iGEM 2022 — Team gyhs

[April](#collapseOne)

* Team members recruiting and leaders election

[May](#collapseTwo)

* Project brainstorm
* Team wiki design studying
* Team logo and uniform design

[June](#collapseThree)

* Hardware design studying
* Kick-off meeting
* Professional theory studying
* Project fundraising
* Final project determination
* Experimental safety learning
* Team leader selection

[July](#collapseFour)

Wet team
:   Experimental principles learning
:   Experimental skills trainning
:   Plasmid construction

Dry team
:   Human practice
    :   HP experts interview, questionnaire design and collection.
    :   HP experts and fieldwork resources to find.
:   Collaboration and partnership
:   Publicity
    :   Promotion video making and posting on the social media
    :   Self-media update
:   Implementation design

[August](#collapse5)

Wet team
:   Experimental skills trainning
:   Chromogenic reaction
:   Summarize experimental scheme

Dry team
:   Publicity
    :   Promotion video making and posting on the social media
    :   Self-media update
    :   Make a comic book manual
:   Wiki design and building
:   Human practice
    :   Online HP Experts Interview
    :   Questionnaire collection
    :   Field research at the Guangdong Institute of Microbiology
    :   Centre Testing International Group Co., Ltd. Online Interview
:   Contribution and Safety first draft
:   Hardware 3D modeling
:   Collaboration and partnership

[September](#collapse6)

* Wiki building
* Experimental data conclusion
* Modeling and hardware design
* Wiki documents writing
* Publicity(Offline publicity activity)

[October](#collapse7)

* Optimization of Wiki
* Presentation preparation
* Submission of final judging form
* Parts registry
* Participation of 2022 giant jamboree

[![↑](https://static.igem.wiki/teams/4430/wiki/arrow.jpg)](#top-box)

[Summary](#list-item-1)
[Procedure](#list-item-2)
[Real sample test](#list-item-3)
[Future work](#list-item-4)

# Summary

In recent years, food safety problems occurred frequently, and food poisoning cases caused by
pathogens are particularly typical. Fast and accurate food safety detection method for pathogenic
bacteria is a good way to quickly confirm the cause of food poisoning. Therefore, our team chose to
design a detection device for potential pathogenic Escherichia coli (E. coli, EC), Staphylococcus
aureus (S. aureus, SA), Vibrio parahaemolyticus (V. parahaemolyticus, VP) and Salmonella enteritidis
(S. enteritidis, SE) in food. Our team built a test kit equipped with core components for the test:
paper-based sensors, small incubators and quick sampling devices needed during the experiment to
achieve household food safety testing.

During the project, we successfully constructed eight plasmids to express four receptor-binding
proteins [RBPs: TFP (tail fiber protein), gp15, CBD(C-terminal cell-binding domain), and TSP
(tailspike protein)] and their corresponding fluorescently labeled GFP-fusion proteins (GFP-RBPs).
TFP, gp15, CBD, and TSP can specifically bind to the target bacteria EC, VP, SA, and SE,
respectively. GFP-fusion RBPs can trace the binding of RBPs with the bacteria via fluorescence and
are used to validate the specificity of RBPs. RBPs are conjugated with nanomagnetic beads (MBs) to
form magnetic-protein complexes (RBP-MBs) to achieve separation and enrichment for specific target
bacteria by magnetic force. The successful construction of RBP-MBs was verified by calculating the
separation rates of RBP-MBs for different bacteria strains.

#### RBPs, GFP-RBPs and RBP-MBs of target bacteria

| Bacteria | RBP | GFP-RBP | RBP-MBs |
| --- | --- | --- | --- |
| EC | TFP | GFP-TFP | MBs-TFP |
| VP | gp15 | GFP-gp15 | MBs-gp15 |
| SA | CBD | GFP-CBD | MBs-CBD |
| SE | TSP | GFP-TSP | MBs-TSP |

[Part list](https://2022.igem.wiki/gyhs/parts)

# Procedure

### Goal 1 : Produce RBPs and identify their activity

#### 1 Plasmid Construction

We designed our functional parts TFP, gp15, CBD, and TSP and respectively cloned them into pET28a
backbone plasmid chemical synthesized by Tsingke Biotechnology Co., Ltd. As mentioned on our design
page, these plasmids are used to express TFP, gp15, CBD and TSP which can respectively bind to and
separate EC, VP, SA, and SE from food samples. For safety concerns, we chose a relatively safe EC
strain K12 MG1655 for laboratory experiments.

We used Gibson assembly method to construct pET28a-TFP, pET28a-gp15, pET28a-CBD, and pET28a-TSP
plasmids.

The gel electrophoresis results (Figure 1 to 3) showed that the gp15 was 561bp, the CBD was 604 bp,
and the TSP was 1993 bp, as expected. In addition, we confirmed the results by sequencing. As for
TFP, we had the plasmid which was already cloned with TFP, so we didn’t electrophorese it.

![](https://static.igem.wiki/teams/4430/wiki/elegp15.png)

Figure 1 Nucleic acid gel electrophoresis results of gp15.

![](https://static.igem.wiki/teams/4430/wiki/elecbd.png)

Figure 2 Nucleic acid gel electrophoresis results of CBD.

![](https://static.igem.wiki/teams/4430/wiki/eletsp.png)

Figure 3 Nucleic acid gel electrophoresis results of TSP.

#### 2 Protein expression test

SDS-PAGE electrophoresis was used to check the expression of four RBPs (TFP: 64.1 kDa, gp15: 25.4
kDa, CBD: 12.8 kDa, TSP: 62.6 kDa). As shown in Figure 4 to 7, the proteins have been successfully
expressed and purified.

![ ](https://static.igem.wiki/teams/4430/wiki/sdstfp.png)

Figure 4 Protein SDS-PAGE electrophoresis results of TFP.

![](https://static.igem.wiki/teams/4430/wiki/sdsgp15.png)

Figure 5 Protein SDS-PAGE electrophoresis results of gp15.

![](https://static.igem.wiki/teams/4430/wiki/sdscbd.png)

Figure 6 Protein SDS-PAGE electrophoresis results of CBD.

![](https://static.igem.wiki/teams/4430/wiki/sdstsp.png)

Figure 7 Protein SDS-PAGE electrophoresis results of TSP.

#### 3 Binding ability test of RBPs

After the expression and purification of four GFP-RBPs, we measured the absorbance of them by optical
density (OD) values at 562 nm (OD562) and converted OD values into concentration of GFP-RBPs by the
function shown in Figure 8. The concentrations of GFP-RBPs are listed in following table .

![](https://static.igem.wiki/teams/4430/wiki/od562.png)

Figure 8 A standard curve of optical density (OD) values at 562 nm versus protein

#### Concentrations of GFP-RBPs

| Fusion protein | concentration |
| --- | --- |
| GFP-TFP | 420μg/mL |
| GFP-gp15 | 722μg/mL |
| GFP-CBD | 210μg/mL |
| GFP-TSP | 425μg/mL |

We mixed the GFP-RBPs with relevant bacteria and measured the fluorescence value of the cells with a
microplate reader. As we expected, GFP-TFP had a good specificity toward EC (Figure 9) and so did
GFP-gp15 toward VP (Figure 10), GFP-CBD toward SA (Figure 11), and GFP-TSP toward SE (Figure 12).
These results confirmed that our RBPs could bind strongly to the corresponding target bacteria
respectively.

![](https://static.igem.wiki/teams/4430/wiki/ecspe.png)

Figure 9 GFP-TFP binds strongly to the cell surfaces of EC.

![](https://static.igem.wiki/teams/4430/wiki/vpspe.png)

Figure 10 GFP-gp15 binds strongly to the cell surfaces of VP.

![](https://static.igem.wiki/teams/4430/wiki/saspe.png)

Figure 11 GFP-CBD binds strongly to the cell surfaces of SA.

![](https://static.igem.wiki/teams/4430/wiki/sespe.png)

Figure 12 GFP-TSP binds strongly to the cell surfaces of SE.

### Goal 2 : Find appropriate chromogenic substrates for bacteria and draw Blue value-concentration curve

#### 1 Literature research

By searching literature, we found out specific enzymes in the four bacteria strains that can catalyze
reactions of certain chromogenic substrates. The chromogenic substrates and their color variation
due to such enzyme catalysis for the four strains are shown below.

#### Chromogenic substrates for bacteria and their color variation due to enzyme catalysis

| Name | Bacteria | Enzyme | Color variation | Concentration |
| --- | --- | --- | --- | --- |
| CPRG | EC | β-gal | yellow→red | 25mg/mL |
| X-β-glu | VP | β-glu | colorless→blue | 100mg/mL |
| pNPG | SA | α-glu | colorless→yellow | 100mg/mL |
| magenta caprylate | SE | esterase | colorless→violet | 100mg/mL |

#### 2 Experiments of chromogenic reactions

For each chromogenic substrate, we mixed it with colistin and a variety of bacteria strains (SA, SE,
ST, VP, PA and EC) to check if the color reaction could specifically identify target bacteria
strain. A control group (C) containing only chromogenic substrate and colistin was set up for each
experiment. The experiments showed that our paper-based sensors based on these chromogenic
substrates could successfully detect EC, SA, and SE. VP experiment failed at first, and by changing
colistin into polymyxin B and making adjustment of experiment conditions, we also successfully had a
sensor with X-β-glu for detecting VP.

#### 2.1 Experiments for identifying EC, SA and SE

Chromogenic substrates CPRG and pNPG showed specific color changes for EC and SA respectively,
distinguishing the target bacteria from other strains (Figure 13 to 14). Although the specificity of
the chromogenic substrate MC was not specific to SE, the specificity of our paper-based sensor can
still be ensured due to the specific separation of the target bacteria by RBP-MBs before sensor
detection (Figure 15).

![](https://static.igem.wiki/teams/4430/wiki/cprg.png)

Figure 13 Specificity of CPRG to EC

![](https://static.igem.wiki/teams/4430/wiki/pnpg.png)

Figure 14 Specificity of pNPG to SA

![](https://static.igem.wiki/teams/4430/wiki/mc.png)

Figure 15 Specificity of MC to SE

#### 2.2 Experiments for identifying VP

For the experiment of chromogenic substrate X-β-glu for VP detection, we failed at first. No color
change was observed after mixing X-β-glu, bacteria and colistin. We proposed several possible
hypotheses for the failure.

##### Hypothesis 1: Volume of reactants was not appropriate.

We changed the volume of colistin, X-β-glu and PBS from 0.1μl,0.1μl and 20μl to 0.2μl, 0.5μl and 10μl
to increase the concentration of colistin and chromogenic substrate. This adjustment did not improve
the results.

##### Hypothesis 2: Reaction time was not enough.

One “failed” tube began to turn blue after one day (Figure 16), we suspected that the reaction
required more time than we expected.

![](https://static.igem.wiki/teams/4430/wiki/oneday.png)

Figure 16 After 1 day, tubes containing X-β-glu and bacteria began to show light blue

##### Hypothesis 3: Quality of chromogenic substrate was poor.

We tested chromogenic substrates from different companies with bacteria handled with ultrasonication,
and found that X-β-glu from Shanghai Yuanye Bio-Technology Co., Ltd and Shanghai Aladdin Biochemical
Technology Co., Ltd. had better chromogenic ability. But although the chromogenic substrate turned
blue in the tubes, both of them failed to turn blue on the paper base. Eventually, chromogenic
substrate from another company (Bide Pharmatech Co., Ltd.) solved the problem.

##### Hypothesis 4: The decomposition agent was inappropriate.

The colistin might have a problem in breaking the cytolemma of VP. We tried several other
decomposition agents for VP, including D-cycloserine, metronidazole, ampicillin, kanamycin,
chloramphenicol, thiamphenicol, erythromycin and polymyxin B (Figure 17). Only metronidazole and
polymyxin B succeeded in turning blue in the reaction. But metronidazole turned the control group
blue as well. We found out that it was due to the hydrolysis of X-β-glu, from Shanghai Aladdin
Biochemical Technology Co., Ltd., in metronidazole, a reaction turning the solution blue without
bacteria. Luckily, polymyxin B didn’t make X-β-glu hydrolyze, so we adopted it as the decomposition
agent for VP.

![](https://static.igem.wiki/teams/4430/wiki/crack.png)

Figure 17 Test of different decomposition agents

##### Hypothesis 5: The activity of enzyme in some breeds of VP didn’t work well.

To take more strains of VP into comparison, we chose VP49, VP123, and VP17082. The results showed
that VP17082 was the most sensitive breed toward polymyxinB + X-β-glu (Figure 18).

![](https://static.igem.wiki/teams/4430/wiki/strain.png)

Figure 18 Chromogenic reactions of different breeds of VP

##### Final solution:

At last we used X-β-glu from Bide Pharmatech Co., Ltd. We mixed 0.5μl of it and 0.2μl of polymyxin B
together with 10μl VP, the solution would turn blue after 2 hours normally (Figure 19). The core
problem of the previous failure was the quality of chromogenic substrate, and we changed the formula
to make sure the bacteria were fully cracked.

![](https://static.igem.wiki/teams/4430/wiki/xbglu.png)

Figure 19 Successful results of chromogenic reactions of VP

#### 3 Experiments for determining the concentrations of bacteria

##### 3.1 Chromogenic reactions of different concentration of bacteria

We noticed that the color variation of chromogenic reactions was correlated to the concentration of
target bacteria, so we analyzed RGB values of the color to determine the concentration of target
bacteria in the samples. Using the RGB values, we were able to draw Blue value-concentration curves
to calculate the bacteria concentrations which are helpful for further application of our product in
reality.

We diluted the solution of bacteria (10⁹ CFU/mL) to 10⁸，5×10⁷，10⁷，5×10⁶，10⁶，...... ，10¹ CFU/mL and
added 10μL of diluted solution to a paper-based sensor one by one, then preserved at 37℃ for 2h. Our
experiments showed that the degree of color change of the sensors for each strain was all positively
related to the concentrations of bacterial solution.

When we detected the RGB value, we found that Blue value was the most sensitive value for the
concentration of bacteria, so we adopted Blue value to draw the curve at last. The curves for four
kinds of bacteria are listed below (Figure 20 to 23). Using the blue value, the lower limit in the
detectable concentration for all bacteria could reach around 10⁶ CFU/mL except for EC whose color
changes were quite apparent from the concentration of 7.5×10⁵ CFU/mL

![](https://static.igem.wiki/teams/4430/wiki/eccur.png)

Figure 20 The blue value-concentration curve for EC

![](https://static.igem.wiki/teams/4430/wiki/vpcur.png)

Figure 21 The blue value-concentration curve for VP

![](https://static.igem.wiki/teams/4430/wiki/sacur.png)

Figure 22 The blue value-concentration curve for SA

![](https://static.igem.wiki/teams/4430/wiki/secur.png)

Figure 23 The blue value-concentration curve for SE

### Goal 3 : Construction of RBP-MBs and measurement of the separation rates

We combined magnetic beads with RBP using condensation reaction of -NH2 on protein and -COOH on
beads. Then we mixed RBP-MBs with bacteria of 103 CFU/mL and separated the bacteria by magnet. After
oscillation and resuspension, we pasted the solution on LB Broth and counted the number of single
colony before separation (sucked bacteria of 103CFU/mL of the same volume and pasted) (Nbefore) and
after separation (Nafter). The separation rate was calculated by Nafter/Nbefore. The results are
shown below. Our results showed that all RBP-MBs successfully separated target bacteria from
samples, with MBs-TFP, and MBs-gp15 having high capture efficiency over 85% in mixed bacteria
solution and over 95% in purified bacteria solution.

Separation rates of RBP-MBs for target bacteria

![](https://static.igem.wiki/teams/4430/wiki/mbs.png)

# Real sample test

After all the components were successfully constructed, we started our whole detection procedure.

The procedure went as following: We mixed the food sample, four RBP-MBs and LB Broth in a tube, and
preserved the mixture at room temperature for 0.5h. We then placed the tube on a magnet separator,
discarded the supernatant and resuspended the precipitate with PBS. We transferred the resuspension
to the paper-based sensors, and preserved the sensors for 2h.
We used canned luncheon meat for the real sample test. We added the four pathogens to the meat to
simulate food pollution. The pathogens spiked in meat were detected by our detection method. The
recovery tests showed that acceptable recovery values ranging from 92% to 101% were obtained, with
relative standard deviation (RSD) values all below 8.28%.

Recovery tests of four foodborne pathogens spiked in canned luncheon meat

![](https://static.igem.wiki/teams/4430/wiki/rst.png)

RSD: relative standard deviation.

# Future work

There are still many problems left in our project. To begin with, the lowest concentration of
bacteria that can be detected is too high compared with other kinds of methods. So the major
improvement of the project is to find chromogenic substrate and decomposition agent with higher
sensitivity or some accelerant which can help the enzyme to decompose the chromogenic substrate. In
addition, the preservation of RBP-MBs can be a problem to low-cost detection. To bring household
food detection into reality, the cost of simplified device should be as low as possible.

Though our original purpose was to detect the pathogens in cans, the ability of our final work is
more than that. For example, it can be used to detect the bacteria in water, excretion, blood sample
and so on. For it is disposable, there’s no concern on maintenance. Imitating the principle used in
the experiment, paper-based sensors designed for more kinds of bacteria can be invented,
contributing to low-cost detection for bacteria.

[![↑](https://static.igem.wiki/teams/4430/wiki/arrow.jpg)](#top-box)

## Protocol

## Medium (For 1L volume)

#### LB Broth

* 10 g Tryptone
* 5g yeast extract
* 10g NaCl
* 1000 mL ddH2O

#### LB agar

* 15g Agar
* 25g LB Broth
* 1000 mL ddH2O

#### MacConkey (MAC) Agar medium

* 55g MAC Agar
* 1000 mL ddH2O

#### 7%NaCl TSA medium

* 40g TSA
* 70g NaCl
* 1000 mL ddH2O

---

## Buffer for protein purification

#### Buffer A

* NaCl 29.25g
* Tris 2.42g
* water 1L

#### Buffer B(pH=8.0)

* NaCl 29.25g
* Tris 2.42g
* water 1L
* imidazole 34.04g

#### PBS

* Dilute 10×PBS to 1×PBS

---

## Plasmid construction

# PCR

#### Material:

* KOD OneTM PCR master Mix
* upstream and downstream primers
* plasmid template
* ddH2O

#### Sequences of primers

| Primers | Sequence(5'-3') |
| --- | --- |
| GFP-TFP-F | GGCATGGATGAGCTCTACAAAGGTGGCAGCGGCATGGCCAACGTGATTAAAACG |
| GFP-TFP-R | GTGGTGGTGGTGGTGCTCGAGTTATTCGTTTTCCACCATGATC |
| gp15-F | ATGGGTCGCGGATCCGAATTCATGACCTTACTAGATGCAATCAATAT |
| gp15-R | AGTGCGGCCGCAAGCTTGttaCTATGTATATAGTGCAGAGCGGA |
| GFP-gp15-F | GGCATGGATGAGCTCTACAAAGGTGGCAGCGGCATGACCTTACTAGATGCAATCAATAT |
| GFP-gp15-R | AGTGCGGCCGCAAGCTTGttaCTATGTATATAGTGCAGAGCGGA |
| CBD-F | ATGGGTCGCGGATCCGAATTCCCGGAAAACGCAACCTT |
| CBD-R | AGTGCGGCCGCAAGCTTGttaTTTAAAAACACCCCATGCA |
| GFP-CBD-F | GGCATGGATGAGCTCTACAAAggtggcagcggtCCGGAAAACGCAACCTT |
| GFP-CBD-R | AGTGCGGCCGCAAGCTTGttaTTTAAAAACACCCCATGCA |

|  |  |
| --- | --- |
| TSP-F | ATGGGTCGCGGATCCGAATTCAAGTTTAAATATAGCGTTAAACTGAG |
| TSP-R | AGTGCGGCCGCAAGCTTGttaCAGGGTTGCCAGGGTCA |
| GFP-TSP-F | GGCATGGATGAGCTCTACAAAggtggcagcggtAAGTTTAAATATAGCGTTAAACTGAG |
| GFP-TSP-R | AGTGCGGCCGCAAGCTTGttaCAGGGTTGCCAGGGTCA |
| X28a-F | taaCAAGCTTGCGGCCG |
| X28a-R | GAATTCGGATCCGCGAC |
| X28a-GFP-F | taaCAAGCTTGCGGCCG |
| X28a-GFP-R | TTTGTAGAGCTCATCCATGCC |
| T7 | TAATACGACTCACTATAGG |
| T7 TER | GCTAGTTATTGCTCAGCGG |

#### Plasmid

| Plasmid | Figure | Plasmid | Figure |
| --- | --- | --- | --- |
| pET28a-TFP | ![](https://static.igem.wiki/teams/4430/wiki/tfpplasmid.png) | pET28a-GFP-TFP | ![](https://static.igem.wiki/teams/4430/wiki/gfp-tfpplasmid.png) |
| pET28a-gp15 | ![](https://static.igem.wiki/teams/4430/wiki/gp15plasmid.png) | pET28a-GFP-gp15 | ![](https://static.igem.wiki/teams/4430/wiki/gfp-gp15plasmid.png) |

|  |  |  |  |
| --- | --- | --- | --- |
| pET28a-CBD | ![](https://static.igem.wiki/teams/4430/wiki/cbdplasmid.png) | pET28a-GFP-CBD | ![](https://static.igem.wiki/teams/4430/wiki/gfp-cbdplasmid.png) |
| pET28a-TSP | ![](https://static.igem.wiki/teams/4430/wiki/tspplasmid.png) | pET28a-GFP-TSP | ![](https://static.igem.wiki/teams/4430/wiki/gfp-tspplasmid.png) |

#### Protocol:

* PCR system:5μl of KOD-one enzyme, 0.3 μl of the upstream and downstream primers, 0.3 μl of the
  template, and up to 10 μl with ddH2O
* Procedure for PCR is shown below.

| Temperature | Stage | Time | cycle |
| --- | --- | --- | --- |
| 98°C | Pre-denaturation | 3 min | - |
| 98°C | Denaturation | 10s | 30 cycle |
| 55°C | Annealing | 5s |
| 68°C | Extension | 25s |
| 68°C | Final extension | 10 min | - |
| 4°C | Preserve | Forever | - |

# Detection of PCR product

#### Material:

* Agarose
* TAE
* nucleic acid dye

#### Protocol:

1. Preparation of electrophoresis gel
   :   Adding 0.5g agarose, 50mL TAE, 2μl of nucleic acid dye and mix.
   :   Heat, then pour it into mold to solidify.
2. Electrophoresis
   :   Loading the prepared agarose gel and putting it into the electrophoresis tank.
   :   Adding 3μl of each PCR product and 3μl marker to the wells of the electrophoresis gel
       respectively.
   :   Electrophoresis (120V) for 20 min.
   :   Comparing the bands with marker to make sure PCR products correspond to the length of
       plasmid.

# Purify the product

#### The kit used in the experiment : FastPure Gel DNA Extraction Mini Kit (Vazyme)

#### Material:

* Buffer GDP
* buffer GW
* ddH2O

#### Protocol:

1. Centrifugate the PCR product in a small centrifuge, and transfer the product to 1.5mL
   centrifugal tube, then supplement to 100μl with ddH2O.
2. Add 500μl buffer GDP to the mixture.
3. Install the adsorption column into the collection tube, pour the mixture into the adsorption
   column, and centrifuge it at 10,000rpm for 30s.
4. Pour out the filtrate, put the adsorption column into the collection tube, add 700μl buffer GW
   (including ethanol) to the adsorption column, centrifuge at 12,000rpm for 30s, and repeat for
   once.
5. Pour out the filtrate and place the adsorption column into the collection tube then centrifuge
   at 12,000rpm for 2min.
6. Put the adsorption column into a 1.5mL centrifuge tube, add 20μl ddH2O, after 2min, centrifuge
   at 12,000rpm for 1min, then discard the adsorption column, save the DNA at -20 °C in the
   centrifuge tube.

# Gibson assembly

#### Material:

* 2×SeamLess cloning Master Mix
* gene fragment
* linearized plasmid fragment
* ddH2O

#### Protocol:

1. Add 5μl of 2×SeamLess cloning Master Mix ,300ng of gene fragment, 100ng of linearized plasmid
   fragment, and up to 10μl using ddH2O.
2. React at 50℃ for 30 min

---

## Plasmid transformation and colony PCR validation

# Preparation of competent cells

#### Material:

* DH5α strains
* LB broth
* LB agar
* 0.1 mol/L CaCl2 solution

#### Protocol:

1. Take the DH5α strain from -80°C refrigerator. Absorb a small amount of the bacterial protection
   liquid and add to LB Broth, paste it on LB agar, then preserve it in 37 ℃ incubator overnight.
2. pick up moist and smooth E.coli single colony from LB agar, add it to 5 mL LB Broth(without
   antibiotics), and incubate overnight at 37℃ until late log growth. Add 2mL suspension to 100mL
   LB Broth. Put it in the shaking table and incubate at 37℃ for 2-3h.
3. Transfer the bacterial solution to a cold centrifuge tube on ice for 10-30min, then centrifuge
   at 4℃, 4000rpm for 10min.
4. Discard the supernatant and reverse it for 1 min to drain the solution
5. Suspend the cells with 10 mL of 0.1 mol/L cold CaCl2 solution, fully mix, and place it on ice.
   After 30 min , centrifuge at 4℃, 4,000 rpm for 10min.
6. Discard the supernatant and reverse it for 1 min to drain the medium.
7. Add 4 mL of 0.1 mol/L cold CaCl2 solution containing 15% glycerol (sterilized), suspend the
   cells and place it on ice for a few minutes. Then store it at-80℃.

# Transfer the plasmid to DH5α（competent)

#### Material:

* DH5α competent cells
* LB Broth

#### Protocol:

1. Take E. coli DH5α competent cells from the -80 °C refrigerator and quickly insert it into the
   ice box to dissolve it. Add the DNA sample and gently mix.
2. After 20min, use 42°C water bath to heat it for 90 seconds, then quickly put it back to the ice.
3. Add 700μl LB Broth(without antibiotics) to the solution and evenly mix.
4. Put it into shaker and incubate it for 45min (37°C，220rpm), and paste 100μl on the LB agar
   containing antibiotics. Then preserve it for one night.

# Identification of the recombinant plasmids

#### Material:

* LB Broth
* 2 × Phanta Flash Master Mix (Dye Plus)
* primers

#### Protocol:

1. PCR: Pick up a single colony from the LB agar, repeatedly blow it into a 1.5 mL centrifuge tube
   containing 1 mL LB liquid medium, then pick up another colony and blow it into PCR tube
   containing 10μl PCR reaction system. The 1.5mL centrifuge tube was incubated in a 37℃ 220 rpm
   shaker for 5 h;
   the PCR tubes were used for reaction amplification. The PCR reaction system and the reaction
   procedure are listed below.

#### Reacion system

| Component | Volume |
| --- | --- |
| 2 × Phanta Flash Master Mix (Dye Plus) | 5 µL |
| T7 F(10 µM) | 0.3 µL |
| T7 TER(10 µM) | 0.3 µL |
| ddH2O | Up to 10 µL |

#### Procedure

| Temperature | Stage | Time | cycle |
| --- | --- | --- | --- |
| 98°C | Pre-denaturation | 3 min | - |
| 98°C | Denaturation | 10s | 30 cycle |
| 55°C | Annealing | 5s |
| 72°C | Extension | 25s |
| 72°C | Final extension | 10 min | - |
| 4°C | Preserve | Forever | - |

- Electrophoresis to test whether the target bands are consistent.
- Selected the suspected positive clone strains, transfer it to LB Broth containing antibiotics,
  and put it in the shaver (37℃ 220 rpm) to culture overnight.
- Extract the plasmid (see IV below for the procedures), and deliver suspected positive
  recombinant plasmid to Shanghai Bioengineering for sequencing. If the sequence is aligned to the
  designed sequence, the result is consistent, then the recombinant plasmid is successfully
  constructed.

# Plasmid extraction

#### FastPure Plasmid Mini Kit (Vazyme) is used

#### Material:

* Mixture of 150μl RNaseA and 30 mL Buffer P1
* Buffer P2, Buffer P3, Buffer PW1, Buffer PW2
* ddH2O
* FastPure DNA mini Columns
* Collection Tubes(2mL)

#### Protocol:

* Transfer the bacterial solution into a centrifuge tube, centrifuge at 10,000 rpm for 1min, and
  discard the culture medium.
* Add 250μl mixture of Buffer P1 and RNaseA and mix with pipette gun
* Add 250μl Buffer P2 and reverse to mix.
* Add 350 μl Buffer P3 and centrifuge at 12,000 rpm for 10min after reversing and mixing.
* Place the FastPure DNA mini Columns into Collection Tubes (2mL), transfer the supernatant to the
  adsorption column, centrifuge at 12,000 rpm for 60s, remove the liquid in collection tubes
  (2mL), and put the adsorption column back to the collection tube.
* Add 600 μl Buffer PW2 (diluted with absolute ethanol), centrifuge at 12,000 rpm for 60s, discard
  the waste liquid, put the adsorption column back to the collection tube, and repeated once.
* Dry the adsorption column by centrifugation at 12,000 rpm for 1min.
* Put the adsorption column into a new 1.5mL centrifuge tube. Add 100μl ddH2O to the center of the
  adsorption column. After 2min, centrifuge at 12,000 rpm for 1min.
* Discard the adsorption column and store the products in the centrifuge tube in the -20℃
  refrigerator.

---

## Protein expression and purification

# Protein expression

#### Material:

* Recombinant plasmid
* E. Coli BL21 (DE3) competent cells
* LB medium
* IPTG
* Buffer A

#### Protocol:

* Transform the recombinant plasmid into E. Coli BL21 (DE3) competent cells. Select the monoclonal
  on the transformation solid medium into LB liquid medium and incubate it overnight at 37℃ and
  220rpm as seed liquid.
* Transfer the seed liquid at 1:50 and incubate at 37℃ and 220 rpm. When the bacteria grew to
  OD600 of 0.6 ~ 0.8, add and express IPTG with a final concentration of 1 mm at 16℃ for 24 hours.

# Cell crushing and sample preparation

#### Material:

* BufferA
* SDS Loading Buffer

#### Protocol:

* After induction and expression, measure the OD600 of the bacterial liquid, centrifugate it at
  8,000rpm for 2min, collect the bacteria, and add Buffer A to resuspend the bacteria. Use a
  high-pressure crusher to crush the cells.
* Suck 80 μ L sample and centrifugate it at 12,000rpm for 1min, then suck 80μl supernatant as
  sample. Abandon the other supernatant, resuspend the deposit by 420μl Buffer A and suck 80μl as
  the bacterial crushed precipitated sample.
* Add 20 μL 5×SDS loading buffer to each sample (2.), mix well, place in boiling water for 10min,
  and centrifuge at 12,000 rpm for 1min.

# SDS-PAGE electrophoresis and staining

#### Material:

* Distilled water,
* 30% ACR BIS
* Tris
* pH8.8
* 10% SDS
* 10% ammonium persulfate
* TEMED,
* staining solution
* decolorizing solution

#### Protocol:

* Preparation of separating glue: assemble four clean thick (1.5 mm) and thin glass plates into
  the glue maker one by one, configure 12% separating glue, inject it between the two glass
  plates, flatten the liquid surface with isopropanol, and stand for 45min until it solidifies.
* Preparation of concentrated gel: pour out isopropyl alcohol, prepare 5% concentrated gel, inject
  it above the separation gel, insert the comb, and preserve for 45min until it solidifies.
* Loading: take out the gel and install it in the vertical electrophoresis tank. Dilute 5×Tris
  Glycine to the working concentration and pour it into the internal and external electrophoresis
  tanks. Carefully remove the comb and spot the sample into the sample hole in sequence. The
  loading amount is 10 μl.
* Gel electrophoresis: after the electrophoresis equipment is installed, conduct electrophoresis
  at 80V for 20min. When the bromophenol blue indicator band of the sample migrates to the
  separation gel, electrophoresis is performed at 120V for 80min.
* Dyeing: take out the gel, put it into the container, add the dyeing solution, and place it in
  the bleaching shaker for dyeing for 30min.
* Decolorization: discard the staining solution, add the decolorizing solution, put it in the
  decolorizing shaker, and change the decolorizing solution every 30min until the protein staining
  band is clear and the background color disappears.

# Protein purification and concentration

#### Material:

* Ni-NTA 6FF（5mL）pre-assembled gravity column
* Buffer A
* Buffer B
* ethanol
* Amicon® Ultra-15 centrifugal filter (Millipore)

#### Protocol:

Use Ni-NTA 6ff (5mL) pre-assembled gravity column on supernatant sample, purify the protein by nickel
affinity chromatography. During purification, the flow rate of the chromatographic column sample
should be less than 5mL/min. The specific purification steps are as follows:

* Discharge the liquid in the chromatography column and wash the chromatography column with ddH2O
  for 5-8 column volumes.
* Balance 5-8 column volumes with Buffer A.
* Inject the protein supernatant sample into the chromatography column, and collect the effluent
  of the chromatography column during the process, and the sample is the permeate.
* Use Buffer A to wash the chromatographic column for 5-8 column volumes and collects the
  discharged liquid, which is the equilibrium liquid.
* Use different concentrations of Buffer B (10%, 20%, 30%, 50%, 100%) for gradient elution, and
  collect the effluent.
* Wash the chromatographic column with ddH2O after elution, and wash 5-8 column volumes.
* Wash 5-8 column volumes with 20% ethanol and store them in 20% ethanol at 4℃.
* Apply the samples to SDS-PAGE gel electrophoresis in order to check the purification effect.
* Place the purified protein in Amicon® Ultra-15 centrifugal filtration unit (Millipore),
  centrifuge it at 4℃ and 4,500×g, discard the filtrate in the collection pipe, and add PBS buffer
  to the upper layer of the device. Repeat several times until the effect of changing protein
  buffer is achieved.
* Gently mix and take out the protein concentrate, and add glycerol to the final concentration of
  15%. Measure the protein concentration with BCA kit. Refer to the instructions for specific
  steps. Use SDS-PAGE electrophoresis to check the concentration effect, and 0.22μM filter
  membrane for sterilization, subpackage and store at - 80℃.

---

## Activation of foodborne pathogenic bacteria and verification of fusion protein activity

#### Material:

* Escherichia coli K12 MG1655
* Vibrio parahaemolyticus ATCC 17802
* Staphylococcus aureus ATCC 43300
* enterica serovar Pullorum str. ATCC 9120
* LB medium.
* Fusion proteins purified from previous experiments.

#### Concentrations of fusion proteins purified from previous experiment

| Fusion protein | concentration |
| --- | --- |
| GFP-TFP | 420μg/mL |
| GFP-gp15 | 722μg/mL |
| GFP-CBD | 210μg/mL |
| GFP-TSP | 425μg/mL |

#### Protocol:

* 1.Take out Escherichia coli K12 MG1655, Vibrio parahaemolyticus ATCC 17802, Staphylococcus
  aureus ATCC 43300, Salmonella enterica subsp. enterica serovar Pullorum str. ATCC 9120 bacterial
  seed from the -80℃ refrigerator and put them on ice for melting. In the biosafety cabinet scribe
  the bacterial liquid on LB solid medium and culture it at 37℃ for 16-24h.
* Select the single colony on the plate, inoculate it into LB liquid medium, and culture it at 37℃
  for 10h.
* Collect 200μl bacteria cultured overnight for 10h, centrifuge at 8,000rpm for 2min, discard the
  medium, wash twice with PBS, and add 200μl LB to resuspend the bacteria.
* Fully mix 100μl fusion protein and 200μl of resuspended bacteria solution, centrifuge it at 37℃
  for 30min at 8,000rpm for 2min.
* Remove the supernatant, wash with PBS and collect the precipitate.
* Add 200μl PBS to resuspend the cell pellet and measure the fluorescence value of the cell with a
  microplate reader.

---

## Manufacturing RBP-MBs(RBP-magnetic beads)

#### Material:

* Carboxyl magnetic beads 1mL(50mg/mL)
* Activation buffer 15mL
* Coupling buffer 15mL
* EDC 25mg
* Sulfo-NHS 25mg
* Sealing liquid 15mL
* Storage solution 15mL

#### Protocol:

* Preparation
  :   Take an appropriate amount of magnetic beads into a clean EP tube, and mix the magnetic
      beads again with a vortex meter for 15s.
  :   Place the EP tube on the magnetic rack for 30s and suck the supernatant with a pipette.
  :   Remove the EP tube from the magnetic rack, add 1mL of activation buffer and mix.
  :   Repeat step 2.
  :   Repeat step 3. and 4. twice.
* Activation
  :   Prepare 50mg/mL EDC and 50mg/mL sulfo NHS with activation buffer.
  :   Add 1.5mL of activation buffer, 0.5mL of EDC and 0.5mL of sulfo NHS into the EP tube
      containing magnetic beads, mix well and store at room temperature for 15min.
  :   Place the EP tube on the magnetic rack for 30s and suck the supernatant with a pipette.

* Coupling
  :   Transfer an appropriate amount of protein into the tube and mix with magnetic beads
      (50μg RBP:100μl magnetic beads), add 2mL of coupling buffer and mix well.
  :   Put it on the rotator and store it at room temperature for 90min.
  :   Place the EP tube on the magnetic force for 30s, and suck the supernatant with a
      pipette.
* Enclosure
  :   Add 5mL of blocking solution into the EP tube, mix well, put it on the rotator and store
      it at room temperature for 30min.
  :   Place the EP tube on the magnetic force for 30s, and suck the supernatant with a
      pipette.
  :   Add 5mL of blocking solution into the EP tube, mix well, put it on the magnetic rack for
      30s, and use a pipette to suck the supernatant.
  :   Add 5mL of preservation solution into the EP tube, mix well, put it on the magnetic rack
      for 30s, and use a pipette to suck the supernatant.
  :   Repeat 3. and 4. twice.
  :   Add 1mL of preservation solution into the EP tube and store it in a 4℃ refrigerator.

---

## Calculation of separation rate of magnetic globin complex

#### Material:

* Pathogenic bacteria after activation (109CFU/mL): EC, VP, SA, and SE bacteria
* LB broth medium
* PBS
* magnetic globin complex

#### Protocol:

* Mix 10μl of pathogenic bacteria and 990μl of PBS.
* (Control group) Shake the solution obtained in step 1 and repeat the step 1 until the bacterial
  solution is diluted to 103CFU/mL.
* (Experimental group - single bacteria) Shake the solution obtained in step 1, mix 10μl of it
  with 100μl of magnetic globin complex evenly and add 890μl LB broth medium.
* (Experimental group - mixed bacteria) Shake the solutions of the four bacteria strains obtained
  in step 1, suck 10μl of each solution, mix them with 100μl of magnetic globin complex evenly and
  add 860μl LB broth medium.
* Put the centrifuge tubes obtained in step 3 and 4 into a 37℃ 220rpm shaker and shake it for half
  an hour.
* (Control group) Shake the final solution of step 2 and suck 100μl of it, smear it on LB medium,
  and put it into a 37℃ incubator for one day and one night.
* (Experimental groups) After the end of the step 5, adsorb the magnetic beads to one side with a
  magnet, suck away the liquid in the centrifuge tube, then add 1,000μl of PBS, use shaker to
  resuspend.
* (Experimental groups) After resuspension, suck 10μl of it and 990μl of PBS to mix, shake and
  mix, suck 100μl of it and paste it on medium, and put it into 37℃ incubator for one day and
  night.
* (Control group) Record the number of single colonies on the obtained medium at step 6 “n” and
  the number of single colonies on the obtained medium at step 8, “M”. Calculate the separation
  rate by the equation: M/n × 100%.

---

## Preparation of paper-base sensor

#### Material:

* Whatman 1442-047(filter paper)
* 5%BSA
* Polymyxin B(10mg/mL)
* ddH2O

#### Chromogenic substrates

| Name | Bacteria | Enzyme | Color variation | Concentration |
| --- | --- | --- | --- | --- |
| CPRG | EC | β-gal | yellow→red | 25mg/mL |
| X-β-glu | VP | β-glu | colorless→blue | 100mg/mL |
| pNPG | SA | α-glu | colorless→yellow | 100mg/mL |
| magenta caprylate | SE | esterase | colorless→violet | 100mg/mL |

#### Protocol:

* Steep Whatman 1442-047 in 5%BSA at 4℃.
* After one night, discard BSA and wash the filter paper with ddH2O until no foam.
* Dry the filter paper at 55℃.
* Take out the dried filter paper and use a punching machine to punch out roundish
  paper(diameter=6mm).
* Use tweezers to place roundish paper into a 96-well plate, add 1μl chromogenic substrate, 1μl
  Polymyxin B (10mg/mL) and 5μl ddH2O.
* Preserve the pretreated paper at 37℃ for 0.5-1h.

---

## Identification of color reaction(without paper base)

#### Material:

* Polymyxin B
* Chromogenic substrate
* PBS
* Bacteria

#### Protocol:

* Add 0.5μl chromogenic substrate, 0.2μl Polymyxin B (10mg/mL) and 20μl bacteria (109CFU/mL,
  resuspended by PBS) to a 0.2mL centrifugal tube.
* Preserve at 37℃ for 2h,then take out the tube to check the color.

---

## Color reaction of different concentration of bacteria

#### Material:

* Paper-base sensor
* PBS
* Bacteria

#### Protocol:

* Dilute solution of bacteria (10⁹ CFU/mL) to 10⁸,5×10⁷,10⁷,5×10⁶,10⁶...... 10¹ CFU/mL.
* Add 10μl of diluted solution to a paper-based sensor, then preserve at 37℃ for 2h.

---

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