iGEM 2022 — Team nwu-china-a

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#### Engineering

[* ![](https://static.igem.wiki/teams/4199/wiki/imag/nav1.png)
  The first iteration](#first)
[* ![](https://static.igem.wiki/teams/4199/wiki/imag/nav1.png)
  The second iteration](#second)
[* ![](https://static.igem.wiki/teams/4199/wiki/imag/nav1.png)
  The third iteration](#third)
[* ![](https://static.igem.wiki/teams/4199/wiki/imag/nav1.png)
  The fourth iteration](#fourth)
[* ![](https://static.igem.wiki/teams/4199/wiki/imag/nav1.png)
  The fifth iteration](#fifth)
[* ![](https://static.igem.wiki/teams/4199/wiki/imag/nav1.png)
  Quote](#Quote)

**The first iteration**

**Design**

Chronic lower gastrointestinal bleeding is a common clinical symptom,
but the traditional detection method of intestinal micro bleeding is more complex and may bring
bad experience to patients. Therefore, we reviewed the relevant literature and established a
pathway for heme transport and visual expression containing four genes in E.coli, that is, ChuA
protein can transfer the extracellular heme to the cell, and the intracellular heme further
combines with the heme binding protein HrtR, changing the HrtR conformation, leading to the
activation of the promoter HrtO, and then it will induce the expression of the downstream
reporter gene cjBlue, producing blue pigment in bacteria.

**Build**

**Plasmid construction**

Because our laboratory has an off-the-shelf source of pET28a (+) vector
and we often use this vector and are most familiar with it, so we decided to use it as the
vector to verify the feasibility of our design, so we constructed pET28a
(+)-ChuA-HrtR-HrtO-cjBlue and transformed it into E. coli BL21 (DE3).

![](https://static.igem.wiki/teams/4199/wiki/engineer/eng1.png)

Figure 1 The plasmid map of pET28a (+)-ChuA-HrtR-HrtO-cjBlue

**Strain establishment**

We u
se
d

E.coli
DH5α to colon
e the
 plasmid and then transformed it into E.coli BL21(DE3) to establish the expression strain.

**Learn**

After
 experimental test
ing
, we found that the expression of pigment was extremely low or even no.
By
 review
ing the
literature and consulting our PI and advisor, we learned that the reason for this phenomenon m
ight
 be that the four genes used in this study
were linked
to a plasmid vector, so that the plasmid was too large and the product
expression was unstable. Therefore, we decided to divide the target gene into two parts and
introduce them into two plasmids, and then transform them into one
s
train at the same time.

**The s****econd iteration**

**Design**

We use
d
 pET28a(+) vector to synthesize
the
pET28a(+)-ChuA
-
HrtR plasmid, and use
d

the
pUC19
-
HrtO and
the
pSB1C3-cjBlue plasmid
in
iGEM 2019 DNA Distribution Kit Plate 5（
[http://parts.igem.org/Part:BBa\_K864404](http://parts.igem.org/Part%3ABBa_K864404)
）
. We used
PCR amplification and enzyme digestion to link the second plasmid pSB1C3-HrtO
-
cjBlue
we needed.

**Build**

**Plasmid construction**

We constructed two recombinant plasmids
:
pET28a(+)-ChuA-HrtR and
pSB1C3-HrtO-cjBlue
.

![](https://static.igem.wiki/teams/4199/wiki/engineer/eng2.png)

![](https://static.igem.wiki/teams/4199/wiki/engineer/eng3.png)

Figure 2 Plasmid Maps of pET28a (+) - ChuA HrtR and pSB1C3-HrtO-cjBlue

**Strain establishment**

We used E.coli DH5α to colone
the
plasmid,
and
then we transformed two plasmids into E.coli BL21 to establish an
expression strain.

![](https://static.igem.wiki/teams/4199/wiki/engineer/eng4.png)

Figure 3
Transformed colony on the LB culture medium

**Test**

**Escherichia coli transformation
experiment**

To confirm the successful construction of the target plasmid
pSB1C3-HrtO-cjBlue, we first transformed the enzyme linked product into E.coli and coated it on
the LB culture medium containing chloramphenicol antibiotic.

![](https://static.igem.wiki/teams/4199/wiki/engineer/eng5.png)

Figure 4
Enzyme linked bacterial colony on LB culture
 medium

**Agarose gel electrophoresis**

Further verify whether the plasmid was successfully
constructed.

**Learning**

In the second iteration, although we successfully
constructed a strain containing pSB1C3-HrtO-cjBlue plasmid, we found mutation in the plasmid
sequence after sequencing verification. Therefore, we would use In-Fusion technology in the
third iteration to reduce non-specific amplification during enzyme digestion and achieved more
efficient connection.

**The t****hird iteration**

**Design**

We used an efficient assembly method——In-Fusion technology to insert
HrtO and cjBlue sequences into a plasmid to confirm the successful construction of the target
plasmid pSB1C3- HrtO-cjBlue.

Due to Covid-19, we could not borrow the instruments needed for
electric transformation in time, so we decided to transform it by ordinary chemical
transformation methods.

**Test**

**E.coli transformation experiment**

In order to confirm the successful construction of the target plasmid
pSB1C3-HrtO-cjBlue, we used seamless cloning technology to insert HrtO and cjBlue sequences into
a plasmid, and coated it on the LB medium containing chloramphenicol antibiotic. E. coli can
only grow normally if it is successfully transferred into the plasmid.

![](https://static.igem.wiki/teams/4199/wiki/imag/test1.png)

Figure 5 The pSB1C3-HrtO-cjBlue plasmids which were successfully
constructed by In-Fusion technology on LB culture medium

Finally, two plasmids PET28A (+)-ChuA-HrtR and pSB1C3-HrtO-cjBlue were
successfully transferred into E. coli, but due to time constraints, we only demonstrated that
heme was indeed able to activate cjBlue expression, so we didn't explore the minimum heme
concentration.

**Agarose gel electrophoresis**

Further verify whether the plasmid was successfully
constructed.

![](https://static.igem.wiki/teams/4199/wiki/engineer/eng62.png)

Figure 6 The running gel of pSB1C3-HrtO-cjBlue plasmid successfully
constructed by In-Fusion technology

![](https://static.igem.wiki/teams/4199/wiki/engineer/eng8.png)

Figure 7 Transformed colony on LB culture medium

**Learn**

Because the strains we built are excreted through feces, and feces
cannot be completely collected and sterilized, which will have some unpredictable effects. So we
decided to add suicide switches inspired by last year's project.

**The fourth iteration**

**Design**

In terms of the design of the suicide switch, we decided to use the
suicide switch that our team had verified last year to reduce the leakage problem and improve
the safety of the sensor. Namely, nMag and pMag are inserted respectively in different domains
of Cas9. They will form a dimer activated by blue light to form a complete Cas9, and then cut
the target gene segment under the guidance of gRNA.

![](https://static.igem.wiki/teams/4199/wiki/engineer/eng9.png)

Figure 8 Blue light activated suicide switch

**Test**

The suicide switch was successfully verified last year (please click
[https://2021.igem.org/Team:NWU-CHINA-A/Implementation](https://2021.igem.org/Team%3ANWU-CHINA-A/Implementation) )

**Learn**

Because our sensor needs to enter the human intestinal tract to play a
role, we chose E.coli Nissle 1917 as the expression strain instead of E.coli BL21. Since there
was no T7RNA polymerase in E.coli Nissle 1917, we planned to use J23119 promoter to replace T7
promoter.

And considering that our suicide switch needs blue light
activation,which is not available in normal homes. By looking through the literature we found
another simple suicide switch , that is, to knock out a specific gene in E.coli.

**The fifth iteration**

**Design**

We imagine replacing the T7 promoter in the plasmid with the J23119
promoter, and since E.coli Nissle 1917 has been proved to be a probiotic that is safe and
harmless to human beings, we wanted to replace the expression strain from E.coli BL21 to E.coli
Nissle 1917.

We found another suicide switch with higher feasibility, that is, to
knock out the dapA gene fragment in the E.coli chromosome. E.coli can only conduct cell wall
biosynthesis and growth when exogenous diaminopimelate (DAP) are added. It is reported that the
concentration of DAP in soil is insufficient to support the growth of cell wall, so as to
achieve biological protection in vivo and environment.

**Build**

**Plasmid construction**

In the first constructed plasmid pET28a(+)-ChuA-HrtR, we updated the T7
promoter to the J23119 promoter, and planned to knock out the dapA gene fragment.

**Strain construction**

We wanted to transform two plasmids into E.coli Nissle 1917 through
electrotransformation to establish an expression strain.

**Test**

**E.coli transformation experiment**

To confirm the successful construction of the target plasmid
J23119-ChuA-HrtR, we transformed the enzyme linked product into E. coli and coated it on the LB
medium.

![](https://static.igem.wiki/teams/4199/wiki/engineer/eng10.png)

Figure 9 Transformed colony on LB culture medium

However, due to Covid-19, our experiment time has been shortened, so
the verification of the introduction of E.coli Nissle 1917 and the design of suicide switch have
not been completed. In the next stage, we will verify it in the laboratory.

**Engineering in
model******Design: Our cooperation with DKU\_China for hardware testing began with a short meeting. During the meeting, we introduced our needs and Shiran Yuan from DKU\_China introduced some basic knowledge and concepts of modeling to NWU-CHINA-A to facilitate communication. Eventually, the two teams jointly selected the parameters for testing and completed the experimental design.******Test: For experimental results and quantitative analysis, please refer to our modeling page.******Learn: We analyzed the results of the first experiment together. The experimental results were enough to indicate that, despite the effects being slightly worse compared to conventional methods, the hardware works well. However, the data indicated that the error is relatively large. Eventually, we concluded that:******1、During calibration, the concentration of strains in the liquid used for calibrating was later found to be inconsistent, therefore indicating that the experimentally tested OD might deviate from the actual values.******2、Due to the problem that some of the team members in charge of operating the experiment lack experience, the operational errors in the experimental results are large.******Design: Learning from the previous cycle, the experimental design was adjusted. The calibration process was standardized, and the operation process was carried out by only one person to minimize operational errors.******Build: The basic experimental design stayed the same.********Test: For experimental results and quantitative analysis, please refer to our modeling page.******Learn: This time, from modeling we learned two useful results which indicate the success of our experiment:******1、The growth curve of the strains fits perfectly with the logistic growth model (theoretical prediction).******2、The hardware works well, and its difference in performance from the conventional hardware is statistically insignificant.******Quote**

[1]
Tan ZiZhu,Li XiaoDan,Kong ChaoDi,Sha Na,Hou YaNan,Zhao KaiHong.
Engineering Bacteria to Monitor the Bleeding of Animals Using Far-Red
Fluorescence.[J]. ACS
sensors,2021.

[2]
Isabella Vincent M,Ha Binh N,Castillo Mary Joan,Lubkowicz David J,Rowe
Sarah E,Millet Yves A,Anderson Cami L,Li Ning,Fisher Adam B,West Kip A,Reeder
Philippa J,Momin
Munira M,Bergeron Christopher G,Guilmain Sarah E,Miller Paul F,Kurtz Caroline B,Falb
Dean.
Development of a synthetic live bacterial therapeutic for the human metabolic
disease
phenylketonuria.[J]. Nature biotechnology,2018,36(9).**************************

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#### Experiment

## **Liquid LB medium (take 1000 mL as an example)**

1. Prepare the following mixture in an Erlenmeyer flask.

|  |  |
| --- | --- |
| Element | Volume or weight |
| NaCl | 10g |
| Peptone | 10g |
| yeast | 5g |
| water | 1000mL |

2. Sterilize in an autoclave.

## **Solid LB medium (take 1000 mL as an example)**

1.Prepare the following mixture in an Erlenmeyer flask.

|  |  |
| --- | --- |
| Element | Volume or weight |
| NaCl | 10g |
| Peptone | 10g |
| yeast | 5g |
| Agar powder | 15g |
| water | 1000mL |

2.Sterilize in an autoclave.

## **Preparation of reagents**

1. Coomassie brilliant blue (500mL): 37.5mL acetic acid. 50mL ethanol. 10mL Coomassie brilliant blue dye solution. 402.5mL water.

## **Agarose gel electrophoresis (take 30mL as an example)**

  1. Prepare a suitable gel plate and comb.

  2. Weigh 0.3g（1%） agarose.

  3. Add 30mL 1×TAE.

  4. Heat in the microwave oven until the agarose is completely dissolved.

  5. Add 3 microliters of nucleic acid staining dye and mix well.

  6. Pour the solution on the flat plate with the comb inserted, and use the pipette tip to suck out all bubbles.

  7. After it solidifies, carefully pull out the comb.

  8. Mix the sample with 6×DNA loading Buffer at a ratio of 5:1. Load an appropriate amount of DNA weight makers and samples into the wells(lanes).

  9. Turn on the power supply, set the voltage to 120V, run the gel until the makers move to the bottom.

10. Remove the gel assembly from the electrophoresis apparatus. Remove the gel from the glass plates using a spatula, and prepare for subsequent analysis.

## **Polyacrylamide gel electrophoresis**

1. Assemble the device and perform leak detection.

2. Prepare separation gel.

|  |  |  |
| --- | --- | --- |
| Gel concentration | 10％ | 12％ |
| water(mL) | 2.08 | 1.75 |
| 30％Acr-Bis(mL) | 1.67 | 2.0 |
| Separation gel buffer(mL) | 1.25 | 1.25 |
| Modified ammonium persulfate（μL） | 20 | 20 |

**Note**: Add modified ammonium persulfate at the end and shake it immediately before pour into the glass plate.

3. Seal the liquid surface with water or isopropanol.

4. Prepare concentration gel.

|  |  |
| --- | --- |
| Gel volume | 2mL |
| water(mL) | 1.14 |
| 30％Acr-Bis(mL) | 0.34 |
| Separation gel buffer(mL) | 0.5 |
| Modified ammonium persulfate(μL ) | 10 |

**Note**: Add modified ammonium persulfate at the end and shake it immediately before pour into the glass plate.

5. Insert the comb and wait for the glue to completely solidify.

6. Preparation of the sample: add a certain amount 5× loading Buffer to the sample .and mix well, heat the sample at 98°C for 12 minutes in theymocycler.

7. After the gel has solidified, pull out the comb, add 1×SDS buffer, and then load them to the wells.

8. Turn on the power supply,  voltage is 120V , run the makers until the makers move to the bottom.

9.Take out the gel, stain with Coomassie Brilliant Blue for 30 minutes, destaining with distilled water washed three times, the gel was used for subsequent analysis.

## **Transformation of bacteria**

1. Thaw a tube of 100μL DH5a Competent cells solution on ice，then add 10ul constructed vectors solution. Carefully flick the tube 4-5 times to mix cells and DNA.

2. Place the mixture on ice for 30 minutes.

3. Heat shock at 42**℃** for 90 seconds.

4. Place the mixture on ice again for 2 minutes.

5. Add 800μl LB culture medium to the mixture and incubate at 37**℃** in the shaking incubator for 1h.

6. Centrifuge the medium at 12，000rpm for 5 min to concentrate the cells, abandon the unnecessary supernatant to leave 100μL solution. mix the cells thoroughly.

7. Take a Erlenmeyer flask containing 100mL LB solid medium which has been sub packed and Sterilized by autoclaving。Heat it in microwave oven for about 5-8min until the medium is transparent and free of colloidal precipitation.

8. Place it on the clean bench. When the medium is cooled to about 50**℃**, add 100μL Kanamycin（or 10μL chloramphenicol or 100μL ampicillin）with a concentration of 100ng / mL and shake it well. Immediately pour it into the sterilized dish. Each Petri dish add about 15-20mL medium. Place them around the alcohol lamp to accelerate the solidification for about 20min.

9. Spread 50-100 μL competent cells onto each sterilized dish and incubate overnight at 37**℃****.**

## **Extract DNA from bacterial culture**

**We used** **omega** **Plasmid Mini Kit I  to extract DNA from bacterial culture.**

Application object: used to extract 1-5 mL of Escherichia coli cultured overnight.

Preparation:

        1. Heat Elution Buffer to 70℃ if plasmid DNA is>10kb.

        2. Add the vial of RNase A to the bottle of SolutionⅠand store at 2-8℃

        3.Dilute DNA Wash Buffer with 100% ethanol as follows and store at room temperature.

**Steps**

1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for 12-16 hours at 37°C with vigorous shaking (12000 rpm). Use a 10-20 mL culture tube or a flask.

      2.Take 1-5mL of bacterial liquid Centrifuge at 12,000 rpm for 1 minute at room temperature.

3. Decant or aspirate and discard the culture media.

4. Add 250 µL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly.

**Note**: RNase A must be added to Solution I before use.

5. Transfer suspension into a new 1.5 mL microcentrifuge tube.

6. Add 250 µL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate Incubate for two or three minutes and avoid vigorous mixing.

7. Add 350 µL Solution III. Immediately invert several times until a flocculent white precipitate forms.

8. Centrifuge at speed (12,000 rpm) for 10 minutes.

9. Transfer the supernatant to a HiBind Miniprep DNA binding column with a 2mL collection tube, centrifuge at the maximum speed for 1 min at room temperature, and pour the filtrate in the collection tube.

10. Reinstall the column into the collection tube, add 500μL HBC Buffer, centrifuge at the maximum speed for 1 min at room temperature, and discard the filtrate.

**Note:** HBC Buffer must be diluted with isopropanol according to the instructions before use .

11. Reinstall the column into the collection tube, add 700μL DNA Wash Buffer, centrifuge at the maximum speed for 1 min at room temperature, and discard the filtrate.

**Note:** DNA Wash Buffer must be diluted with absolute ethanol according to the instructions before use.

12.Discard the filtrate and repeat step 10 once .

      13. Discard the filtrate, reinstall the column back into the collection tube, and centrifuge the empty column at maximum speed for 2 minutes to spin dry the column matrix. Put the column in a clean 1.5mL centrifuge tube, add 30-100μl Elution Buffer to the column matrix, let it stand for 1 min, and centrifuge at 13,000xg for 1 min to elute the DNA.

14. Store the eluted DNA at -20°C.

## **Recover and purify DNA fragments from agarose gel**

**We successively used two kits to Recover and purify DNA fragments from agarose gel.**

**Preceding Kit: TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0**

**Preparation before Use**

1. add 56 ml of 100% ethanol to Buffer WB.

2. Confirm the Buffer GM is still yellow.

**Steps**

1. Excise the agarose gel slice containing the DNA fragment with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove residual buffer.

2. Cut the gel into small pieces by cutting the gel.

3. Weigh the gel pieces and calculate the volume of gel. 1 mg of gel is equivalent to 1μL volume.

    Add Buffer GM to gel for melting. The amount of Buffer GM is shown in the table below.

|  |  |
| --- | --- |
| gel concentration | Buffer GM |
| 1.0％ | 3X sample volume |
| 1.0 - 1.5％ | 4X sample volume |
| 1.5 - 2.0％ | 5X sample volume |

4. Mix well and melt the gel . warm at 37℃. accelerate gel solubilization.

**Note**: Gel must be completely dissolved.

5. After the gel has been completely molten, check the color of the solution. when the color becomes orange or pink from yellow, add 10 μL of 3M sodium acetate (pH 5.2) to the solution and vortex well untill the solution return to yellow.

6. Set a Spin Column into Collection Tube.

7. Transfer the solubilized agarose from Step 7 into the column. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through. For improvement the recovery rate of DNA, transfer the flow-through to Spin. Column again and centrifuge again.

8. Add 700 μL of Buffer WB into the Spin Column. Centrifuge at 12,000 rpm for 30 seconds. Discard the flow-through.

9. Repeat Step 10.

10. Place the Spin Column back into Collection Tube. Centrifuge at 12,000 rpm for 1 minute.

11. Place the Spin Column back into Collection Tube. Centrifuge at 12,000 rpm for 1 minute.

12. Centrifuge at 12,000 rpm for 1 minute to elute the DNA.

**Subsequent Kit : TIANprep Mini Plasmid Kit**

**Steps**

1. Column equilibration steps: put the adsorption column into the collection tube, add 500μL of the balance solution BL to the adsorption column CB2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Cut a single target DNA band from the agarose gel into a clean centrifuge tube, weigh it.

3. Add an equal volume of PC solution to the gel block, place it in a 50℃ water bath for about 10 minutes, and turn the centrifuge tube up and down gently.

4. Add the solution obtained in the previous step to an adsorption column CB2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

5. Add 600μL rinsing solution PW (preliminarily added absolute ethanol) to the adsorption column CB2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

6. Repeat step 5.

7. Put the adsorption column CB2 into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove the rinse solution. Put the adsorption column at room temperature for a few minutes and let it dry thoroughly.

8. Put the adsorption column CB2 into a clean centrifuge tube, and drop an appropriate amount of sterile water into the middle of the adsorption membrane, and leave it at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution.

# **Infusion**

**Steps**

1. PCR: Use PCR method to amplify HrtOand pSB1C3-cjBlue, and useTaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 to recover and purify.

2. Reaction sy stem.

|  |  |
| --- | --- |
|  | Adding  amount |
| Purified PCR snippet | 50ng |
| Linear  vector | 200ng |
| Infusion Ligase（5\*） | 2μL |
| Deionized water | 10μL |

HrtO : pSB1C3-cjBlue  ===  2 : 1

3. Incubate at 50 degrees Celsius for 15 minutes before placing on ice.

4. Transformation.

4.1 Thaw the connected product on ice.

4.2 Add 2.5μL for conversion.

4.3 Place on ice for 30 minutes.

4.4 42**℃** heat shock 45S.

4.5 Place 90s on ice.

4.6 Add 500μL SOC medium (heated to 37**℃** in advance).

4.7 Shake at 37**℃** for 1h.

4.8 Centrifuge the remainder of each conversion reaction at 6000 rpm for 5 minutes. Discard the supernatant and re suspend each particle at 100 μ L In fresh SOC medium.

4.9 Disperse each sample on a separate LB plate containing appropriate antibiotics.

4.10 Incubate all dishes overnight at 37 ° C.

4.11 The next day, single colony was selected from each experimental plate and plasmid DNA was isolated. To determine the presence of the insert, DNA was analyzed by restriction enzyme digestion or PCR screening.

## **Polymerase chain reaction (PCR)**

1. Mix individual components prior to use .

 **Q5 High-fidelity PCR kit**

|  |  |
| --- | --- |
| Components | Volume for 50μL reaction |
| Q5 High-fidelity 2X Master Mix | 25μL |
| 10 uM Forward primer | 2.5μL |
| 10 uM Reverse primer | 2.5μL |
| Template DNA | 5μL |
| Nuclease-Free Water | 15μL |

**Taq PCR Mix**

|  |  |
| --- | --- |
| Components | Volume for 50μL reaction |
| 2X Taq PCR Mix | 25μL |
| 10 uM Forward primer | 2.5μL |
| 10 uM Reverse primer | 2.5μL |
| Template DNA | 5μL |
| Nuclease-Free Water | 15μL |

2.Gently mix the reaction and collect the liquid at the buttom of a PCR tube with a quick spin.

3. Transfer reaction quickly to a preheated thermocycler , run with the following thermocycling conditions.

**Q5 High-fidelity PCR kit**

|  |  |  |
| --- | --- | --- |
| Step | temperature | time |
| Initial Denaturation | 98**℃** | 30 seconds |
| 32 Cycles | 98**℃** | 5-10 seconds |
| 50-72**℃** | 10-30 seconds |
| 72**℃** | 20-30 seconds |
| Final Extension | 72**℃** | 2 minutes |
| Hold | 4-10**℃** |  |

**Taq PCR Mix**

|  |  |  |
| --- | --- | --- |
| Step | temperature | time |
| Initial Denaturation | 94**℃** | 3 minutes |
| 30 Cycles | 94**℃** | 30 seconds |
| 55**℃** | 30 seconds |
| 72**℃** | 1 minute |
| Final Extension | 72**℃** | 5 minutes |
| Hold | 4**℃** |  |

## **Restriction Enzyme Digestion**

1. Mix components on ice.

|  |  |
| --- | --- |
| Components | Volume for 30μL reaction |
| 10\* NEBBuffer | 3μL |
| EcoRⅠ-HF Restriction enzyme | 1μL |
| XbaⅠRestriction enzyme | 1μL |
| DNA solution | 24μL |
| Deionized distilled water | 1μL |

2. Incubate the mixture in thermostat water bath at 37**℃** for 4h.

3. Run gel electrophoresis to check the length of the digested DNA fragment.

## **DNA ligation**

1. Set up the following reaction in a microcentrifuge tube on ice, the T4 DNA Ligase should be added last.

|  |  |
| --- | --- |
| Components | Volume for 20μL reaction |
| T4 DNA Ligase Buffer (10X) | 2μL |
| Vector DNA | 50 ng (0.020 pmol) |
| Insert DNA | 37.5 ng (0.060 pmol) |
| Nuclease-free water | 20μL |
| T4 DNA Ligase | 2μL |

**NOTE**: The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. We run the gel electrophoresis to estimate the DNA moles by comparing the luminance with DNA ladder.

2. Gently mix the reaction by pipetting up and down and incubate at 16**℃** for 16 hours or connect overnight at 4℃

## **IPTG induction**

1. Pick a colony from a plate and grow in LB liquid medium with Kanamycin and chloramphenicol.

incubate overnight at 37**℃**.

2. Dilute the overnight culture to 100ml (1:100 dilution) and incubate the cells at 37**℃** until the OD600 reaches 0.6-0.8.

3. Add IPTG (1μL/mL,) to the culture and incubate at 37**℃** for 12-16 hours in a shaker.

4. Store at 4**℃**.

## **Competent Cell Preparation**

1. Pick a single colony from the E. coli BL21 (DE3) plate and attach it to a shaker tube of 3 ml LB liquid medium and incubate at 37 °C for overnight concussion.

2. Take 0.5 ml of bacterial liquid and transfer it to a LB liquid medium containing 50 ml Erlenmeyer flask and incubate at 37 ℃ for 2-3 h (at this time, OD600 reaches 0.6-0.8, the number of cells must be < 108/ml, which is the key to the success of the experiment)

3. Transfer the bacterial liquid to a 1.5 ml centrifuge tube on ice; place for 10 min.

4. 4 °C 8000r/min, centrifugate for 1 min.

5. Decant the culture medium.(aspirate as much as possible with a pipette)

6. Suspend the pellet with 1.0 ml of ice-cold 0.1 mol/ml CaCl2. Centrifuge at 4 °C (6000r/min) for 1 min, recycle cells

7. Suspend the pellet with 1.0 ml of ice-cold 0.1 mol/ml CaCl2, immediately put on ice to act for more than 15 min.

8. Centrifuge at 4 °C (6000r/min) for 1 min

9. Aspirate 850 -900 microliters of CaCl2 solution, and use the remaining 100-150 microliters of ice-cold 0.1mol/ml CaCl2
to suspend cells (operate on ice). This cell is the competent cell.

### **Ultrasonic wave breaking cell**

1. After induction, the bacterial solution is transferred to the centrifuge tube after balancing at 4000r for 5min (use a fourth-degree centrifuge because heat generation will occur during centrifugation, which will lead to protein denaturation).

2. Discard the supernatant to leave the precipitate and add the same volume of PBS buffer to wash the thalli 1-2 times (shaking can be done).

3. Centrifuge at 4000r for 5min, then discard the supernatant to retain the bacteria.

4.Suspend the thallus with 10ml PBS buffer and add the buffer according to the ratio of 10ml buffer for 1g thallus.

5. Insert the ultrasonic rod and then cover the surrounding with ice water.

6. Set the ultrasonic pulverizer to work at 100-150W for 1s and rest for 2s for 10min at a temperature control of 30°C.

7. Centrifuge at 12000r for 20min at 4°C.

8. 40ul of crude protein solution in supernatant was used as supernatant sample.

### **Make fecal simulators**

**1. Material:**

Flour: We use red flour, white flour and yellow flour as basic raw materials.

Normal saline :0.9% sodium chloride (NaCl) is isotonic solution, which will not destroy the form of tangible components such as cells and bacteria.

AGAR powder: 0.1g.

Anticoagulant blood: waste blood samples after routine blood determination, 2 ml samples with increased white blood cells are generally selected.

Vegetables:A leaf of spinach，green vegetables or cabbage.

Meat: minced pork or mutton 0.1g.

Flora: Recombinant strains

Liquid medium :0.3% beef extract, 1% peptone, 0.5%NaCl, distilled water.
Mortar: One set.

**2. Method:**

First, mix the three kinds of flour and make a paste with distilled water, which serves as a substrate. Put the vegetables and minced meat in a mortar, add a small amount of distilled water for grinding, make it into a uniform puree, add AGAR powder, vegetable puree, meat puree in the matrix, stir with bamboo skewer, make evenly mixed in the matrix.

The anticoagulant blood was centrifuged (1 500 r/min, 10min), and an appropriate amount of red blood cell layer was absorbed into the dropper and added to the specimen.

After amplification, the bacterial solution was added to the specimen and mixed well to make the positive simulated specimen for experiment.

{% endblock %}

{% extends "base.html" %}
{% block page\_content %}

#### Results

**Plasmid Construction**

We constructed two basic plasmids pET28a(+)-ChuA-HrtR and pSB1C3-HrtO-cjBlue and finally transferred them together into E.coli sucessful ,synthesizing our target engineered strain to detect small amounts of intestinal bleeding. (Click [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) to kn ow more)

![](https://static.igem.wiki/teams/4199/wiki/mod/pro/res1.png)![](https://static.igem.wiki/teams/4199/wiki/mod/pro/res2.png)

The plasmid profiles of pET28a(+)-ChuA-HrtR and
pSB1C3-HrtO-cjBlue

After transferring them into E.coli BL21(DE3), we
observed the recombinant plasmid transformed colonies on the LB
culture medium.

![](https://static.igem.wiki/teams/4199/wiki/mod/pro/res3.png)![](https://static.igem.wiki/teams/4199/wiki/mod/pro/res4.png)

![](https://static.igem.wiki/teams/4199/wiki/mod/pro/res5.jpg)

Transformed colony on the LB culture medium

We then successfully verified the correctness of
our plasmid by agarose gel electrophoresis:

![](https://static.igem.wiki/teams/4199/wiki/mod/pro/res6.png)

The a
garose gel electrophoresis
results of the strain containing two plasmids

**Expression of****ChuA and HrtR**

After repeated experiments, we searched for the
optimal IPTG induction time and obtained the target protein by
ultrasonic cell crusher, and finally successfully observed the band
by SDS-PAGE.

![](https://static.igem.wiki/teams/4199/wiki/imag/proof-1.jpg)

Figure 1. SDS-PAGE result of supernatant of bacteria broken by ultrasound.

![](https://static.igem.wiki/teams/4199/wiki/imag/proof-2.jpg)

Figure 2. SDS-PAGE results of resuspended precipitate after ultrasonic fragmentation of bacteria.

**Supporting** **Hardware** **Device**

**Collecting and** **O****scillating Device**

Users can collect and treat their own faeces under non-experimental
conditions with our device, and judge whether blood is present in the
faeces by observing the apparent results. (Click [here](https://2022.igem.wiki/nwu-china-a/hardware) to know more)

![](https://static.igem.wiki/teams/4199/wiki/mod/pro/res8.png)

Hardware entity
diagram

**Biosafety** **Box**

We use thermal insulation materials for 3D printing, which is conducive to the stable culture of bacteria. The small pore region is
used for culturing the strain, and the large pore region is used for
releasing the reagent tube to ensure the safe transport of the strain.

![](https://static.igem.wiki/teams/4199/wiki/mod/pro/res9.png)

Biosafety
box

{% endblock %}