if the total volume of gel glue is more than 800 μl., it can be added step by step to the recovery
column. After each addition, centrifuge 30-60s and discard the waste liquid until all the gel solutions
are added. Each recovery column 1 can exceed 1g of agarose gel.

⑤ Optional: this step is suitable for the recovered DNA to be directly used for sequencing. 100μl of
binding solution was added to the genejet recovery column. Centrifuge for 1 minute, pour out the waste
liquid and put the column into the recovery header.

⑥ Add 700 μl of rinse solution to the genejet recovery column. Centrifuge for 1 minute, pour out the
waste liquid and put the column into the recovery header.

⑦ Centrifuge the empty recovery column for another minute to completely remove the residual rinse
solution. Note: this step is to avoid residual ethanol in the recovered DNA. If ethanol remains in DNA,
it may inhibit the downstream enzymatic reaction.

⑧ Add 30 μl DD aqueous solution to the center of the membrane, put it into a new EP tube, centrifugate
for 1 min at 12000 RMP, repeat once, and store in - 20℃.

**3.6 Medium configuration**

Material: DH5α (competentcell）

① Put the competent cells on ice to melt.

② Add 5μl DNA to the competent cell suspension, rotate the centrifuge tube gently to mix the contents,
and place in ice bath for 30 min.

③ Place the tube in a 42°C water bath for 60-90 seconds, then quickly transfer to an ice bath for 2-3
minutes, taking care not to shake the tube.

④ Add 500ul sterile non-resistant LB medium into the centrifuge tube, and shake the culture at 180rpm at
37°C for 1 hour.

⑤ Coate 100μl transformed competent cells with LB plate containing chloromycetin and cultured upside down
at 37°C for 12-16 hours.

**3.7 Yeast transformation**

① Add 5 μl predenaturated Carrier DNA (YT0003), 50 μl yeast receptor cells, and 350 μl LiAc/PEG3350
mixture (YT0006) into a 1.5 ml sterile centrifuge tube containing about 1 μg plasmid, and mix
gently.

② Take water bath at 30 ℃ for 30 min, gently mix it upside down every 10 min.

③ Add 20 μl DMSO to each tube, mix gently upside-down.

④ 42 ℃ water bath heat shock for 15 min, gently mixing every 5 min upside down.

⑤ Supernatant was discarded after centrifugation at 3,000 RMP for 5 min.

⑥ The thall deposits were suspended again with 1 ml YPD Plus Liquid Medium, and shaken on a 30 ℃ shaker
for 30-60 min.

⑦ Centrifuged at 3,000 RMP for 5 min, the supernatant was discarded and 1 ml sterile 0.9% NaCl
suspension was added.

⑧Diluted 10 times and 100 times, respectively, then coated on the corresponding defective medium
plate.

⑨The plate was inverted at 30 ℃ for 3-5 days.

### 4.Verification tests

**4.1 plasmid extraction**
Material: Omega Plasmid Mini  Kit Ⅰ

① Inoculate E.coli with plasmids in 5ml LB/ antibiotic medium.,Culture on 37°C shaker for 12 to
16h。Pipette 1.0-5.0 ml of bacterial
culture,Harvest 1-4 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm（～10000x g) in a conventional.

② Discard medium.Add 250μl SolutionI/RNaseA mixtrue,Vortex to make the cells are completely suspended.

③ Resuspend pelleted bacterial cells in 250 μl Solutionl .Gently invert and mix 4-6 times.This operation
avoids severe mixing of cracking fluid and the cracking reaction should not exceed 5 minutes.

④ Add 350μl SolutionlI,Mild reversal several times to form white flocculent precipitation.

⑤ Table-top microcentrifuge for 10 min at room temperature.

⑥ Transfer the supernatant to HiBindo Mini prep DNA columns with a 2ml collection tube，Centrifuge at room
temperature for 1min at maximum speed, and pour the filtrate from the collection tube.

⑦ Reinstall the column in the collection tube,add 500uL HBC Buffer,Centrifuge at room temperature for
1min at maximum speed, and discard the filtrate.

Note:HBC Buffer must be diluted with isopropyl alcohol in accordance with the instructions before use.

⑧ Reinstall the column in the collection tube,add 700μl DNA Wash Buffer,Centrifuge at room temperature
for
1min at maximum speed, and discard the filtrate.

Note: The DNA Wash Buffer must be diluted with anhydrous ethanol in accordance with the instructions
before use.

⑨ Discard the filtrate and repeat step.

⑩ Discarded the filtrate and Reload the column into the collection tube, and the empty column was
centrifuged at a maximum speed of 2min to dry the column matrix.

⑪ Install the column on a clean 1.5ml centrifuge tube, Add 30-100μl Elution Buffer to the matrix of the
column, stood for 1min, and centrifuged at 13.000xg for 1min to Elution DNA.

⑫ Store Elution DNA at -20°C.

**4.2 Bacteria liquid PCR**

10μl system (spotting on the ice)

| Component | Volume |
| Bacteria liquid | 1μl |
| 2×Mix buffer | 10μl |
| primer F | 2μl |
| primer R | 2μl |
| ddH2O | 5μl |

PCR procedure

| Temperature | 94℃ | 94℃ | 54℃ | 72℃ | 72℃ | 16℃ |
| Time | 10min | 30s | 30s | 2min | 10min | Forever |
| Cycle | 1 | 35 | 35 | 35 | 1 |  |

**4.3 Yeast DNA extraction**
Add 5 μl of logarithmic phase bacterial solution to 20 μl 0.02M sodium hydroxide solution. Pyrolysis at
99 ℃ for 10 minutes. 1 microliter supernatant was taken and used as PCR template.

**4.4 Yeast DNA extraction**

① Yeast are cultured overnight at Take 1 to 1.5 ml of culture in 2 ml centrifuge tube, 5,000 xg
centrifugal 5 centrifuges.Clocks collect yeast cells and carefully absorb culture.

② Add 300pl Buffer SE, 10pl 2-hydro-hydro-based ethanol and 30μl Lyticase to the sample, vortex
re-suspended bacteria,37℃Shaking incubating for 30 to 60 minutes.5,000xg centrifugation 5 minutes to
collect yeast progenitor, carefully absorb the liquid.

③ Add 300uL Buffer ATL to the precipitation, vortex resuspending precipitation.To remove RNA, add 10 μl
RNase A (ordered separately) to the digestive fluid and leave to remove RNA at room temperature for 15
minutes.

Optional: Add up to 300mg glass beads and 2μl ReagentDX to the sample and mix at high speed on the
vortex 5 min Further lyse the yeast cells.

④ Join 300μl Buffer DL and 10pi Proteinase K, vortex mix, 70℃ water bath for 10 minutes.

⑤ 10,000 xg centrifugation for 3 min removes undigested impurities and transfers 500 μl of liquid to the
new centrifuge tube.

⑥ Add 250 μl waterless ethanol to the supernormal liquid and mix the vortex for 10 seconds.

⑦ Place the DNA binding column in the collection tube. Transfer the mixture to the DNA binding column.
10,000 xg centrifugal 30 to 60 seconds.

⑧ Discard the discharge fluid and reload the column back into the collection tube. Add 500μl Buffer GW1
(diluted with ethanol)to the post. 10,000 xg centrifugation 30-60 seconds.

⑨ Discard the filter and reload the column back into the collection tube. Add 600 pl Buffer GW2 (diluted
with ethanol) in the column, 10,000 xg centrifuge for 30-60 seconds.

⑩ Discard the filter and put the column back into the collection tube. 13,000 xg centrifugal 2 min dry the
post.

⑪ Carefully transfer the post to the new 1.5 ml centrifuge tube. Add 30 to 50 μl to preheat to 65 ℃Buffer
AE to the center of the membrane of the column. Leave for 3 minutes. 10,000 xg centrifugation for 1
minute.

⑫ Discard the DNA binding column and store the DNA at 2-8 ℃, and the long-term preservation should be
kept at -20 ℃ or -80 ℃.

**4.5 Gas Chromatography**

Yeast inverters were selected and added into the corresponding resistant medium, and cultured at 30℃ and 180rpm for 3 days. 6 ml of bacterial solution was added into aMicro-Reaction Vial and detected by gas chromatography.

**4.6 *E.coli* part**

① Inoculate 3 ml *E.coli* culture into 100ml co-culture medium.Repeat 3 times.

② Measure the initial OD600 value, then measure the OD600 value per 2 hours.(Halve the interval
measurement time if there is a large difference between the two OD600 values.

③ The first bottle add Neroli and linalool at stationary phase.The second bottle add Neroli and linalool
at the end of exponential phase.The third bottle is left untreated.

④ Draw the OD600 curve.

**4.7 Yeast part**

① Inoculate 3 ml yeast culture into 100 ml co-culture medium.Repeat 3 times.

② Measure the initial OD600 value, then measure the OD600 value per 2 hours.(Halve the interval
measurement time if there is a large difference between the two OD600 values.

③ The first bottle add Neroli and linalool at stationary phase.The second bottle add Neroli and linalool
at the end of exponential phase.The third bottle is left untreated.

**4.8 Coculture part**

① Inoculate 3 ml yeast and 3 ml yeast culture into 100 ml co-culture medium.Repeat 3 times.

② Measure the initial OD600 value, then measure the OD600 value per 2 hours.(Halve the interval
measurement time if there is a large difference between the two OD600 values.

③ The first bottle add Neroli and linalool at stationary phase.The second bottle add Neroli and linalool
at the end of exponential phase.The third bottle is left untreated.

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# Team:FAFU-CHINA/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)