iGEM 2021 — Team SHSID

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# Team:SHSID/Notebook

SHSID

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NOTEBOOK

6/26

Team ice-breaking
Project Discussion

![](https://static.igem.org/mediawiki/2021/4/4d/T--SHSID--NOTEBOOK01.jpg)

6/27

Laboratory Safety Training

![](https://static.igem.org/mediawiki/2021/2/26/T--SHSID--NOTEBOOK02.jpg)

Media preparation
Transfer and culture E. coli and the yeast
PCR to amplify the target genes, AsADC、speB and pYES2

6/28

![](https://static.igem.org/mediawiki/2021/8/82/T--SHSID--NOTEBOOK03.jpg)

Plasmid Extraction: pYES2-X-1-XI-4
PCR verification

6/29

One-day off

6/30

Prepare the competent cells of the yeast
PCR to amplify the vector

![](https://static.igem.org/mediawiki/2021/1/15/T--SHSID--NOTEBOOK04.jpg)

7/1

Repeat PCR

7/2

Overlap PCR
PCR verification

![](https://static.igem.org/mediawiki/2021/5/56/T--SHSID--NOTEBOOK05.jpg)

2kM 1 2 3 4 5 6 7 8

**All lanes are correct.**

7/3

Team Photo Shooting
DNA gel extraction
Overlap PCR

![](https://static.igem.org/mediawiki/2021/1/13/T--SHSID--NOTEBOOK06.jpg)

2kM 1 2 3 4

**All lanes are correct.**

7/4

Overlap PCR
PCR verification

![](https://static.igem.org/mediawiki/2021/b/bf/T--SHSID--NOTEBOOK07.jpg)

15kM 1 2 3 4 5 6 2kM

**Gel electrophoresis diagram. Lane 1, 3, and 5 represent DNA segment 45678, and
lane 2, 4, and 6 represent segment 678.**

7/5

KOD FX PCR to amplify the terminator CYC1
Transformation into E. coli

7/6

Check the transformants
Repeat the transformation

![](https://static.igem.org/mediawiki/2021/1/1d/T--SHSID--NOTEBOOK08.jpg)

7/7

Repeat: KOD FX PCR to amplify the terminator CYC1
Overlap PCR
Transformation

7/8

Colony PCR

![](https://static.igem.org/mediawiki/2021/e/ee/T--SHSID--NOTEBOOK09.jpg)

September

Complete the plasmid construction

![](https://static.igem.org/mediawiki/2021/d/d9/T--SHSID--NOTEBOOK10.jpg)

Send for sequencing

![](https://static.igem.org/mediawiki/2021/2/2b/T--SHSID--NOTEBOOK11.jpg)

Transformation into the yeast

10/1-10.7

Fermentation test
Send out the samples for measuring the putrescine yield by LC-MS/MS

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# Team:SHSID/Engineering

SHSID

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Engineering

Background

Kl tropane alkaloids (TAs) refers to a kind of alkaloids containing the tropane
alkyl skeleton formed by the combination of pyrrole ring and piperidine ring in structure. It is a natural
product of plant and has a long history and important medicinal value. tropane alkaloids have great market
demand and often appear in global shortages. A method that can produce Tas in scale is expected. Using
synthetic biology to create a microbial cell factory to produce TAs is a highly potential strategy.

Design

The Tropine part of Tropane alkaloids (TAs) is obtained from arginine to putrescine
(1,4-butanediamine, putrescine), and then through a series of chemical reactions. In our project, natural
genes involved in arginine metabolism and polyamine biosynthesis was designed to overexpress in yeast. The
engineer strains that produced excess putrescine. The following genes were used to improve putrescine
production:

(1) SPE1 gene: Overexpression of the SPE1 gene can increase the expression of Spe1p
which converts ornithine to putrescine.

(2) AsADC and speB gene: Arginine is decarboxylated by arginine decarboxylase (ADC)
to agmatine (Agmatine), agmatine hydrolase ( AUH) can remove the guanidine group from agmatine and directly
produce putrescine. In this study, ADC from oats (AsADC) and AUH from E. coli (speB) were selected, both of
which have been shown to be active in Saccharomyces cerevisiae.

Build

The three genes SPE1, speB, and AsADC were inserted into yeast expression vector
pYES2. different promoters and terminators were applied for the transcription of the three genes,
respectively (Figure 1).

![](https://static.igem.org/mediawiki/2021/5/52/T--SHSID--engineering01.jpg)

Test

Plasmids pYES2-AsADC-SPE1-SpeB were transferred to BY4741 chemically competent
yeast cells and screened on YPD/Hyg plates. Transformants were picked into YPD/Hyg medium for activation.
Metabolite production tests were carried out in YNB-SC fermentation medium (containing 10 times arginine raw
material). 48h metabolites (supernatant) were collected and the putrescine analyze metabolites by LC-MS.

![](https://static.igem.org/mediawiki/2021/2/24/T--SHSID--engineering02.jpg)
Figure 3. The peak of putrescine detected by LC-MS.

The results showed that at 32 min, the peak pattern of putrescine appeared (as
shown in the figure 3), indicating that the engineered strain we constructed successfully produced
putrescine.

Learn

The three genes that can improve putrescine production were successfully introduced
into the yeast strain. The yeast strain generates putrescine during fermentation experiment. What is
unsatisfactory is the peak area in LC-MS is small. The yield of putrescine is far below the factory mass
production level. It is likely that the protein expression is insufficient and the metabolic pathway is
small. In the future, the expression of the three proteins needs to be further optimized.

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# Team:SHSID/Experiments

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Experiments

LB Media Preparation

Materials:

1. Yeast extract 5g/L (0.5%)
2. Tryptone 10g/L (1%)
3. NaCl 10g/L (1%)
4. Add agar 15g/L (1.5%)
5. Distilled water 600mL
6. 4 150mL Beakers
7. Graduated Cylinder

Methods:

1. Measure 600 mL of distilled water & add into a big beaker
2. Measure 6g of tryptone, add to beaker
3. Measure 6g of NaCl, add to beaker
4. Measure 3g of yeast extract, add to beaker
5. Stir and dissolve completely using a stirring rod
6. Prepare 4 150 mL beakers
7. Measure and add 2.25 g of agar (per flask) to two of the four flasks
8. Distribute solution to the four flasks, 150 mL each.
9. Seal using brown paper & rubber band as lid
10. Put on sterilization tape
11. Autoclave for with the setting of 121oC for 30 minutes (Total duration 2 hours)

YPD Media Preparation

Materials:

1. Yeast extract 20g/L
2. Tryptone 40g/L
3. Glucose (independent liquid, 40g/L medium concentration)
4. If making solid, add agar 15g/L
5. Distilled water 500mL
6. 2 100mL beakers, 1 300mL beaker

Methods:

1. Measure 500ml water, add to beaker
2. Measure 20g tryptone, add to beaker
3. Measure 10g yeast extract, add to beaker
4. Mix all substances to dissolve, use magnetic stirring if needed
5. Distribute solution to 300ml, 100ml, and 100ml triangular flask
6. Add 1.5g agar to 100ml to make solid
7. Use brown paper to seal the triangular flask, wrap with rubber band
8. Put on sterilization tape
9. Autoclave at 121oC for 30min, total time 2hrs

Prepare competent cell solution

Materials:

\*\*\* glycerol 20% \*\*\* total 80ml solution

1. glycerol 16ml
2. Distilled water 64ml

Methods:

1. Measure 64ml water, add to cylinder
2. Add oil 16ml
3. Add water until solution reaches 80ml
4. Mix thoroughly, transfer to bottle with cap, seal with sterilization tape

PEG 3350 Solution

Materials:

1. PEG 25g
2. Distilled water 15ml

Methods:

1. Measure 25g PEG, add to cylinder
2. Add distilled water 15ml
3. Stir then heat until dissolve (becomes transparent solution)
4. Transfer to bottle and seal
5. Put on sterilization tape
6. Autoclave at 121oC for 30min, total time 2hrs

Lithium Acetate Solution

Materials:

1. 5.1g of lithium acetate

Methods:

1. Measure 5.1g of lithium acetate
2. Measure 50mL of distilled water
3. Add lithium acetate in water, stir and dissolve.
4. Close apparatus lid, making sure that the lid is not completely sealed
5. Autoclave for 30 minutes at 121oC (total duration 2 hours)

Culture Medium

1. YPD → yeast
ingredients: yeast extraction(20g/L); tryptone (40g/L); glucose (200g/L originally and 40g/L，added 400µg)

2. LB → e. coli

a. ingredients: yeast extraction(5g/L); tryptone (10g/L); sodium chloride (10g/L)
Fill in Petri dish
combine with 2 antibiotics
LB uses amp（100mg/mL, to dilute to 100 µg /mL we pour 150 µL using pipet）
YPD uses HYG (200/mL, to dilute to 400 µg /mL we pour 200 µL using pipet）

Inoculum and culture

1. speb (E coli)
2. Yeast plasmid (pYES2) (4mL of broth, 母液100mg/mL target 100 µg /mL, pipet 4 µL)
3. by4741 (10 µL yeast)
shaker bed incubator 30⁰C 200rpm overnight

PCR

1. DNA replication
2. In vivo DNA replication process:

central dogma: DNA → RNA → protein
semi-conservative replication
    one parent strand, one new strand
1. helicase: unzips two strands of DNA
2. primase: synthesize RNA primer for DNA polymerase III to attach
3. DNA polymerase III: add complementary nucleotides at the 3' end
4. DNA polymerase I & DNA ligase: replace primer & combine okazaki fragments

3. In vitro DNA replication process:

Denaturing: heat to 95-98⁰C, break hydrogen bonds and separate DNA into two strands
Annealing: DNA sample is cooled to 54 ⁰C (generally 50-60), depending on the length of primers,
allowing
forward and reverse primers attach to opposite ends of the target DNA sequence
    ATCG (Adenine, Thymine, Cytosine, Guanine)
    number of hydrogen bonds (2, 3 → different temperature)
Extending: A heat-tolerant DNA polymerase (Taq) added nucleotides at 72 ⁰C
    5' end to 3' end
    2^30 DNA by 30 rounds of replication

4. system

2x PCR mix 10 µL includes:
     buffer
    dNTP, ATP, TIP, CTP, GTP
    Taq, DNA polymerase
primer - F 0.2 µL
primer - R 0.2 µL
template 1 µL
ddH2O 8.6 µL

5. program

![](https://static.igem.org/mediawiki/2021/d/d2/T--SHSID--Experiments01.jpg)

Plasmid Extraction

![](https://static.igem.org/mediawiki/2021/7/77/T--SHSID--Experiments02.jpg)

Agarose gel el electrophoresis

Materials:

1. agarose N
2. TAE buffer
3. Loading buffer
4. nucleic acid dye
5. DNA samples after PCR

Methods:

1. Dissolve 1g agarose in 100 mL TAE buffer (1% w/v), microwave for about 2 minutes until mixture turns
transparent
2. Put small comb and medium-sized comb for identifying DNA and large comb for recycling DNA
3. Pour the agarose solution in slot, completely covering the large comb and covering half of the small
comb
4. Wait for 30 minutes to cool and solidify
5. During waiting: prepare DNA sample
    a. Loading buffer + 1 µL nucleic acid dye to visualize DNA under UV and sink to the
bottom of gel under TAE
buffer solution
    b. 5 µL DNA sample + 1 µL buffer solution
6. Pour more TAE buffer in electrophoresis device so that the liquid covers the whole sample
7. Put the sample with appropriate order in each hole (First two are markers)
8. Set electrophoresis at 185V for 15 minutes
9. Use UV to visualize the DNA strand

Nanodrop

1. used nanodrop to measure the concentration of yeast inoculation in yeast solution
    a. follow nanodrop steps as described in 6/28
2. undiluted yeast solution should have an Optical Density (OD) >10
    a. OD estimated how much DNA you have in solution by measuring the absorbance at a
certain wavelength
        i. the most common wavelength used is 600 nm, which is one
we used in our experiment
    b. we first diluted the solution from 0 and found that our yeast solution's OD was 2

![](https://static.igem.org/mediawiki/2021/f/f3/T--SHSID--Experiments03.jpg)

3. then we add glucose to the yeast solution for dilution

![](https://static.igem.org/mediawiki/2021/b/bf/T--SHSID--Experiments04.jpg)

Prepare Yeast Competent Cell

1. culture yeast cells in 3mL test tubes
2. measure OD
    a. pipette 100µL of yeast culture into 4mL of YPAD in culture flask
3. culture yeast strains for 3.5 hours, measure OD again (new OD is 0.3)
4. centrifugation
    a. 4500 rpm for 5 minutes
5. use ddH2O to wash and re-suspend the cells
    a. washing process: add ddH2O → mix together → centrifuge → pour away the fluid
section
6. add frozen competent cell solution (FCCS) (1% of total volume)
    a. ingredients

![](https://static.igem.org/mediawiki/2021/a/a4/T--SHSID--Experiments05.jpg)

b. purpose：to preserve the yeast in the fridge
7. separate yeast culture to 100 µL tubes at -20 ℃

Overlap PCR

**procedure as follows**

![](https://static.igem.org/mediawiki/2021/1/1c/T--SHSID--Experiments06.jpg)

Gel Recycle

Measurements:

![](https://static.igem.org/mediawiki/2021/2/24/T--SHSID--Experiments07.jpg)

Methods:

1. measure 2g agarose powder
2. measure 200ml TAE
3. add two components in a triangular flask
4. heat the flask in a microwave for 2 min at medium heat, continue until substance all dissolved
5. prepare gel models with large sample holes
6. add the agarose TAE solution to the gel models
7. set aside to cool

Gel Extraction

1. Add 8µL loading buffer into three 4-7 tubes (50µL each)
2. gel electrophoresis: 185V 22min
a) add 8.3µL of loading buffer (10x) to PCR sample (total 60 µL)
b) 15k marker
3. collect DNA and add 500 µL of NTI buffer
i) 4-7 first tube: 0.06g
ii) 4-7 second tube: 0.23g
iii) 4-7 third tube: 0.20g
c) Heat it at 50℃ until all the contents melt
4. centrifuge for 30sec at 12000rpm
5. Pour the liquid in microcentrifuge tube back to the column and centrifuge for 30sec at 12000rpm
6. add 700 µL of NT3 to wash silica membrane, then centrifuge for 30sec at 12000rpm (twice)
7. Dry the membrane by centrifuging for 1min at 12000rpm
8. add 50℃ of ddH2O 20µL and 30sec 12000rpm
9. Use nanodrop to check concentration
a. 4-7 first tube: 23.8
b. 4-7 second tube: 22.4
c. 4-7 third tube: 26.4
We decided to use 4-7 first and second tube.

Reflection

Due to the lack of results (unsuccessful E. Coli culture) from previous lab, we made 2 changes

1. double the amount of DNA fragment
    a. decrease water volume since the total volume must maintain 20µL
2. decrease number of fragments for gel electrophoresis
    a. previous lab: 3 fragments
    b. current lab goal: 2 fragments

Experiment:
double the amount DNA fragments

1. remake DNA solution (specifics as follow)

![](https://static.igem.org/mediawiki/2021/4/40/T--SHSID--Experiments08.jpg)

2. add mixture(5µL) into E. Coli template (dH5a)
a. ratio 1:10
3. heat shock in dry bath
a. 40℃ for 90 seconds
b. principle: cell membrane becomes semi-permeable under sudden exposure to higher temperature allowing
the DNA fragments to enter in E.coli template
4. add LB culture (600µL)
a. principle: makes e. coli competent stronger
5. put mixture in rotary shaker for 60 minutes at 2200rpm
6. Remove 700µL of E.coli culturing solution
7. Vortex the remaining solution with residue
8. Open a prepared Petri dish with LB culture
9. Evenly transfer the remaining solution to the Petri dish with a pipette
10. Discard the initial pipette head and change a new one, bend the new head
11. Gently spread the culturing solution with the bent Petri dish head until the surface is even and
dry
12. Close the Petri dish lid and seal with laboratory film, transfer Petri dish to incubator

3 combinations of DNA segments for constructing plasmid

goal: reduce the number of fragments by homologous recombination to improve plasmid construction
    elongation time is based on length
    successful combination allows total fragment number to reduce from 3 to 2, improve
the probability of plasmid constructions' success
combinations
    0123+45, length:5k
    4567+678, length:3.7k---success
    45+678, length: 3.7k
materials are as following

![](https://static.igem.org/mediawiki/2021/0/02/T--SHSID--Experiments09.jpg)

E.coli Culture

Methods:

1. Remove 700microliter of E.coli culturing solution
2. Vortex the remaining solution with residue
3. Open a prepared Petri dish with LB culture
4. Evenly transfer the remaining solution to the Petri dish with a pipette
5. Discard the pipette head and change a new one, bend the new head
6. Gently spread the culturing solution with the bent Petri dish head until the surface is even and
dry
7. Close the Petri dish lid and seal with laboratory film, transfer Petri dish to incubator
8. Leave it overnight and check results in the morning

Putrescine Yield Measurement

Methods:

1. At least 3 repeated small-scale metabolite production tests were carried out in YNB-SC medium (containing
metabolic raw materials)
2. The yeast colonies were inoculated into 300 μL of culture solution and incubated on a shaker at 30°C, 460
rpm and 80% relative humidity for 48 hours.
3. The culture was pelleted by centrifugation at 3500 × g for 5 minutes at 12°C, and 100–200 μL of the
supernatant (containing putrescine) was removed for analysis.
4. The production of metabolites was analyzed using Agilent 1260 Infinity binary HPLC and LC-MS/MS.

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# Team:SHSID/Results

SHSID

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Results

Plasmids containing target genes, AsADC, SPE1, and speB
are successfully synthesized from 10 DNA segments. Segments 0 to 2 contain promoter PPGK1, target gene
AsADC, and terminator TADH1. Segments 3 to 5 contain promoter PTEF1, target gene SPE1, and terminator TTEF1.
Segments 6 to 8 contain promoter PTDH3, target gene speB, and terminator TCYC1. Segment 9 is the backbone
plasmid pYES2. Transforming this plasmid into yeast will increase the yield of putrescine.

The first round of PCR
The first round of PCR amplifies DNA segments 0 to 7 separately.

![](https://static.igem.org/mediawiki/2021/6/65/T--SHSID--Results01.png)

segment 3. Lane 4 is segment 4. Lane 5 is segment 5. Lane 6 is segment 6. Lane 7 is segment 1. Lane 8 is
segment 7.

On this gel electrophoresis diagram, all DNA segments were successfully amplified, and the band is on the
corresponding position to its number of base pairs.

The second round of PCR

The second round of PCR overlapped two DNA segments from 0 to 7.

![](https://static.igem.org/mediawiki/2021/2/2d/T--SHSID--Results02.png)

Figure 2: Gel electrophoresis diagram. Lane 1 is segments 0 and 1. Lane 2 is segments 2 and 3. Lane 3 is
segments 4 and 5. Lane 4 is segments 6 and 7.

This result shows that the overlap PCR between two DNA segments was successful because the bands are in
their right position corresponding to the number of base pairs and relative to the ladder.

The third round of PCR
The third round of overlap PCR connects DNA segments 01 with 23, and 45 with 67.

![](https://static.igem.org/mediawiki/2021/7/71/T--SHSID--Results03.png)
Figure 3: Gel electrophoresis diagram. Lane 1 is DNA segments 0 to 3. Lane 2 is DNA segments 4 to 7.

Lane 1 shows a correct and bright band for segments 0 to 3. However, although the band for DNA segments 4 to
7 in lane 2 has the correct band, the band is very weak, which means that the amount of this segment is not
enough to carry out the construction of plasmid. This is probably because segments 4 to 7 is longer than
segments 0 to 3, and the primer is not designed very well. More round of overlap will be needed to obtain
more segments of 4 to 7.

The fourth round of PCR

The fourth round of PCR amplifies segments 8 and 9, and overlapped segments 45 with 67 again.

![](https://static.igem.org/mediawiki/2021/c/c9/T--SHSID--Results04.png)
Figure 4: Gel electrophoresis diagram. Lane 1 to 3 is segments 4567. Lane 4 to 6 is segment 8. Lane 7
to 9 is segments 9.

This result shows that the overlap PCR between segments 45 and 67 was successful. The PCR of segment 9 is
also successful. However, there are no clear bands shown in lane 4 to 6, indicating that the PCR of segment
8 is not successful.

Segment 8 was amplified using PCR again.

![](https://static.igem.org/mediawiki/2021/4/47/T--SHSID--Results05.png)

Figure 5: Gel electrophoresis diagram. Lane 1 to 3 is segment 8.

This result shows that there are bands on the right position corresponding to the number of base pairs, but
they are not clear and bright enough. The product of this round of PCR can be used in the construction of
plasmid.

The fifth round of PCR

The fifth round of PCR overlapped segment 67 with 8, and segment 4567 with 8.

![](https://static.igem.org/mediawiki/2021/6/63/T--SHSID--Results06.png)
Figure 6: Gel electrophoresis diagram. Lane 1, 3, 5 are segment 45678. Lane 2, 4, 6 are segment 678.

This figure shows that the overlap PCR of segments 67 and 8 was successful, while segments 4567 and 8 was
not successful. This is because segment 45678 is too long, which is difficult to carry out accurate overlap
PCR.

The sixth round of PCR

The sixth round of overlap PCR connected segments 4567 with 678, which is a modified version of overlap PCR
between 4567 and 8 in previous round.

![](https://static.igem.org/mediawiki/2021/d/d3/T--SHSID--Results07.png)
Figure 7: Gel electrophoresis diagram. Lane 1 is segment 45678.

This result shows that the overlap PCR between segments 4567 and 678 was successful. This is because these
two segments have more overlapping base pairs, which ensures higher efficiency during overlap PCR.

Homologous recombination of 0 to 9 fragments

In order to obtain our target plasmid, there are multi-fragment assembly plan A and B. In plan A, PCR
amplification products of 0123, 456, 678 and 9 were recovered from gel and the corresponding recombinant
plasmids were transformed into competent cells for resistance screening of kana. In plan B, the sequence
fragments were 0123, 4567, 8 and 9. Five single colonies in plan A and one single colony in plan B were
picked up for further cultivate and plasmid extraction. Identification by electrophoresis showed that
plasmid 1,4,5 of plan A and 6 of plan B were the candidate with correct size, in which plasmid 5 of plan A
was confirmed by sequencing.

![](https://static.igem.org/mediawiki/2021/b/b5/T--SHSID--Results08.png)

Figure 8: Gel electrophoresis diagram. Line 1-5 is plasmids 1-5 on plan A plate, in which the size of 1, 4,
and 5 are correct. Line 6 is plasmid 6 on the plan B plate with correct size. Line 7 is a negative control
plasmid.

![](https://static.igem.org/mediawiki/2021/4/40/T--SHSID--Results09.jpg)

Figure 9: Blast DNA sequences with theoretical sequences and actual sanger sequencing documents of
pYES2-ASADC-SPE1-SpeB. The blast result shows that the plasmid is constructed successfully.

And then, Plasmid 5 was transferred into BY4741 and AQ competent cells. For BY4741, the initial OD600 of
seed cells was 0.3, and then cells were collected when OD600 reached 0.9 after 3h of culture. The
transformed plasmid pYES2-ASADC-SPE1-SpeB was cultured on YPD20 with hygromycin B plates for 3 days.

Fermentation test

The clones were picked up into YPD20/Hyg test tube for cultivate and activation. In order to observe the
growth of fermentation strains, we set up negative control and test group, referred as NC and Test,
respectively. Each of them also includes a putrescine production group referred as NC+Arg\*10 and
Test+Arg\*10, respectively, whose medium was supplemented with 10 times Arg.

![](https://static.igem.org/mediawiki/2021/9/9d/T--SHSID--Results10.png)
Figure 10. Growth curve of fermentation strains

As shown in the figure, the growth trend of the four groups was similar. It took 24 hours to reach the
stationary phase of growth and this stable period last up to 96 hours. Specifically, the overall growth of
NC+Arg\*10 and Test+Ar\*10g group was a little lower than that of NC and Test group perhaps due to the
pressure of high concentration of Arg.

At least 3 repeated small-scale metabolite production tests were carried out in YNB-SC medium (containing 10
times arginine raw material). 2ml supernatant (containing putrescine) of the 48h-metabolites after
centrifugation were analyzed by LC-MS.

![](https://static.igem.org/mediawiki/2021/1/1c/T--SHSID--Results11.jpg)

Figure 11. The peak of putrescine detected by LC-MS.

The results showed that the peak of putrescine appeared at 32 min (as shown in the figure11), indicating
that the engineered strain we constructed successfully produced putrescine. However, the peak area is small,
and the output of putrescine is far below the factory mass production level. It is likely that the protein
expression is insufficient and the metabolic pathway is limited. In the future, the expression of the three
proteins needs to be further optimized.

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