iGEM 2021 — Team Shanghai_Metro_HS

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# Team:Shanghai Metro HS/Notebook

Shanghai\_Metro\_HS

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![](https://static.igem.org/mediawiki/2021/a/a4/T--Shanghai_Metro_HS--Team_Members01.png)

8.16.21

Orientation day: each member of the Ceres team introduced themselves to the whole
team.
we also had an ice-breaking activity which is a game that requires lots of discussions and got everyone
involved.

Then, our coach explained our project to us and our plan for the next few days.
Afterward, we went on to team name/logo generation and researching for ideas.

![](https://static.igem.org/mediawiki/2021/2/28/T--Shanghai_Metro_HS--NOTEBOOK01.jpg)
![](https://static.igem.org/mediawiki/2021/b/ba/T--Shanghai_Metro_HS--NOTEBOOK02.jpg)

8.17.21

Wet Lab:
Our coach firstly explained our lab routine for the next few days and the scientific mechanism of the
design.
1. We prepared both liquid state and solid-state culture mediums which will be used in the future for the
growing of our engineer bacteria.
2. We also underwent restriction enzyme(Sah 1, Xho 1) cutting to expose sticky ends on the gene PKC-op and
plasmid PET 25b.
3. obtained the recombinant DNA by connecting cut gene PKC0-op with cut plasmid PET 25b using T4 ligase.

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

8.18.21

1. transfer the recombinant DNA into our BL21 competent cell, then culture the
cells via the shaking incubator inside liquid state LB culture medium which has no ampicillin inside.
afterward spread the cells evenly on the solid-state culture medium which has ampicillin in, culture the
cells at 37 degree celsius for 24 overnight (24 hours).

2. prepare ampicillin solution for future use, sterilise.

![](https://static.igem.org/mediawiki/2021/4/41/T--Shanghai_Metro_HS--NOTEBOOK04.jpg)
![](https://static.igem.org/mediawiki/2021/7/79/T--Shanghai_Metro_HS--NOTEBOOK05.jpg)

8.19.21

there was no visible growth in all petri dishes, our bacteria failed to grow.
Therefore we underwent the same process as before again to try to grow our bacteria.

1. to make sure that we obtained the correct DNA fragment, we ran electrophoresis
on the cut gene.

8.20.21

1. extract recombinant plasmids from our grown e.coli bacteria(BL21 competent cell)
via alkaline lysis

2. test the concentration of plasmids extracted via nanodrop. then carryout the
restriciton enzyme cutting to the recombinant plasmid in order to obtain individual gene which we will run
electrophoresis on later.

3. We ran electrophoresis on the DNA fragments obtained from restriction enzyme
cutting, however there were no sensible results visible, eventually we sent our DNA fragments to sequencing.

4. After failing the electrophoresis, because our instructor's bacteria were grown
well, we cultured those bacteria and planned to use those for future experiments.

![](https://static.igem.org/mediawiki/2021/6/6c/T--Shanghai_Metro_HS--NOTEBOOK06.jpg)
![](https://static.igem.org/mediawiki/2021/b/b6/T--Shanghai_Metro_HS--NOTEBOOK07.jpg)

8.22.21

it was a day of break

8.22.21

1. Results of sequencing were not valid, therefore we changed our testing approach
of the DNA fragment. Instead of extracting the recombinant plasmid and cutting it using a restriction
enzyme, we used PCR to verify our grown bacteria, then ran electrophoresis on it. There were sensible
results however it was not strong enough (bands at 1000bp were quite shallow). Hence we have to just extract
the recombinant plasmid from the bacteria and send it to sequencing for further proof.

2. We transferred the original gene PUC57-PKC-OP which the company has into our
BL21 competent cell, then cultured it via a shaking incubator and solid-state culture medium which has
ampicillin in at 37 degrees celsius for 24 hours. (Similar to the processes on 8.18)

![](https://static.igem.org/mediawiki/2021/d/d5/T--Shanghai_Metro_HS--NOTEBOOK08.jpg)
![](https://static.igem.org/mediawiki/2021/7/71/T--Shanghai_Metro_HS--NOTEBOOK09.jpg)

8.23.21

1. We prepared IPTG and glucose which will be used for the Induction of cellulase
protein expression and chromogenic reaction

2. We used PCR to extract the DNA(PKC-op) that we wanted.
we used PCR to amplify pkc-op, then we ran electrophoresis on it, we saw sensible results. (bands were
visible at 1000bp）

3. We recycled pkc-op that is in the gel

4. Meanwhile, we continued culturing bacteria that have PUC57-PKC-OP gene inside
it.

![](https://static.igem.org/mediawiki/2021/c/cf/T--Shanghai_Metro_HS--NOTEBOOK10.jpg)
![](https://static.igem.org/mediawiki/2021/5/59/T--Shanghai_Metro_HS--NOTEBOOK11.jpg)

8.24.21

1. We extracted and stored plasmid PET 25b for future use.

2. We tested the OD600 value for the cultured bacteria that has gene PUC57-PKC-OP,
we found that we over cultured those bacteria hence we could not use them for next steps

3. Therefore we restart the culturing process for bacteria that have gene
PUC57-PKC-OP, and we test their OD600 value every 30 minutes. Eventually we obtained bacteria in the
logarithmic growth phase, then we put it in ice for storing.

4. At the same time, we underwent restriction enzyme cutting on pkc-op(that we
recycled from the gel from 8.23) and pet 25b(from step 1). Using two restriction enzymes Sah I and Xho I.

5. Ran electrophoresis on cut pkc op and pet 25b, recycle them afterwards and store
them at -20 degrees celsius for the transformation on the next day.

![](https://static.igem.org/mediawiki/2021/3/3e/T--Shanghai_Metro_HS--NOTEBOOK12.jpg)

8.25.21

1. We connected two cut genes using T4 ligase.

2. Transferred the recombinant plasmid into our BL 21 competent cell, culture the
bacteria just like before. (shaking incubator, then spread on solid culture medium with ampicillin in, 37
degrees celsius in the incubator for 24 hours.)

3. Bacteria colonies that have the PUC57-PKC-OP gene were induced by IPTG under
conditions: 4 hours, 8 hours, 12 hours, 16hours, and 0.01mmol/L, 0.5mmol/L, 1mmol/L. we underwent ultrasonic
cell fragmentation centrifugation to all bacteria in concentrations of 4 hours and 8 hours. Due to the fact
that there is no signal peptide-guided cellulase expression, the location of expressed cellulase is
uncertain. Hence we tested the protein expression levels of both clear liquid and bottom bacteria colonies
of the two centrifuges. In theory, we should find cellulose in the sunk bacteria colony or in the clear
liquid (depending on the solubility of the cellulose) of the second time centrifuge. The results will be
displayed through SDS Page electrophoresis, the results will be available on the next day.

![](https://static.igem.org/mediawiki/2021/4/41/T--Shanghai_Metro_HS--NOTEBOOK13.jpg)

8.26.21

1. Underwent PCR on the bacteria that has gene PET 25b-PKC OP that we grew for 24
hours, afterwards verify the gene using electrophoresis.

2. React different concentration of glucose (0mmol/L, 0.2mmol/L, 0.4mmol/L,
0.6mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L, 1.4mmol/L, 1.6mmol/L) with SDS to carryout chromogenic reaction,
then use nanodrop to test OD540 which enable us to draw a standard graph presenting the correlation between
the OD540 value and the concentrations of glucose.

3. Results from SDS Page electrophoresis shows that there is protein in the broken
bacteria which sinked. There were also shallow bands shown in the clear liquid of second centrifuges. Which
means cellulose cannot easily dissolve in environmental solutions. Therefore we underwent testing using
suspended the rest of the sunken bacteria colonies and added filter paper (made of cellulose) and xylan
(hemicellulose), DNS was added and chromogenic reaction was performed by Nanodrop measurement OD540.
Together with the graph obtained in step2, we are able to obtain data of enzyme activity in different
culture conditions. In theory both filter paper and xylan can be decomposed and filter paper will be
decomposed more in particular. However we only obtained the results for when bacteria were grown for 4 hours
and 8 hours, we think we can only get the most accurate and reliable outcome when we obtain the results for
bacteria that were grown for 12 hours and 16 hours.

8.27.21

The team Ceres had a presentation drill to present our findings to the audience.

Sept.

We repeat the plamid construction experiments through Homologous recombination and
finally obtained the pET-25b-PCK-OP E. coli BL21 strains.

![](https://static.igem.org/mediawiki/2021/0/0c/T--Shanghai_Metro_HS--NOTEBOOK14.jpg)
![](https://static.igem.org/mediawiki/2021/0/08/T--Shanghai_Metro_HS--NOTEBOOK15.jpg)

CONTACT INFO

Email: igemteamceres@gmail.com

*WeChat Official Account: iGEM Ceres*

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

# Team:Shanghai Metro HS/Engineering

Shanghai\_Metro\_HS

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Background

In China, animal husbandry in many places has become a pillar industry of the rural
economy and the main source of increasing farmers’ income. With the continuous expansion of the scale of
animal husbandry production, the demand for high-quality feed has also increased significantly. Silage is a
kind of natural plant feed. After cutting the stems and leaves of plants with a moisture content of 65%-75%,
under airtight hypoxia conditions, the fermentation effect of anaerobic lactic acid bacteria is used to
inhibit the reproduction of various miscellaneous bacteria. And it is mainly used to feed ruminants (such as
cattle, sheep, alpacas, and deer). Compared with fresh feed, silage is more durable in storage, has stronger
nutrients than dry feed, and is rich in nutrients, which is conducive to long-term preservation. It is an
excellent source of feed for livestock.

Silage contains a lot of cellulose, which is the main structural component of plant
cell walls. It is usually combined with hemicellulose, pectin and lignin, which is difficult to digest and
absorb. Enzymes that can degrade cellulose can be added to the silage to improve the ruminant's ability to
obtain monosaccharides. We can prepare cellulase through experiments and explore the best conditions for
cellulose degradation. Use it as a silage additive and add it to feed to promote appetite of livestock,
increase and supplement the content of various digestive enzymes in the animal’s body, prevent and treat
some common gastrointestinal diseases of livestock and improve the quality of livestock products.

Design

The alkaline cellulase gene from Pseudomonas aeruginosa PKC-001 was selected and
inserted into the pET-25b vector which contains a pelB signal peptide. The recombinant plasmid then was
transformed it to E. coli BL21(DE3) strain to produce cellulase.

Build

The pET-25b-PKC expression plasmid was successfully constructed by homologous
recombination PCR technology (Figure 1).

![](https://static.igem.org/mediawiki/2021/5/53/T--Shanghai_Metro_HS--Engineering01.jpg)
Figure 1. Schematic map of pET-25b-PKC expression plasmid.

Test

Different induction conditions were tested for protein expression. As the pET-25b
vector contains a pelB signal peptide, the engineered strain would secret the protein into the medium.
Therefore, we collected the culture supernatant after induction and ran SDS-PAGE for verification (Figure
2).

![](https://static.igem.org/mediawiki/2021/7/79/T--Shanghai_Metro_HS--Engineering02.jpg)
Figure 2. SDS-PAGE analysis of culture supernatant under different induction conditions.

The theoretical molecular weigh of the PKC cellulase is 45.6 kDa. As seen from the
SDS-PAGE (Figure 2), there is a wide band just above 40 kDa and it indicates that we have obtained the PKC
cellulase.

Learn

In this experiment, we successfully prepared genetically engineered bacteria which
produce PKC cellulase.

CONTACT INFO

Email: igemteamceres@gmail.com

*WeChat Official Account: iGEM Ceres*

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

# Team:Shanghai Metro HS/Experiments

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# Team:Shanghai Metro HS/Results

Shanghai\_Metro\_HS

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![](https://static.igem.org/mediawiki/2021/d/d6/T--Shanghai_Metro_HS--results01.jpg)
Figure 1. The result of enzyme cutting and PCR

This picture is the nucleic acid electrophoresis result of
enzyme cutting and
PCR(By performing PCR, we can obtain the desirable PKC-OP’s gene segment.). Column “marker” is a column that
is used to show the position of different lengths of genes. In this step, our aim is to verify whether these
results are the desired ones.
In figure 1, the number labeled above figure 1 represents different results. To begin with, number 1 and 2
is the result for pET-25b after enzyme cutting. Additionally, number 3 is the pET-25b without enzyme
cutting. At last, number 4 and 5 is the PKC-OP after PCR. As our experiment moves on, the data of our PKC-OP
and pET-25b are 233.2ng/μL and 17.8ng/μL. Lastly, we use the homologous combination to combine them.

![](https://static.igem.org/mediawiki/2021/6/61/T--Shanghai_Metro_HS--results02.jpg)
Figure 2 E. coil having the desired pET-25b and PKC-OP

The pET25b-PKC-OP was constructed.

The plate shows monoclonals of pET25b-PKC-OP constructs

![](https://static.igem.org/mediawiki/2021/b/b3/T--Shanghai_Metro_HS--results03.jpg)
Figure 3. The result of Apa1 enzyme digestion validation

Column “marker” is a column that is used to show the position of different lengths of genes. Number 1 to 10
is the result for recombinant plasmid pET25b-PKC-OP after Apa1 enzyme digestion. We get two bands of 4712bp
and 1894bp. It further indicates that the obtained monoclonals were positive monoclonals containing the
recombinant plasmid.

Future plan: Testing the effectiveness of producing
cellulase of positive
recombinant bacteria. This is important because we need to know how much bacteria is needed to be used to
effectively help animals digest fodder. In this experiment, we can control the time that positive
recombinant bacteria grow and use the OD test to set the numbers of bacteria. Then put different numbers of
bacteria and cellulose filter paper together. OD test can be used again to see the glucose concentration.

![](https://static.igem.org/mediawiki/2021/5/55/T--Shanghai_Metro_HS--chgt3.jpg)

![](https://static.igem.org/mediawiki/2021/7/78/T--Shanghai_Metro_HS--chgt4.jpg)

We designed several different induction conditions for protein expression. As the vector we chose to
construct the plasmid contains a pelB signal peptide, the engineered strain would secret the protein into
the medium. Therefore, we collected the culture supernatant after induction and ran SDS-PAGE for
verification.
According to the calculation by Snapgene, the expected protein molecular shall weigh 45.6 KDa. As seen from
the SDS-PAGE map (Fig. 4), there is a wide band just above 40 KDa and it indicates that we have obtained the
PKC-OP protein.

Future Plan

Testing the effectiveness of producing cellulase of
positive recombinant bacteria.
This is important because we need to know how much bacteria is needed to be used to effectively help animals
digest fodder. In this experiment, we can control the time that positive recombinant bacteria grow and use
the OD test to set the numbers of bacteria. Then put different numbers of bacteria and cellulose filter
paper together. OD test can be used again to see the glucose concentration.

CONTACT INFO

Email: igemteamceres@gmail.com

*WeChat Official Account: iGEM Ceres*