iGEM 2023 — Team subcat-shanghai

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**Date:**  08/04/2023
**Event:**  1st
online theory training for whole members
**Agenda:**iGEM
competition background information training

**Date:**  15/04/2023
**Event:**  2nd
online theory training for wet lab
**Agenda:**Academic
frontier of genetic engineering - Literature reading and analysis of classical routes of genetic
engineering.

**Date:**  22/04/2023
**Event:**  3rd
online theory training for dry lab
**Agenda:**How
to tap into market demand - questionnaire design and market research methods.

**Date:**  29/04/2023
**Event:**  4th
online theory training for wet lab
**Agenda:**Essential
for Introduction to Biological Experiments - Basic Research Methods of Modern Molecular Biology 1.

**Date:**  06/05/2023
**Event:**  5th
online theory training for dry lab
**Agenda:**Hands-on
teaching you to complete a high-quality interview - expert interview tips and examples.

**Date:**  13/05/2023
**Event:**  6th
online theory training for wet lab
**Agenda:**Advanced
Teaching of Biological Experiments-Basic Research Methods of Modern Molecular Biology 2.

**Date:**  20/05/2023
**Event:**  7th
online theory training for dry lab
**Agenda:**How
to creatively disseminate scientific information - popular science education activities and
materials.

**Date:**  27/05/2023
**Event:**  8th
online theory training for whole members
**Agenda:**Use
synthetic biology to "build blocks" - biological elements and gene circuits.

**Date:**  03/06/2023
**Event:**  9th
online theory training for whole members.
**Agenda:**From
0 to 1: The Road to Product Commercialization - Business Planning and Product Implementation
Strategy

**Date:**  10/06/2023
**Event:**  10th
online theory training for whole members

**Agenda:**"Speak"
with data-modeling methods and diagrams to teach.

**Date:**  17/06/2023
**Event:**  11th
online theory training for whole members

**Agenda:**Demystifying
The Rule Off iGEM Challenge I

**Date:**  24/06/2023
**Event:**  12th
online theory training for whole members

**Agenda:**Demystifying
The Rule Off iGEM Challenge Ⅱ

**Date:**  20/07/2023
**Event:**  offline training with both wet
and dry lab

**Agenda:**Self-introduction,
reserve captain campaign, background explanation of the topic. Team discussion: team name, logo, Slogan,
VI, Team, Mission, uniform, social platform account.

**Date:**  21/07/2023
**Event:**  offline training with
separated wet and dry lab

**Agenda:** 1.
Learn the safety rules.

2.
Configure LB solution.

3.
Complete the process of PCR system.

4.
Prepare target gene fragment CBD and MS18 solution.

5.
Prepare target plasmid PET28A samples.

**Date:**  22/07/2023
**Event:**  offline training with
separated wet and dry lab

**Agenda:**1.
Extract empty target plasmid.

2.
Add restriction enzymes.

3.
Finish the process of homologous recombination.

4.
Extract target DNA gel.

5.
Transform recombinant products.

6.
Coat the plate with bacteria.

**Date:**  23/07/2023
**Event:**  offline training with
separated wet and dry lab

**Agenda:**1.
Pick monoclonal bacteria and culture it by shaking.

2.
Finish the process of PCR of bacteria solution.

3.
Configure stock liquid of Tris Buffer, NaCl solution, imidazole solution.

4.
Configure SDS-page gel.

**Date:**  24/07/2023
**Event:**  offline training with
separated wet and dry lab

**Agenda:**1.
Configure LB solution.

2.
Shake bacteria solution.

3.
Configure SDS-page gel.

4.
Configure Tris-HCl.

5.
Configure His Buffer A and His Buffer B.

6.
Measure absorbance of samples by Microplate Spectrophotometer

7.
Extract the plasmid and convert the validated recombinant plasmid into BL21.

**Date:**  25/07/2023
**Event:**  offline training with
separated wet and dry lab

**Agenda:**1.
Expand cultivation

2.
IPTG induced protein expression（Overnight）

**Date:**  26/07/2023
**Event:**  offline training with
separated wet and dry lab

**Agenda:** 1.
Extract the target protein by high-speed centrifugation and ultrasonic crushing;

2.
protein purification by nickel column (6x His).

3.
Dialysis (overnight)

**Date:**  27/07/2023
**Event:**  offline training with
separated wet and dry lab

**Agenda:** 1.
Determination of protein concentration;

2. SDS
PAGE analysis;

3.
designing experiments to map out the optimal reaction conditions of the system: different concentrations
of TMB and different enzyme-catalyzed reaction times (model)

**Date:**  28/07/2023
**Event:**  offline training with
separated wet and dry lab

**Agenda:**1. System
function test: detect different concentration gradients of bacterial solutions

2. draw
standard curves through the results, as the basic data of the app for detection.

**Date:**  30/07/2023
**Event:**  offline training with both wet
and dry lab

**Agenda:**1. Write
lab reports and related text materials.

2.Prepare
for the internal presentation rehearsal.

**Date:**  31/07/2023
**Event:**  offline training with both wet
and dry lab

**Agenda:**Internal presentation rehearsal

**Competition Milestones**

**Date:** 14/09/2023

**Event:** Team
Project Promotion Video Submission

**Date:** 18/09/2023

**Event:**Preliminary
Judging Form  Submission

**Date:** 12/10/2023

**Event:** 1.
Wiki Freeze.

            2.
Judging Form Freeze.

            3.
Project Attributions.

            4.
Form Freeze.

            5.
Registry Part Pages Freeze.

            6.
Project Title and Abstract.

**Date:** 19/10/2023

**Event:**1.
Presentation Video Submission

**Date:** 2~5/11/2023

**Event:** Grand
Jamboree

**Date:**
16/11/2023

**Event:** Wiki
Thaw.

**Date:** 14/12/2023

**Event:**1.
Wiki Archive.

            2.
2023 Competition Closing

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Introduction

Foodborne
pathogens
are primarily transmitted via contaminated food and water. Their ingestion can lead to intestinal illness
or toxicity. Therefore, rapid real-time detection of foodborne pathogens is crucial for food
safety. Colorimetric
assays have gained research attention for their simplicity, intuitive interpretation, and field
deployability. In this study, we developed smartphone-based colorimetric detection1 of
*Bacillus
cereus* (BC)
and*Vibrio
parahaemolyticus* (VP)
using bacterial binding proteins. Smartphone quantification of color changes enabled the determination of
bacterial levels in test samples, meeting the criteria for point-of-care testing (POCT).

![](https://static.igem.wiki/teams/4908/wiki/engineering/figure-1.jpg)

**Figure 1 Design diagram of this
project.Drawing under the guidance of Mr. Hong**

Cycle 1: AutolysinCBD-pET28a ([**BBa\_K4908003**](https://parts.igem.org/Part%3ABBa_K4908003))

**Design**

In order to study the colorimetric
of *Bacillus
cereus* (BC)
binding proteins labeled
with anti-His-HRP antibody and TMB substrate, we aimed to constructed a
His-tagged BC autolysin cell wall binding domain (CBD) with the vector pET-28a.

The target
gene fragment CBD sequence comes from the
C-terminal cell wall binding domain of *Bacillus cereus ATCC 14579
autolysin*.*Bacillus
cereus*,
also known as cactus bacillus, is a Gram positive bacterium, β Hemolytic rod-shaped bacteria. Often
found in soil and food, some strains can cause Fried Rice Syndrome.2 The
fragment we used is a characteristic sequence of BC and was synthetic by the company Genscript.

The
experimental design of this study is as follows:

1. Construction
of plasmids pET28a-His-autolysinCBD

2.
Expression and purification of autolysinCBD

3.
Detection system optimization

4. Colorimetric
detection with autolysinCBD

**Build**

We
designed a plasmid encoding the BC autolysin cell wall binding domain (CBD) *i.e.*,
pET28a-His-autolysin CBD. To construct this plasmid,
we first amplified the autolysin CBD fragment from the synthetic plasmid and obtained the pET28a vector by
enzymatic digestion reaction at *Nde*I
and*Xho*I
sites.
Then, we homologous recombined the autolysin CBD with the pET28a vector to generate the
pET28a-His-autolysin CBD (Figure 2).

![](https://static.igem.wiki/teams/4908/wiki/engineering/figure-2.jpg)

**Figure
2****Mapping of
pET28a-His-autolysinCBD plasmid.**

The
constructed plasmid was transformed into *E. coli* and
plated on LB agar containing kanamycin. The plates were incubated at 37°C overnight. On
the next day, it was observed that transformants grew
on the plate, and we
selected the transformants for colony PCR and sequencing. The colony PCR (autolysinCBD: 402
bp) and sequencing results verified
the successful construction of the
plasmid (Figure 3).

![](https://static.igem.wiki/teams/4908/wiki/engineering/figure-3-1.png)
![](https://static.igem.wiki/teams/4908/wiki/engineering/figure-3-2.png)

**Figure 3 Colony PCR result****and sequencing****of pET28a-His-autolysinCBD
transformants.**

**Test**

**Test 1: Expression
and purification of autolysinCBD**

We
inoculated the correctly sequenced transformant and subjected it to an expanded culture. When the cultures
grew to OD600 0.6-0.8,
they were induced with IPTG for 24 h. We lysed the cells by sonication, obtained the protein supernatant
by centrifugation, and purified it by Ni-affinity chromatography. As shown in Figure 4, we successfully
obtained purified autolysin CBD (15.3 KDa).

![](https://static.igem.wiki/teams/4908/wiki/engineering/figure-4.jpg)

**Figure 4 Expression and
purification result of autolysinCBD.**

**Test
2:****Detection system optimization**

To
improve the detection and to obtain optimal reaction conditions, we explored the two most important
experimental conditions in the autolysinCBD detection system, *i.e.*,
the catalytic reaction time and the TMB substrate concentration. We first diluted the bacterial suspension
to 106 CFU/mL
and used different concentrations of TMB 0.01-0.05% (w/v) as the colorimetric substrate. The concentration
of bacteria was determined at different times (0.5-2 h). The
results showed that the optimal assay conditions were at 0.01% TMB for 1 h for autolysinCBD (Figure
5).

![](https://static.igem.wiki/teams/4908/wiki/engineering/figure-5.jpg)

**Figure
5****Optimal reaction
conditions for the detection system.**

**Test
3:****Detection of different concentrations of
bacterial fluids**

For
testing the ability of the system to detect BC. We
diluted overnight bacterial cultures of BC to 102-107 CFU/mL
using PBS, and then 1 mL of each concentration was taken for the assay. The bacterial solutions were
reacted under previously determined optimal conditions (Figure 5).

We
performed curve fitting of the detection results, and the experimental results are shown in Figure 6. For
BC bacteria, the fitted curve of the detection was y = 0.1922x - 0.1994 (R² = 0.9423), The detection
values showed a favorable
linear relationship with the concentration of the bacteria. However, it was also seen that when the
concentration of the bacterium reached 107 CFU/mL,
the detection value decreased instead, which suggested that we should control the concentration of the
bacterium between 102-106 CFU/mL
to obtain the best detection results when using this detection system.

![](https://static.igem.wiki/teams/4908/wiki/t11.jpg)

**Figure 6
T****esting****r****esults of different
concentrations of bacteria.**

**Learn**

To detect BC pathogens using a colorimetric assay based on
smartphone and bacterial binding proteins, we first recombinantly expressed and purified a bacterial
binding protein (His-autolysinCBD). Per the results mentioned above, we can validate our theory as a
success of the colorimetric detection by the binding proteins of  BC. In order to implement our
detection method, we need to conduct more experiments, including investigating the limit concentration
of detection and the quantity relationship between RGB values of the test samples and the bacteria
concentrations per the app development requirement.

Cycle 2:MS18-pET28a ([**BBa\_K4908002**](https://parts.igem.org/Part%3ABBa_K4908003))

**Design**

In order to study the colorimetric
detection of *Vibrio
parahaemolyticus* (VP)
binding proteins labeled
with anti-His-HRP antibody and TMB substrate, we aimed to constructed a
His-tagged VP bacteriophage
tail fiber protein (TFP)  with the vector pET-28a.

The gene fragment TFP we used comes from the tail fiber protein (TFP) MS18 of
*Vibrio parahaemolyticus phage VPMS1*.
*Vibrio parahaemolyticus* is a marine bacterium that mainly comes from seafood such as fish,
shrimp, crab, shellfish and seaweed. Consumption of food containing this bacterium can lead to food
poisoning, also known as halophilic food poisoning. Clinically, acute abdominal pain, vomiting, diarrhea
and watery stools are the main symptoms.3  The
fragment we used is a characteristic sequence of VP and was synthetic by the company Genscript.

The
experimental design of this study is as follows:

1. Construction
of plasmids pET28a-His-MS18

2.
Expression and purification of MS18

3.
Detection system optimization

4. Colorimetric
detection with MS18

**Build**

We
designed plasmids encoding the VP
phage tail fiber protein (MS18)
*i.e.*,
pET28a-His- MS18. To construct this plasmid,
we first amplified the MS18 fragment from the synthetic plasmid and obtained the pET28a vector by
enzymatic digestion reaction at*BamH*I
and *Xho*I sites.
Then, we homologous recombined the MS18 with the pET28a vector to generate the pET28a-His- MS18 (Figure
7).

![](https://static.igem.wiki/teams/4908/wiki/engineering/figure-7.jpg)

**Figure
7****Mapping of pET28a-His-MS18
plasmid.**

The
constructed plasmid was transformed into *E. coli* and
plated on LB agar containing kanamycin. The plates were incubated at 37 °C overnight. On
the next day, it was observed that transformants grew
on the plate, and we
selected the transformants for colony PCR and sequencing. The colony PCR (MS18: 495
bp) and sequencing result verified
the successful construction of the plasmid (Figure 8).

![](https://static.igem.wiki/teams/4908/wiki/engineering/figure-8-1.png)
![](https://static.igem.wiki/teams/4908/wiki/engineering/figure-8-2.png)

**Figure 8 Colony PCR
result****and sequencing****of pET28a-His-MS18
transformants.**

**Test**

**Test 1: Expression
and purification of** **MS18**

We
inoculated the correctly sequenced transformant and subjected it to an expanded culture. When the cultures
grew to OD600 0.6-0.8,
they were induced with IPTG for 24 h. We lysed the cells by sonication, obtained the protein supernatant
by centrifugation, and purified it by Ni-affinity chromatography. As shown in Figure 9, we successfully
obtained purified MS18.

![](https://static.igem.wiki/teams/4908/wiki/t9.jpg)

**Figure 9 Expression and
purification result of MS18.**

**Test
2:****Detection system optimization**

To
improve the detection and to obtain optimal reaction conditions, we explored the two most important
experimental conditions in the MS18 detection system, *i.e.*,
the catalytic reaction time and the TMB substrate concentration. We first diluted the bacterial suspension
to 106 CFU/mL
and used different concentrations of TMB 0.01-0.05% (w/v) as the colorimetric substrate. The concentration
of bacteria was determined at different times (0.5-2 h). The
results showed that the optimal assay conditions were at 0.04% TMB for 0.5 h for MS18 (Figure 10).

![](https://static.igem.wiki/teams/4908/wiki/engineering/figure-10.jpg)

**Figure 10 Optimal reaction
conditions for detection systems.**

**Test
3:****Detection of different concentrations of
bacterial fluids**

For
testing the ability of the system to VP. We
diluted overnight bacterial cultures of VP to 102-107 CFU/mL
using PBS, and then 1 mL of each concentration was taken for the assay. The bacterial solutions were
reacted under previously determined optimal conditions (Figure 10).

We
performed curve fitting of the detection results, and the experimental results are shown in Figure 11. For
VP bacteria, the fitted curve of the detection was y = 0. 1797x - 0.1067 (R² = 0.9775), The detection
values showed a favorable
linear relationship with the concentration of the bacteria. However, it was also seen that when the
concentration of the bacterium reached 107 CFU/mL,
the detection value decreased instead, which suggested that we should control the concentration of the
bacterium between 102-106 CFU/mL
to obtain the best detection results when using this detection system.

![](https://static.igem.wiki/teams/4908/wiki/t6.jpg)

**Figure 11
T****esting****r****esults of different
concentrations of bacteria.**

**Learn**

The same as the cycle 1, it
can be seen that this pathogenic bacteria detection system based on smartphone and bacterial binding
protein has a good detection effect for both BC and VP pathogenic bacteria. It should not be overlooked
that although the results of our experiments have a certain degree of accuracy and reliability, they still
cannot be used in products directly based on current progress.
In the detection of bacterial concentration, we initially obtained the upper and lower limits of the
detection system, but this part of the results still needs to be repeatedly verified by
experiments.

In addition, we also had a review of the whole
experiment during which we
encountered some problems. For example, the concentration of the plasmids we extracted was always very
low, which could be caused by not using fresh enough bacteria. Another example is that the recovery rate
of DNA fragments is low, to optimize this part of the experiment, we can obtain better and more gene materials for further research
use.

Reference

1.
Hong Bin, Li Yanmei, Wang Wenhai, Ma Yi, Wang Jufang. Separation and colorimetric detection of Escherichia
coli by phage tail fiber protein combined with nano-magnetic beads[J]. Microchimica
Acta,2023,190(6).

2.
Ivanova Natalia, Sorokin Alexei, Anderson Iain, Galleron Nathalie, Candelon Benjamin, KapatralVinayak,
Bhattacharyya Anamitra, Reznik Gary, Mikhailova Natalia, Lapidus Alla, Chu Lien, Mazur Michael, Goltsman
Eugene, Larsen Niels, D'Souza Mark, Walunas Theresa, Grechkin Yuri, PuschGordon, Haselkorn Robert,
Fonstein Michael, Ehrlich S Dusko, Overbeek Ross, KyrpidesNikos. Genome sequence of Bacillus cereus and
comparative analysis with Bacillus anthracis. [J]. Nature,2003,423(6935).

3.
Ramírez-Orozco Martín, Serrano-Pinto Vania, Ochoa-Álvarez Norma, Makarov Roman,
Martínez-Díaz Sergio F. Genome sequence analysis of the Vibrio parahaemolyticus lytic
bacteriophage VPMS1. [J]. Archives ofvirology,2013,158(11).

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![](https://static.igem.wiki/teams/4908/wiki/experiments.png)

I.Preparation

1. LB
liquid medium

Make
500 mL LB medium by adding 5 g tryptone, 2.5 g yeast powder, 5 g NaCl, and water until LB broth is 500 mL
and mixing thoroughly. Sterilize
the two LB mediums at 121
°C for 20 min.

2. LB
solid medium

Make
200 mL solid LB medium by adding 2 g tryptone, 1 g yeast powder, 2 g NaCl, 3 g agar powder, and
water until solid LB is 200 mL and mixing thoroughly. Sterilize
the two LB mediums at 121
°C for 20 min. Allow medium to cool to 50-60 °C after autoclaving. Aseptically pour plates in a
laminar flow hood. Allow to solidify.

3. His
Buffer A:

Add
1.78 g of Na2HPO4·2H2O,
14.6 g of NaCl, and 1.02 g of imidazole. Adjust the pH to 7.4 by adding concentrated hydrochloric
acid.

4. His
Buffer B:

Add
0.89 g of Na2HPO4·2H2O,
7.3 g of NaCl, and 8.5 g of imidazole. Adjust the pH to 7.4 by adding concentrated hydrochloric
acid.

5. PBS
Buffer:

Add
16 g of NaCl, 0.4 g of KCl, 2.84 g of Na2HPO4,
0.48 g of KH2PO4,
and 2 L of water.

6. SDS-PAGE
gel

1) Prepare
resolving gel by mixing 4.9 mL water, 6.0 mL 30% acrylamide, 3.8 mL 1.5 M Tris pH 8.8, 0.15 mL 10% SDS,
0.15 mL 10% ammonium persulfate (APS), and 6 μL TEMED in a tube.

2) Pour
the resolving gel solution into the assembled cassette.

3) Prepare
stacking gel by mixing 6.8 mL water, 1.7 mL 30% acrylamide, 1.25 mL 1.5 M Tris pH 6.8, 0.1 mL 10% SDS, 0.1
mL 10% APS, and 0.01 mL TEMED.

4) Pour
stacking gel solution on top of polymerized resolving gel. Insert comb and allow stacking gel to
polymerize.

II.Plasmid extraction (pET28a)

1. Equilibrate
the adsorption column

1) Add
500 μL of Buffer S to the adsorption column and place a collection tube below it.

2) Centrifuge
at 12,000 ×*g* for
1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the
collection tube.

2. Transfer
the *E. coli* culture into
a 2 mL centrifuge tube, centrifuge at 12,000 ×*g* for
2 min to collect the bacteria. Discard the supernatant and retain the precipitate.

3. Add
250 μL of Buffer SP1 to the 2 mL centrifuge tube, and thoroughly suspend the precipitate.

4. Add
250 μL of Buffer SP2, and gently invert the tube 5-10 times to mix the solution.

5. Add
350 μL of Buffer SP3 and mix the solution by gently inverting the tube 5-10 times.

6. Centrifuge
at 12,000 ×*g* for
5-10 min. Transfer 700 μL of the supernatant to the adsorption column, centrifuge at 8,000 ×*g* for
30 sec, and discard the filtrate in
the collection tube.

7. Add
500 μL of Wash Solution, centrifuge at 9,000 ×*g* for
30 sec, and discard the filtrate in
the collection tube.

8. Repeat
wash step.

9. Centrifuge
the empty column at 9,000 ×*g* for
1 min.

10. Place
the column into a clean 1.5 mL centrifuge tube, and add 25 μL of Elution Buffer to the center of the
adsorption membrane. Incubate
for 1 min at room temperature,
centrifuge at 9,000 ×*g*,
and keep the DNA solution in the tube.

III.Polymerase chain reaction (PCR)

To
construct the pET28a-His-autolysinCBD and pET28a-His-MS18 plasmids, we used the synthesized plasmids
containing the His-autolysinCBD or His-MS18 fragments as templates for PCR amplification of the target
fragments.

The
reaction system for PCR amplification is shown
in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| 2´ Phanta Max Master Mix | 25 |
| primer F (10 μM) | 2 |
| primer R (10 μM) | 2 |
| DNA template | 1 |
| ddH2O | 20 |
| Total | 50 |

The
reaction procedure for PCR amplification is shown in the table below:

|  |  |  |
| --- | --- | --- |
| Step | Temperature ( ℃) | Time (sec) |
| 1 | 95 | 180 |
| 2 | 95 | 15 |
| 3 | 55 | 15 sec/kb |
| 4 | 72 | 30 |
| 5 | 72 | 300 |
| 6 | 4 | ¥ |

Notice:
Repeat step 2 to step 4 for 30 times.

IV.Double enzyme digestion of vectors

1. The
digestion system of the pET28a vector (for autolysinCBD) is shown in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| pET28a vector (for autolysinCBD) | 20 |
| *Nde*I | 1 |
| *Xho*I | 1 |
| Buffer (CutSmart) | 5 |
| ddH2O | 23 |
| Total | 50 |

The
enzyme digestion systems are incubated at 37 ℃ for 3 h.

2. The
digestion system of the pET28a vector (for MS18) is shown in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| pET28a vector (for MS18) | 20 |
| *BamH*I | 1 |
| *Xho*I | 1 |
| Buffer (CutSmart) | 5 |
| ddH2O | 23 |
| Total | 50 |

V.Agarose gel electrophoresis

1. Preparation
of agarose gel

1) Add
50 mL 1× TAE buffer and 0.5 g agarose to a flask, and place it in a microwave oven on high for 2
min.

2) Take
it out after the solution turns transparent and shake it evenly.

3) Add
2 μL nucleic acid dye GelRed to
the molten agarose.

4) Assemble
the gel tray and insert the comb into the mold.

5) Pour
the liquid agarose into the mold, avoiding air bubbles.

6) Wait
for the solidification of the gel at room temperature.

7) Carefully
remove the comb and transfer the
solidified
gel to the electrophoresis tank.

8) Completely
submerge the gel in 1´ TAE
buffer.

2. Loading
samples

1) Add
DNA
loading buffer
into samples to dye them blue, then load into wells sequentially.

2) Add
10 μL of Trans15K DNA Marker into a lane.

3. Electrophoresis

1) Place
wells at the negative electrode and cover the tank.

2) Turn
on the power, and run the agarose gel at 120 V for 15 min.

4. Visualizing
DNA bands

1) Turn
off the power, and remove the gel.

2) Place
the gel under the Ultra Slim LED Illuminator to visualize the results.

3) Take
out the gel, and put it under the gel-imaging system to save data.

VI.Gel extraction

1. Excise
the DNA band of interest under UV light using a clean scalpel.

2. Transfer
the gel slices into centrifuge tubes, and
measure the weight of the gel slices.

3. Add
3 times of sample volume of Buffer B2 into the centrifuge tubes, and mix until the gel completely
dissolved.

4. Transfer
the solution into
the absorption column, and centrifuge at 9000 ×*g* for
30 sec. Pour off the filtrate into
the collection tube.

5. Add
500 μL of Wash Solution and centrifuge at 9000 ×*g* for
30 sec. Pour off the filtrate into the collection tube.

6. Repeat
wash step.

7. Centrifuge
the empty column at 9000 ×*g* for
1 min.

8. Place
the absorption column into a clean 1.5 mL centrifuge tube, add 15-40 μL Elution Buffer to the center of
the absorption membrane, and centrifuge at 9000 ×*g* for
1 min. Store the DNA solution in the tube.

VII.Homologous recombination of fragments and vectors

To
recombine the pET28a-His-autolysinCBD and pET28a-His-MS18 plasmids, we homologous recombined the target
fragment (autolysinCBD or MS18) after gel recovery with the corresponding pET28a vector. We used the
ClonExpress Ultra One Step Cloning Kit, and the reaction system is listed below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| Fragment (autolysinCBD or MS18) | 2.5 |
| pET28a vector | 2.5 |
| 2× ClonExpress Mix | 5 |

Incubate
at 50 °C for 5 min; cool to 4 °C or place on ice immediately.

VIII.Transformation of recombinant plasmids to E. coli BL21(DE3)

1. Add
5 μL of plasmids into 50 μL of *E. coli* BL21(DE3)
competent cells, respectively. Incubate
the mixture on
ice for 30 min.

2. Heat
shock at 42 °C for 45 sec, and put the mixture to the ice for 2
min.

3. Add
the LB culture into the tubes to 0.7 mL, and shake the tubes at 37 ℃, 200 rpm for 60 min.

4. Centrifuge
it at 5000
rpm for
1 min, remove 600
μL of supernatant, and resuspend the cells in the remaining LB culture.

5. Spread
50 μL cells onto LB agar plates containing kanamycin
and
incubate overnight at
37 ℃.

IX.PCR identification of transformants

1. Place
50 μL of Lysis Buffer for Microorganism to Direct PCR in a sterilized microtube. Pick a single colony
with a sterilized toothpick or lance tip, place it in the microtube, and stir a few times before removing
it.

2. After
heat denaturation at 80 °C for 15 min, centrifuge at low speed and take 1~5 μL of supernatant after
lysis as PCR template.

The
PCR reaction system is shown in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| Lysis supernatant | 1 |
| Premix Taq (Ex Taq Version 2.0) | 10 |
| Forward Primer (10 μM) | 1 |
| Reverse Primer (10 μM) | 1 |
| sterilized water | 7 |

The
PCR reaction condition is shown in the table below:

|  |  |  |
| --- | --- | --- |
| Step | Temperature (˚C) | Time (sec) |
| 1 | 94 | 600 |
| 2 | 94 | 30 |
| 3 | 55 | 30 |
| 4 | 72 | 120 |
| 5 | 72 | 300 |
| 6 | 4 | ¥ |

Notice:
Repeat step 2 to step 4 for 28 times.

3. Take
positive transformants and add 3 mL LB culture medium containing kanamycin,
shake bacteria, and incubate at 37 ℃ at 220 rpm for 12-16 h.

X.Protein expression and disruption

1. Inoculate
overnight cultures 1: 100 into 200 mL fresh LB culture medium containing kanamycin in
conical flasks.

2. Incubate
at 37°C, 200 rpm until OD600 reaches
0.6.

3. After
the temperature of the LB bacterial medium is reduced to 16 ℃, add IPTG to a final concentration of 1 mM.

4. Induction
at 16 ℃, 200 rpm for 24 h.

5. Harvest
cells by centrifugation at 10000 rpm for 5 min at 4 °C. Discard supernatant.

6. Resuspend
the pellet in lysis buffer by adding lysis buffer (containing Tris-HCl
with 25 mM imidazole).

7. Sonication
of the bacterial resuspension is performed using φ6 probes at 50% power for 50 min.

XI.Protein purification by nickel affinity chromatography

1. Equilibrate
1 mL nickel column with 10 column volumes of ultrapure water, followed by 10 column volumes of His Buffer
A at ~1 mL/min flow rate.

2. Load
filtered cell lysate supernatant containing target proteins onto the column at 1 mL/min.

3. Wash
loaded column with 10 column volumes of His Buffer A at 1 mL/min.

4. Elute
proteins with imidazole gradient buffer at 1 mL/min while collecting 1 mL fractions. Store eluates
temporarily at 4 °C.

5. Wash
the column with His Buffer B, His Buffer A, water, and 20% ethanol sequentially. Store the column in 20%
ethanol.

XII.Detection of purified protein

1. SDS-PAGE

1) Combine
4 μL loading buffer with 16 μL each purified protein fraction in tubes.

2) Assemble
the gel cassette and apparatus.

3) Perform
a leak test by adding water to the cassette gap and observing for leaks.

4) Load
protein samples into wells of polymerized gel.

5) Run
the gel at 80 V for 30 min, then 120 V for 1 h.

6) Stain
gel with Coomassie blue. Destain with water and visualize protein bands.

2. BCA
Protein Quantification

1) Mix
BCA Reagent A with BCA Reagent B at a 50:1 ratio to create the working reagent.

2) Add
25 μL each for all samples and standards into the microplate wells.

3) Add
200 μL of the working reagent into each well.

4) Tap
the microplate to mix each well.

5) Cover
the microplate with foil to minimize light exposure.

6) Incubate
the mixture at 37 °C for 30 min.

7) Measure
the absorbance at 562 nm with the microplate reader.

8) Make
a graph plotting the concentrations and absorbances

XIII.Detection system optimization

1. Prepare
106 CFU/mL
*Bacillus cereus* and
*Vibrio
parahaemolyticus* suspensions
(*i.e.* OD600=1
and then dilute 1000 times).

2. Test
different TMB concentrations from 0.01-0.05% (w/v) as substrate.

3. Select
enzyme reaction times of 0.5 h, 1 h, 1.5 h and 2 h.

XIV.Smartphone-based colorimetric detection with bacteria-binding proteins

1. Dilute
overnight cultures of *Bacillus
cereus* and
*Vibrio
parahaemolyticus* in
PBS from 102 to
107 CFU/mL.

2. Add
1 mL of each bacterial dilution or PBS control to tubes.

3. Add
equal volumes of autolysinCBD
or MS18 binding proteins and 1 mg/mL anti-His HRP conjugated antibody.

4. Incubate
at 37 °C for 30 min with shaking.

5. Centrifuge
at 8000 rpm for 5 min. Wash the pellet with PBS.

6. Resuspend
labeled bacteria in 50 μL TMB substrate and transfer to a 96-well plate.

7. React
at 37 °C for optimized time based on modeling.

8. Scan
colorimetric results with a smartphone app and analyze RGB values.

9. Construct
a standard curve of R/B and G/B ratios. Import into the app.

10. App
scan of test samples directly outputs bacterial concentration.

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![](https://static.igem.wiki/teams/4908/wiki/results.png)

Overview

Conclusion

Overview

In
this study, we developed a new detection strategy for *Bacillus
cereus* (BC)
and *Vibrio
parahaemolyticus* (VP)
based on the colorimetric detection of smartphones and bacterial binding proteins. This assay enabled the
detection of BC and VP at low concentrations without pre-enrichment. Bacterial levels could be determined
by the naked eye or quantified by smartphone image analysis, meeting the criteria for point-of-care
testing (POCT). Our key experimental findings were as follows:

1. Construction
of plasmids pET28a-His-autolysinCBD
and pET28a-His-MS18

2.
Expression and purification of autolysinCBD
and MS18

3.
Detection system optimization

4. Colorimetric
detection with bacteria-binding proteins

5. Mobile app development

**1. Construction of plasmids**

We
designed plasmids encoding the BC autolysin cell wall binding domain (CBD) and VP phage tail fiber protein
(MS18), *i.e.*,
pET28a-His-autolysinCBD and pET28a-His-MS18. To construct these plasmids, we first amplified the
autolysinCBD and MS18 fragments from the synthetic plasmids and obtained the pET28a vector by enzymatic
digestion reaction (Figure 1.1 A-B). Then, we homologous recombined the target fragment (autolysinCBD or
MS18) with the corresponding pET28a vector. The constructed plasmids were transformed into
*E.
coli* and
plated on LB agar containing kanamycin. The plates were incubated at 37°C overnight (Figure 1.1
C-D).

![](https://static.igem.wiki/teams/4908/wiki/result/figure-1-1.jpg)

**Figure 1.1 Construction of
plasmids pET28a-His-MS18 & pET28a-His-autolysinCBD.**

(A)
Double enzyme digestion result of pET28a.

(B)
Amplification result of MS18 (495 bp) and autolysinCBD (402 bp).

(C-D)
Transformation results of recombinant plasmids.

We
selected the transformants grown on solid plates for colony PCR, and the results showed that both
pET28a-His-autolysinCBD and pET28a-His-MS18 showed the correct bands, which initially indicated that our
constructs were successful (Figure 1.2).

![](https://static.igem.wiki/teams/4908/wiki/result/figure-1-2.jpg)

**Figure 1.2****Colony PCR results of
transformants.**

To
further confirm that the plasmid was constructed successfully, we sent the transformants to the company
(Aznta) for sequencing. As shown in Figure 1.3, we did obtain the correct recombinant plasmid.

![](https://static.igem.wiki/teams/4908/wiki/result/figure-1-3.jpg)

**Figure 1.3 Sequencing
results** **of****transformants.**

**2. Expression and
purification of the target protein**

Two
plasmids were transformed into *E. coli* BL21(DE3)
for protein production. Cultures were grown to OD600 0.6-0.8
and induced with IPTG for 24 h. Cells were lysed by sonication and His-tagged proteins were purified by
Ni-affinity chromatography. SDS-PAGE analysis demonstrated successful expression and purification of
autolysinCBD (15.3 KDa) and
MS18 (17.84 KDa) in Figure
2.

![](https://static.igem.wiki/teams/4908/wiki/result/figure-2.jpg)

**Figure 2 SDS-PAGE result of
autolysinCBD and MS18.**

**3. Detection system
optimization**

To
improve the detection and to obtain optimal reaction conditions, we explored the two most important
experimental conditions in the autolysinCBD and MS18 detection system, *i.e.*,
the catalytic reaction time and the TMB substrate concentration. We first diluted the bacterial suspension
to 106 CFU/mL
and used different concentrations of TMB 0.01-0.05% (w/v) as the colorimetric substrate. The concentration
of bacteria was determined at different times (0.5-2 h).

The
results showed that for MS18, the optimal assay conditions were the optimal conditions for the 0.04% TMB
substrate concentration for 30 min; for autolysinCBD, the optimal assay conditions were at 0.01% TMB for 1
h (Figure 3).

![](https://static.igem.wiki/teams/4908/wiki/result/figure-3.jpg)

**Figure 3 Optimal reaction
conditions for detection systems.**

**4. Colorimetric detection
with bacteria-binding proteins**

For
testing the ability of the system to detect *Bacillus
cereus* (BC)
or *Vibrio
parahaemolyticus* (VP). We
diluted overnight bacterial cultures of BC or VP to 102-107 CFU/mL
using PBS, and then 1 mL of each concentration was taken for the assay. The two bacterial solutions were
reacted under previously determined optimal conditions (Figure 3).

We
performed curve fitting of the detection results, and the experimental results are shown in Figure 4. For
BC bacteria, the fitted curve of the detection was y = 0.1922x - 0.1994 (R² = 0.9423), and for VP
bacteria, the fitted curve of the detection was y = 0. 1797x - 0.1067 (R² = 0.9775). The detection
values of the two bacteria showed a favorable
linear relationship with the concentration of the bacteria. However, it was also seen that when the
concentration of the bacterium reached 107 CFU/mL,
the detection value decreased instead, which suggested that the optimal detection interval of the
bacteria concentration shall be between
102-106 CFU/mL when
using this detection system.

![](https://static.igem.wiki/teams/4908/wiki/result/figure-4.jpg)

**Figure 4
T****esting****r****esults of different
concentrations of bacteria.**

5. **Mobile App Development**

In order to facilitate our bacteria detection method, we also
worked on developing a mobile app in which people can use their phone camera to scan the color of the
test sample to obtain the value of the bacteria concentration directly. The app installation package
(not fully finished) is available [***here***](file:///C%3A/Users/1/Desktop/4-1%20%E6%8E%8C%E4%B8%8A%E7%BB%86%E8%8F%8C%E6%8E%A2%E6%B5%8B%E4%BB%AA/4-1%20%E6%8E%8C%E4%B8%8A%E7%BB%86%E8%8F%8C%E6%8E%A2%E6%B5%8B%E4%BB%AA/wiki%E7%BB%93%E6%9E%9C%E5%9B%BE%E7%89%87/8%E3%80%81Result/Handy%20ECD%20app.apk.1.1) for showcase only.

![](https://static.igem.wiki/teams/4908/wiki/result/5-1.jpg)
![](https://static.igem.wiki/teams/4908/wiki/result/5-2.jpg)
![](https://static.igem.wiki/teams/4908/wiki/result/5-3.jpg)

**Figure 5 Screenshots of the mobile app from left to right:
Homepage; Select Bacteria Type; Test Scanning Results (including RGB values and the detected bacteria
concentration)**

Conclusion

In
conclusion, we have successfully developed and optimized a novel smartphone-based detection strategy for
the detection of major foodborne pathogens BC and VP.

Currently, we
have produced a prototype of the mobile app with the curve of bacteria concentration and OD450
only,
and in the future, we will further investigate the limit of the detection the quantity
relationship between RGB values and the bacteria concentration of the test sample by obtaining more
experimental data to upgrade our model so as to develop the mobile app, besides evaluating the detection
sensitivity for refinement.

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