iGEM 2021 — Team Shanghai_HS_ID

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# Team:Shanghai HS ID/Notebook

Shanghai\_HS\_ID

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  + [Proof Of Concept](https://2021.igem.org/Team%3AShanghai_HS_ID/Proof_Of_Concept)
  + Notebook
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* Parts

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* Human Practices

  + [Integrated Human Practice](https://2021.igem.org/Team%3AShanghai_HS_ID/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AShanghai_HS_ID/Communication)
  + [Fundraising](https://2021.igem.org/Team%3AShanghai_HS_ID/Fundraising)
* [Implementation](https://2021.igem.org/Team%3AShanghai_HS_ID/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3AShanghai_HS_ID/Entrepreneurship)
* [Model](https://2021.igem.org/Team%3AShanghai_HS_ID/Model)
* Team

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![](https://static.igem.org/mediawiki/2021/c/c3/T--Shanghai_HS_ID--banner.jpg)

NOTEBOOK

27th June: We had our first team meeting and discussed our topic

28th June: We made the work distribution and finalized the designs of
the team name, logo, and merch. Besides, we conducted the interview with an expert.

![](https://static.igem.org/mediawiki/2021/5/5f/T--Shanghai_HS_ID--Notebook01.jpg)

**Wet team agenda**

28th June:

• Lab safety training

![](https://static.igem.org/mediawiki/2021/2/2a/T--Shanghai_HS_ID--Notebook03.jpg)

30th June- 2nd July:

• Construction of sgRNA, upstream and downstream homologous arms
• Overlap PCR and electrophoresis
• Preparation of mediums
• Bacteria culture

![](https://static.igem.org/mediawiki/2021/a/a9/T--Shanghai_HS_ID--Notebook04.jpg)

3rd July:

• pLCNICK enzyme digestion
• Connection of sgRNA and pLCNICK
• Transformation of the recombinant plasmid into E.coli

![](https://static.igem.org/mediawiki/2021/6/66/T--Shanghai_HS_ID--Notebook05.jpg)

4th July:

• Culture L. casei cells
• Preparation of competent L. casei cells

![](https://static.igem.org/mediawiki/2021/2/2a/T--Shanghai_HS_ID--Notebook06.jpg)

5th July:

• Picked and shakes L. casei
• L. Casei PCR and electrophoresis

![](https://static.igem.org/mediawiki/2021/8/8d/T--Shanghai_HS_ID--Notebook08.jpg)

6th-7th July :

• Verification of the plasmid
• Electrotransformation of the plasmid into L. casei ATCC 533
August an September
• Repeat the plamid construction and electrotransformation
• L. casei cultivation

![](https://static.igem.org/mediawiki/2021/7/74/T--Shanghai_HS_ID--Notebook09.jpg)

Dry team agenda

28th June:

• Published the first article of our Wechat official account: introducing us and
our topic.

![](https://static.igem.org/mediawiki/2021/d/d9/T--Shanghai_HS_ID--Notebook10.jpg)

30th June:

• Discussed the script of our promotion video
• Listed survey questions and interview questions

![](https://static.igem.org/mediawiki/2021/f/f4/T--Shanghai_HS_ID--Notebook11.jpg)

1st July :

• Organized and analyzed the records of the 1st Expert Interview
• Gathered information for our topic background
• Prepared a question list for the site visit to Bright Dairy & Food company in the next day
• Assigned tasks for the next day's site visit

2nd July :

• Data analysis on survey results
• Integrated all interview records of the past few days
• Prepared questions for the 2nd Expert Interview
• Drafted a new article of our public Wechat account for the site visit
• Ordered merch: notebooks
• Designed post-it notes
• organize stuff for the article for the merch sale

![](https://static.igem.org/mediawiki/2021/f/f4/T--Shanghai_HS_ID--Notebook12.jpg)

3rd July :

• Drafted self-introductions
• Drafted integrated human practices (transcription and translation of interview records, data of our Wechat
official account and survey results)
• Booked another interview
• Listed interview questions
• Publish the second article of our Wechat official account, which was focused on our site visit to Bright
Dairy & Food company.

4th July :

• Drafted inspiration
• Discussed to determine the lecture location and target audience
• Promotion video filming
• Roll Screen design
• Visit BFC for merch-sale site pre-check

![](https://static.igem.org/mediawiki/2021/4/4a/T--Shanghai_HS_ID--Notebook13.jpg)

5th July :

• Finalized IHP and inspiration
• Finalized details and publicize charity sale
• Started writing implementation and business plan
• Promotion video filming

![](https://static.igem.org/mediawiki/2021/f/f0/T--Shanghai_HS_ID--Notebook14.jpg)

6th July :

• Drafted attributions, business plan
• Initially completed the script of our promotion video

![](https://static.igem.org/mediawiki/2021/5/5b/T--Shanghai_HS_ID--Notebook15.jpg)

7th July :

• Planed for another charity sale
• Started writing a new article for merch sale to be published in our Wechat official account
• Collaboration activity
• Built our cost structure
• Finalized the script for promotion video
• First merch sale: yielded a profit of 50 RMB around and got 13 more followers

![](https://static.igem.org/mediawiki/2021/c/cd/T--Shanghai_HS_ID--Notebook16.jpg)

8th July :

• Drafted future plan, safety, public engagement, and education
• Organized what we have done and drafted the first version of the presentation PowerPoint
• Fundraising

![](https://static.igem.org/mediawiki/2021/c/c9/T--Shanghai_HS_ID--Notebook17.jpg)

11th July :

• Published the article about our merch sale in Wechat official account
• Determine the sale day online for our merch

Follow us on WeChat!

![](https://static.igem.org/mediawiki/2021/c/c3/T--Shanghai_HS_ID--qrcode.png)

*WeChat Official Account: 乳酸LAB*

Contact Email：*jhxchen@gmail.com*

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

# Team:Shanghai HS ID/Engineering

Shanghai\_HS\_ID

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  + [Proof Of Concept](https://2021.igem.org/Team%3AShanghai_HS_ID/Proof_Of_Concept)
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Engineering

Background

Lactobacillus casei is commonly used in the production of cheese, yogurt, and other
products. Studies have shown that L. casei can regulate the intestinal flora, lower blood pressure and
cholesterol, enhance immunity, prevent cardiovascular disease and fight cancer. The unique probiotic
function of L. casei and its wide application in the field of food and medicine make the study of the
molecular mechanism of L. casei have great significance, and the in-depth study of it involves the transfer
of foreign DNA into L. casei.

Research showed that the transfermation efficiency of foreign DNA in L. casei is
much lower than the model microorganism Escherichia coli. This will restrict the research on the molecular
mechanism of L. casei. Therefore, in order to realize the heterologous expression of foreign genes, it is
first necessary to realize the efficient transformation of L. casei.

Design

In this study, the CRISPR-Cas9 gene editing method was used to knock out the
restriction enzyme LSEI-2094 gene of L. casei ATCC 334. The gene knockout strains were selected by
antibiotics resistance, PCR and sequencing. The yield L. casei strain is of great significance to the
development of L. casei genetic engineering.

Figure 1 shows the design of a CRISPR-Cas9-based gene knockout vector in L. casei
ATCC 334. Single plasmid CRISPR-Cas system is applied, namely gRNA, Cas effector protein, and repair
template on one plasmid.

![](https://static.igem.org/mediawiki/2021/c/c2/T--Shanghai_HS_ID--engineering01.jpg)

Figure 1. CRISPR-Cas9-based gene knockout vector design in Lactobacillus casei ATCC
334. P is the promoter (promotor); T is the terminator (terminator); pNCas9 is the Cas effector protein;
HA-L and HA-R are the left homology arm and the right homology arm, respectively.

Build

![](https://static.igem.org/mediawiki/2021/9/95/T--Shanghai_HS_ID--engineering02.jpg)

Figure 2. A: DNA profile of the plasmid PLCNICK; B: DNA Profile of LSEI-2094+
upstream and downstream homologous arms.

Test

**(1) Improvement of transformation efficiency**

Plasmid pIB165 was transformed the as the foreign DNA to test the transformation
efficiency of our modified L. casei. It took several days to culture and finally observed the comparison
results. As shown in Figure 3, the modified L. casei (KO) has higher transformation efficiency with
remarkably more colonies than the wild strain.

![](https://static.igem.org/mediawiki/2021/1/1b/T--Shanghai_HS_ID--engineering03.jpg)

Figure 3. Comparison between the wild L. caseil (left) and modified L. casei
(right, LSEI-2094 knocked out) in transformation.

![](https://static.igem.org/mediawiki/2021/5/53/T--Shanghai_HS_ID--engineering04.jpg)
Figure 4. Histogram comparison between wild strain and KO strain.

In addition, we measured OD600 of these strain groups which were pre-spreaded
plates with different volumes of bacteria solutions so as to quantify the transformation results as showing
above (Figure 4).

**(2) model build**

In order to scientifically determine the transformation efficiency of our
modified L. casei (KO) and the wild L. casei (Wild), we collected the colony cultured which were pre-spread
plates with different volumes of bacteria solutions and measured their OD600 after cultured for the same
hours.
In the meantime, we also aimed to explore the optimal condition for our modified L. casei’ growth by
applying
different amounts of the bacteria seed solution.

![](https://static.igem.org/mediawiki/2021/d/dd/T--Shanghai_HS_ID--engineering05.jpg)

According to the scatter plots, we chose to use the quadratic polynomial equation
to
build the model :

![](https://static.igem.org/mediawiki/2021/3/30/T--Shanghai_HS_ID--engineering06.jpg)

After calculation, below are the constants of the solved quadratic polynomial
equations
of the KO group and Wild group, respectively.

Table 2. Model results
![](https://static.igem.org/mediawiki/2021/1/14/T--Shanghai_HS_ID--engineering07.jpg)

![](https://static.igem.org/mediawiki/2021/f/fa/T--Shanghai_HS_ID--chgt2.jpg)
Figure 5. Comparison between two fitting curves of KO group(red) and Wild group(blue).

In figure 5, we can clearly see that the modified L. casei shows much higher
transformation efficiency than the wild L. casei especially when the volume of the initial bacteria seed
solution is used less than 100 uL when the difference between them is remarkable.

Besides, the equation model we built for the modified L. casei as showing below,
could
be used to analyze the relationship between the volume of the bacteria seed solution and its
OD600,
it could be
used as a reference when we conduct the expression efficiency tests in the future. If we could further build
the
relationship between the expression level and OD600, we could adjust the volume of the bacteria
seed
solution
for culturing accordingly.

**Model for the modified L. casei:**

![](https://static.igem.org/mediawiki/2021/b/bf/T--Shanghai_HS_ID--engineering09.jpg)

Learn

In conclusion, it indicates that our modified L. casei has much higher efficiency
of
the foreign plasmid transformation than the wild and the modified L. casei has great potential to be used as
the
recombinant carrier in various areas.

Follow us on WeChat!

![](https://static.igem.org/mediawiki/2021/c/c3/T--Shanghai_HS_ID--qrcode.png)

*WeChat Official Account: 乳酸LAB*

Contact Email：*jhxchen@gmail.com*

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

# Team:Shanghai HS ID/Experiments

Shanghai\_HS\_ID

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

  + [Description](https://2021.igem.org/Team%3AShanghai_HS_ID/Description)
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  + [Proof Of Concept](https://2021.igem.org/Team%3AShanghai_HS_ID/Proof_Of_Concept)
  + [Notebook](https://2021.igem.org/Team%3AShanghai_HS_ID/Notebook)
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  + [Fundraising](https://2021.igem.org/Team%3AShanghai_HS_ID/Fundraising)
* [Implementation](https://2021.igem.org/Team%3AShanghai_HS_ID/Implementation)
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* [Model](https://2021.igem.org/Team%3AShanghai_HS_ID/Model)
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Experiments

I. General Protocols

**1. MRS media preparation**

10g Tryptone
2g Meat extract
4g Yeast extract
20g Glucose
2g K2HPO4
5g Sodium acetate trihydrate
2g (NH4)2 citrate
0.2g MgSO4-7H2O
0.05g MnSO4-H2O
1g Tween-80
With up to 2L distilled water

**2. LB media preparation**

10g Tryptone
5g Yeast extract
10g NaCl
15g Agar
With up to 1L distilled water

**3. Obtaining the Lactobacillus Casei colony**

• Culture Lactobacillus. casei by using the streak plate method to get a single bacterial colony
• Incubate in 37℃ conditions overnight

**4. Kanamycin solution preparation**

• Combine 0.15g Kanamycin with 3mL water to prepare a 50μg/mL medium.
• Use vortex mixer to melt the Kanamycin
• Sterilize the solution with a 0.22μm aperture filter paper
• Suck up the kanamycin solution into the syringe
• Remove the needle and put it on the filter
• Squeeze the solution into a 15ml PCR tube
• The complete sterilization procedure should be done in the ultra-clean cabinet

**5. Resuscitation media preparation**

• MRS (liquid form)
• 50 uM glucose
• 20 uM magnesium chlorides
• 2 uM calcium chlorides

II. Construction of the recombinant plasmid

To achieve our goal, we need to construct a recombinant plasmid pNCas9-LSEI-2094
that contains the sgRNA,
which guides the Cas9 protein to locate the target gene. This construction has 5 main steps and some other
auxiliary experiments, as presented below.

**A.** Amplification of sgRNA and homologous arms,
**B.** Enzyme digestion that cuts the circular plasmid into a linear form
**C.** Connection of the plasmid and sgRNA
**D.** Transformation
**E.** Verification

**1. Amplify right and left homology arms, sgRNA through PCR**

•Amplification of upstream and downstream homology arms
•Design primers (around 1000 bp for each homology arm)
•Go to website NCBI to obtain the DNA sequence of L.casei ATCC 334
•Use online software to design the primers for upstream and downstream homology arms of LSEI-2094 gene
•ATCC 334 bacterium solution as a template
•334 2094-up-F/ 334 2094-up-R as primers for upstream homology arm
•334-2094-down-F / 334 2094-down-R as primers for downstream homology arm
•PrimeStar is used as the DNA polymerase

PCR protocol:

（1）95℃ 2min
（2）98℃ 10s
（3）53℃ 15s
（4）72℃ 90s
（2）-（4）repeat for 30 times
（5）72℃ 5min
（6）12℃ ∞（preserve in a low temperature）

PCR reaction system:

![](https://static.igem.org/mediawiki/2021/a/a1/T--Shanghai_HS_ID--Experiments01.jpg)

• Amplification of sgRNA
• Design primers
• Go to website NCBI to search for the DNA sequence of LSEI-2094 gene in L.casei ATCC 334
• Design sgRNA and its primer
• Amplify sgRNA1
• pLCNICK as a template
• Use 334 2094-sgRNA-F/ 334 2094-sgRNA-R1 as primer to amplify sgRNA1
• Amplify sgRNA2
• sgRNA1 as a template
• Use BSAI-334 2094-sgRNA-F/ BSAI-334 2094-sgRNA-R2 as primer to amplify sgRNA2

PCR protocol: same as the one for homology arm amplification
PCR reaction system:

![](https://static.igem.org/mediawiki/2021/1/13/T--Shanghai_HS_ID--Experiments02.jpg)

**2. Enzyme digestion to cut the plasmid into linear form**

Use enzymes XbaI and ApaI for double enzyme digestion
Enzyme digestion reaction system:

![](https://static.igem.org/mediawiki/2021/d/d1/T--Shanghai_HS_ID--Experiments03.jpg)

The electrophoresis result of the product after double enzyme digestion

![](https://static.igem.org/mediawiki/2021/8/8c/T--Shanghai_HS_ID--Experiments04.jpg)

1-4 are pLCNICK after enzyme digestion, 5 is pLCNICK plasmid before enzyme
digestion, 6-7 are upstream
homologous arms after PCR, 8-9 are downstream homologous arms after PCR.
Use gel extraction to recycle the homologous arms and plasmids)

**3. Homologous recombination**

• The mass of the carrier is the length of the carrier (1200bp) times 0.02, giving a result of 240 μg. As
it’s out of the available range (50-200 μg ), we instead use the minimum of 50μg
• The DNA mass we need is the length of the DNA ( 2200 bp) times 0.04, 88 μg, which can be used directly.
The volume of the carrier and DNA is obtained from the formula mass/ concentration measured ( unit: μL
)
• The connection system is 20 μL, which contains :
• Carrier ( 50/concentration )
• DNA ( 88/concentration )
• Exnase II enzyme ( 2 μL )
• CE II buffer ( 5\* concentration ) 4μL
• Make up the rest with water
• (the steps are all done on ice)

Reaction system:

![](https://static.igem.org/mediawiki/2021/7/7a/T--Shanghai_HS_ID--Experiments05.jpg)

Use pipette to blend, collect the liquid after brief centrifugation

**4. Transform the constructed plasmid into DH5α and add kanamycin to the LB
medium to culture the
DH5α**

• Take out E. coli DH5α competent cells from -80 ℃ refrigerator
• Add reconstructed plasmids for every 100 μL competent cells
• Ice-bath for 30 min
• Water-bath at 42 ℃ for 45 s, and immediately place it on ice for 3 min
• Add 700 μL liquid LB medium
• Shake culture at 37 ℃ for 1 hour
• Spread coat 100μL of the mixture on the LB plate that has kanamycin (50μg/mL)
• Inverted culture at 37 ℃ for 12-16 hours

**5. Extract the plasmid from the cultured DH5α**

• Column balancing steps: Add 500 μL of BL buffer into the adsorption column with a collection tube,
centrifuge for 1 min ( with a centrifugal force of 12000 g ), pour out the waste liquid in the collection
tube.
• Add (1-5mL) of the cultured DH5α into the centrifugal tube, centrifuge for 1 min with 12000g of the
centrifugal force to make the bacteria precipitate on the pipe wall, and empty the supernatant.
• Add P1 buffer (250 μL) into the centrifugal tube which contains the precipitate of bacteria, use vortex
mixer to make bacteria suspend
• Add P2 buffer (250 μL) into the centrifugal tube, flip upside down gently to fully lyse the bacteria ( a
vigorous shock may break the DNA )
• Add P3 buffer (350μL) into the centrifugal tube, flip upside down gently for 6-8 times. At this point, a
milky precipitate may appear (the lysate of the bacteria), then centrifuge for 10 min with a centrifugal
force of 12000g
• Remove the supernatant from the last step into an adsorption column (which contains a collection tube),
avoid the precipitate, centrifuge for 1 min, and pour out the waste liquid in the collection tube.
• Add 600μL of PW already containing alcohol into the adsorption column, centrifuge for 1 min, pour out the
waste liquid in the collection tube. (twice)
• Centrifuge for 2 min to remove the remaining buffer. Open the lid of the adsorption column and let stand
for 2-3 mins at room temperature to dry the remaining buffer completely, in case the alcohol may affect the
later experiment.
• Put a new EP tube below the absorption, aim at the adsorption column membrane to add the EB buffer
(100μL), let stand for 2 mins at room temperature/ centrifuge for 2 mins to make the plasmid enter the EP
tube.
• Testify the plasmids
• Pick multiple colonies and add them into 10μL LB medium. Use it as a template for PCR. Use 334
2094-down-F/ 334 2094-down-R as primers testify through PCR
• Electrophoresis result:

![](https://static.igem.org/mediawiki/2021/b/bb/T--Shanghai_HS_ID--Experiments06.jpg)

• Add the colonies that are tested positive into 3 mL
liquid LB medium that has 50 mg/L kanamycin, 200rpm, and shake the culture at 37℃

**6. Test whether the reconstruction of the plasmid is successful**

• Deliver the plasmids to Tsingke Biotechnology for DNA sequencing
• Primer: Nickcas-seq-F/Nickcas-seq-R
• Compare and analyze the result according to the sequencing result on software Snapgene

III. Obtainment of the modified L. casei strains

After the construction of the plasmid, we need to get the modified L.casei strains
by transforming the
plasmids into more L.casei, which needs electroporation.

**1. Preparation of competent cell for L.casei（all procedure should be done on
ice）**

• Culture the single colony of L.casei in the constant temperature shaker until its value of OD600 reaches
approximately 0.6 (at which the bacteria has reached the plateau developmental phase)
• Pour the bacterium solution into centrifugal tubes of 50mL (pre-cooled), centrifugate (4℃, 5000rpm,
10min)
• Pour out the supernatant, add 100mL of double-distilled water (pre-cooled) and mix properly to let
bacteria re-suspense (avoid using the vortex mixer because huge mechanical force might break the bacteria).
Repeat this step.
• Pour out the water, add 2mL of water (pre-cooled), mix it to re-suspense, then distribute the bacterium
solution into a centrifugal tube of 1.5mL, centrifugate (4℃, 13000rpm, 3min), pour out the water.
• Add 1mL of water in each tube, mix it gently, centrifugate (4℃, 13000rpm, 3min), pour out the water, do
this step twice to ensure that there are no impurities
• Add 500μL of PEG-8000 (30% concentration), re-suspense, centrifugate (4℃, 13000rpm, 3min)
• Add 250μL of PEG-8000 (pre-cooled) each tube, re-suspense, then distribute to pre-cooled centrifugal tubes
(100μL per tube)

**2. Electroporation**

• Unfreeze the L.casei competent cells and the constructed plasmids at room temperature
• Add 150 mL H2O into competent cells and place the mixture at room temperature for 30 minutes
• Centrifugate the mixture (4 Celsius, 13000rpm, 3 minutes)
• Add 500 uL PEG-8000 and centrifugate the mixture again (4 Celsius 13000rpm, 3 minutes)
• Add 100 uL PEG-8000
• Add 10 uL plasmid and do electroporation
• Electric capacity: 25 uV
• Voltage: 2000V
• Resistance: 400 ohms
• Add 900 uL resuscitation medium and shake it for 3 to 4 hours
• Take 100 µL mixture and spread coat it on solid MRS medium, culture it in the incubator at 37 ℃ for 3-4
days, observe its growth

**3. Use PCR to testify**

• Pick a colony of L.casei and add it into 10 µL of MRS
medium, use 1 µL as template for PCR

![](https://static.igem.org/mediawiki/2021/f/f2/T--Shanghai_HS_ID--Experiments07.jpg)

• Conduct electrophoresis

**4. DNA sequencing**

• Deliver the plasmids to Tsingke Biotechnology for DNA sequencing
• Primer:2094-seq-F / 2094-seq-R
• Compare and analyze the result according to the sequencing result through NCBI/BLAST

IV. Result

Compare the DNA transformation rate of wild type L.casei and L.casei with LSEI-2094 gene being knocked out

1. Prepare ATCC 334 competent cells for wild type and LSEI-2094 defect type
2. Electroporation: add plasmids (10 µL plasmid for 100 µL bacterium solution)
3. Cultivate L.casei with resistance cell culture

• Place the bacteria into the incubator at 37 ℃ For 3-4 hours
• Use a pipette to draw two types of competent cells and spread coat them on MRS medium with 3 µg/mL
erythromycin
• Cultivate them for 4 days

![](https://static.igem.org/mediawiki/2021/b/b0/T--Shanghai_HS_ID--Experiments08.jpg)

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# Team:Shanghai HS ID/Results

Shanghai\_HS\_ID

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Results

1. DNA Profiles

![](https://static.igem.org/mediawiki/2021/5/54/T--Shanghai_HS_ID--results01.jpg)
Figure 1. DNA profile of the plasmid PLCNICK

![](https://static.igem.org/mediawiki/2021/c/c7/T--Shanghai_HS_ID--results02.jpg)
Figure 2. DNA Profile of plasmid PTB165 which was electrotransformed into L. casei for transformation
efficiency test

![](https://static.igem.org/mediawiki/2021/0/0f/T--Shanghai_HS_ID--results03.jpg)
 Figure 3. DNA Profile of LSEI-2094+ upstream and downstream homologous arms

2. Electrophoresis result after PCR

![](https://static.igem.org/mediawiki/2021/a/a8/T--Shanghai_HS_ID--results04.jpg)
Figure 4. Electrophoretogram of pLCNICK PCR resiult

1-4 are pLCNICK after enzyme digestion, 5 is pLCNICK plasmid before enzyme
digestion, 6~7
are upstream
homologous arms after PCR, 8~9 are downstream homologous arms after PCR.

3. Verification result of colony PCR

![](https://static.igem.org/mediawiki/2021/2/29/T--Shanghai_HS_ID--results05.jpg)
Figure 5. PCR verification result

As showing above, there are bands (1~3, 6, 8~12) at 1000 bp around which are
consistent with the DNA
profile of the downstream homologous arms. Therefore, it indicated that we have successfully transformed the
plasmid in E. coli.

4. Transformation test result

![](https://static.igem.org/mediawiki/2021/b/b9/T--Shanghai_HS_ID--results06.jpg)
Graph 1. Comparison between the wild L. caseil and modified L. casei in transformation

After we electrotransformed the plasmid pIB165 as the foreign DNA to test the
transformation efficiency of our modified L. casei, it took several days to culture and finally saw the
comparison results. As showing above, we can see that the modified L. casei (KO) has higher transformation
efficiency with remarkably more colonies than the wild (Wild).

![](https://static.igem.org/mediawiki/2021/d/d8/T--Shanghai_HS_ID--results07.jpg)
Figure 6. Histogram comparison between wild strain and KO strain

In addition, we measured OD600 of these strain groups which were pre-spreaded plates with
different volumes of bacteria solutions so as to quantify the transformation results as showing above (Fig.
6).
In conclusion, it indicates that our modified L. casei has much higher efficiency of the foreign plasmid
transformation than the wild and the modified L. casei has great potential to be used as the recombinant
carrier in various areas.

5. Future plan and our concern

As we have primarily tested the transformation effiency of our modified L. casei, we would think about to
further explore the expression performance of the foreign plasmid as well. Besides, we have built the
partnership with EV71 Terminator (2021 iGEM team Shanghai\_Metropolis ) who we will work together to develop
a HFMD Oral Vaccines using L. casei as the live carrier. Therefore, in next step we will electrotransform
the plasmid constructed by EV71 Terminator into our modified L. casei and test the expression performance of
this recombinant L. casei.
About our modified L. casei as well as its derived recombinant bacterium, we also take the safety and
stability into acount, it would be important for us to do more literature research and seek more
professional advice on future development of our product before we step into the real application.

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