iGEM 2021 — Team BUCT-China

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# Team:BUCT-China/Notebook

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* [Home](https://2021.igem.org/Team%3ABUCT-China)
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* Project
  + [Description](https://2021.igem.org/Team%3ABUCT-China/Description)
  + [Design](https://2021.igem.org/Team%3ABUCT-China/Design)
  + [Engineering](https://2021.igem.org/Team%3ABUCT-China/Engineering)
  + [Notebook](https://2021.igem.org/Team%3ABUCT-China/Notebook)
  + [Proof of Concept](https://2021.igem.org/Team%3ABUCT-China/Proof_Of_Concept)
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* [Model](https://2021.igem.org/Team%3ABUCT-China/Model)
* Human Practices
  + [Integrated Human Practices](https://2021.igem.org/Team%3ABUCT-China/Human_Practices)
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* Parts
  + [Part1](http://parts.igem.org/Part%3ABBa_K3742000)
  + [Part2](http://parts.igem.org/Part%3ABBa_K3742001)
  + [Part3](http://parts.igem.org/Part%3ABBa_K3742002)

* [Synthesis](#h1)
* [Hydroxylation](#h2)
* [Expression](#h3)
* [Cell culture](#h4)

## Notebook

### I.Experiment Record-----Synthesis of Myristoleic Acid

Experimental time: July 23-30, 2021
Experimental staff: Siying Yang
Experimental content: Amplification bacteria each four tubes ready to extract plasmids: T-C and C
Steps:
1.Remove the Glycerin tubes for T-C and C in the refrigerator
2.Eight LB liquid media were added with 4ulcana antibiotics each
3.Each tube takes 10ul of bacteria in the glycerin tube, T-C and C each add 4 tubes
4.37 degrees C shake culture

Experimental time: August 3-5, 2021
Experimental staff: Siying Yang
Repeat to C to do double enzyme cutting verification, found that always can not cut down, give up this group, do TU plasmids.

Experimental time: August 6, 2021
Experimental staff: Siying Yang
Experimental content: Refetching T-C plasmids

Experimental time: August 10, 2021
Experimental staff: Siying Yang
Experimental content: T-C running glue, glue recycling
Experimental results: running glue results have problems, cut incomplete

Experimental time: August 11, 2021
Experimental staff: Siying Yang
The TC sequencing results are as follows, correct

![](https://static.igem.org/mediawiki/2021/0/0b/T--BUCT-China--notebook0.png)

Company resoes (T-C) are connected
Connecting T-C and U (operation see T4 connection operation above)

Experimental time: August 17, 2021
Experimental staff: Siying Yang
Convert and Coating

Experimental time: August 19, 2021
Experimental staff: Siying Yang
The board grows colonies,Pick the gusty pipe and refrigerate.

![](https://static.igem.org/mediawiki/2021/0/0e/T--BUCT-China--notebook1.png)

### II. Experiment Record-----Hydroxylation and Polymerization of Myristoleic Acid

**[1]**
Experiment time: Friday, July 16, 2021
Experimental staff: Jin Shuming, Wang Ting, Zhang Hanyi
Experimental content: Enlarging Fadl, P450 BM3 plasmid copy-transformation

1. To prepare plasmid solution, the company synthesized 4mg of FadL-pUC57-Mini and P450 BM3-pET28a plasmids, centrifuged at 120,000g for 2min, added 40μl of ddH2O, and centrifuged again with a small centrifuge.

2. For transformation, take 2 tubes of XL10 competent cells on an ice box, add 2μl of a plasmid respectively, place on ice for 30 minutes, then take out in a water bath at 42°C for 90 seconds, and place on ice for 3 minutes. Add 500μl of KmR-resistant LB medium (100ml of LB medium with 100μl of kanamycin) on the ultra-clean table, and then place it on a shaker for 1 hour.

3. Coat the plate, after transforming and incubating for 1h, take out the EP tube 4000g and centrifuge for 2min, spread two plates on the ultra-clean workbench, and place them in a constant temperature incubator at 37℃ for constant temperature culture (colonies will be visible after 10h, and colonies will be seen after 16-18h. Too mature cannot be picked).

4. Primer design, according to P450 BM3-pET28a upstream and downstream design primer company synthesis, plan to obtain a large number of target gene fragments by PCR for double digestion.

**[2]**
 Experiment time: Sunday, July 17, 2021

Experimental staff: Jin Shuming, Wang Ting, Zhang Hanyi

Experimental content: Fadl, P450 BM3 plasmid copy-picking culture

1. Take the plate coated on July 17, Fadl has only one single colony on one plate, and P450 BM3 has many single colonies on the other plate. On a clean bench, pick one each with a pipette tip and add them to two tubes of 5ml LB medium with 50μl kanamycin.

2. Incubate in a shaker at 37°C for 12 hours.

3. Take out the test tube, add 500μl of bacteria solution to 500μl of 50% glycerin aqueous solution, and store the bacteria in the refrigerator at -80℃.

**[3]**
 Experiment time: Tuesday, July 18, 2021

Experimental staff: Jin Shuming, Wang Ting, Zhang Hanyi

Experimental content: Enlarging Fadl and P450 BM3 plasmid copies-inoculation culture

1. Take Fadl and P450 BM3 LB test tubes from the refrigerator, and add 100μl each to 4 tubes of 5ml LB medium (4μl kanamycin has been added) on a clean bench.

2. Place in a shaker overnight。

**[4]**
 Experiment time: Wednesday, July 19, 2021

Experimental staff: Jin Shuming, Wang Ting, Zhang Hanyi

Experimental content: Fadl and P450 BM3 plasmid extraction
1. Take out the Fadl and P450 BM3 LB test tubes that were expanded on the 18th, transfer the bacterial solution to a 2ml EP tube and centrifuge at 12000rpm for 1min.
2. Discard the supernatant, add 250μl solution I (stored at 4°C), and resuspend the bacterial solution.
3. Add 250μl solution II, turn it upside down and rotate evenly, and let it stand for 2 minutes.
4. Add another 350μl solution III, and mix upside down immediately after adding.
5. Centrifuge at room temperature, 12000rpm, 10min.
6. Aspirate the supernatant and add it to the adsorption column, centrifuge at room temperature, 12000rpm, 1min.
7. Discard the filtrate, add 500μl Buffer HB to the adsorption column, centrifuge at 12000rpm for 1min, and wash the protein.
8. Discard the filtrate, add 700μl DNA wash Buffer to the adsorption column and centrifuge, 12000rpm, 1min, wash salt .
9. Repeat step 8 .
10. Centrifuge the empty adsorption column at 13000 rpm for 2 minutes to further dry the adsorption column.
11. The adsorption column is placed in the oven, and the lid is opened and dried for 5 minutes.
12. Take 30μl of EB touch the adsorption membrane with a pipette tip, add it to the adsorption column, leave it for 1-2min, and centrifuge at 12000rpm for 1min.
13. Add the eluate from the previous step to the adsorption membrane again in the same way, leave it for 1-2 minutes, and centrifuge at 12000 rpm for 1 minute.
14. At this time, the liquid at the bottom of the centrifuge tube is the extracted plasmid solution.

**[5]**
 Experiment time: Monday, July 20, 2021
Experimental staff: Jin Shuming, Wang Ting, Zhang Hanyi, Shan Qi
Experimental content: P450 BM3 plasmid PCR running gel, Fadl double enzyme digestion and running gel.
1. Fadl double enzyme digestion and running gel and enzyme digestion system 50μl, 6 tubes in total
2. ddH2O 38.5μl, buffer 5μl, Fadl plasmid 3.5μl, NotI 1μl, NcoI 1μl, the order of addition is the same, calculate the amount of plasmid added according to the plasmid concentration, the amount of plasmid is required to be 100ng, and the concentration of Fadl plasmid is 330ng/μl.
3. Digestion at 37°C for 90 minutes
4. Glue 90ml TAE, 0.9g agarose.
5. Prepare new loading buffer: 100μl 6X loading buffer, 100μl ddH2O, 0.4μl gold view
6. Prepare a new maker: 100μl ddH2O, 20μl 6X loading buffer, mix well, aspirate 24μl solution and discard, then add 24μl maker to it.
7. The last maker to add samples is to take 4μl of the new maker and 10μl of the new loading and mix the solution.
8. For every 50μl system, add 20μl of Loading buffer and 14μl of Marker.
9. Running rubber 160v, 20mins .
10. Comparing with Maker after running the glue, double enzyme digestion is successful, and the glue is cut and recovered.
1) Fadl double enzyme digestion and gel recycling.
1. Under a long-wave UV lamp, use a clean blade to cut off the Fadl strips that need to be recycled. The smaller the size of the glue block, the better.
2. Put the cut gel into a 1.5ml centrifuge tube and weigh it.
3. Add the buffer solution Bingding Buffer according to the glue block: Bingding Buffer=1:1000.
4. Water bath at 56℃ for 10min until the colloid is completely dissolved, shake it every 2-3min to speed up the dissolution.
5. Add the solution obtained in the previous step to the adsorption column, leave it at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube.
6. After adding 300μl Bingbuffer, centrifuge at 12000rpm for 1min, and discard the waste liquid in the centrifuge tube.
7. After adding 700μl of WB buffer, centrifuge at 12000rpm for 1min, and discard the waste in the centrifuge tube.
8. Repeat step 7.
9. Put the adsorption column into a new 1.5ml centrifuge tube, add 15μl EB, leave it at room temperature for 1-2min, and centrifuge at 12000rpm for 2min.
10. Add the liquid obtained in the previous step to the adsorption column again, and centrifuge at 12000rpm for 1min.
11. The liquid collected at the bottom of the tube is the recovered Fadl after double digestion.
2) P450 BM3 plasmid PCR running gel
1. PCR system 50μl, 4 tubes.
2. ddH2O 17μl, 2X buffer 25μl, upstream primer 1μl, downstream primer 1μl, P450 BM3 plasmid 2μl, dNTP 1μl, Pfu enzyme 1μl. The order of sample addition is the same. Adjust the amount of water according to the amount of plasmid. The water is added first and not last.
3. Prepare gel with 45ml TAE and 0.45g agarose to prepare gel.
4. Add 4μl of gold view to the PCR product and then spot, add 6μl of 10000bp at the end maker.
5. Run rubber 160v, 20min. After running the gel, put the gel under the ultraviolet lamp to observe the band. After comparing with the maker, the band is normal, and proceed to the next step-cutting and recycling.
3) P450 BM3 cut rubber recycling
1. Under a long-wave UV lamp, use a clean blade to cut off the P450 BM3 strips that need to be recycled. The smaller the size of the glue block, the better.
2. Put the cut gel into a 1.5ml centrifuge tube and weigh it.
3. According to the glue block: Bingding Buffer=1: 1000, add the buffer Bingding Buffer.
4. 56℃ water bath for 10min until the colloid is completely dissolved, shake it every 2-3min to speed up the dissolution.
5. Add the solution obtained in the previous step to the adsorption column, leave it at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube.
6. After adding 300μl Bingbuffer, centrifuge at 12000rpm for 1min, and discard the waste liquid in the centrifuge tube.
7. After adding 700 μl of WB buffer, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the centrifuge tube.
8. Repeat step 7.
9. Put the adsorption column into a new 1.5ml centrifuge tube, add 30μl EB, leave it at room temperature for 1-2min, and centrifuge at 12000rpm for 2min.
10. Add the liquid obtained in the previous step to the adsorption column again, and centrifuge at 12000rpm for 1min.
11. The liquid collected at the bottom of the tube is the recovered P450 BM3 after double digestion.

### III.Experiment Record-----The expression of hydroxylated collagen

##### Heat transduction (Transduction of plasmid into bacteria)

Experiment time:July 19, 2021
Experimental staff: Shuming Jin, Xiao Xiao, Sijia Huang
pET28a-hCOL3 Kanamycin antibiotics
pET16b-L593 AmpR antibiotics
1. LB solid medium(0.5% 300ml sodium chloride. Yeast 0.5% peptone 1% agar 1.5%),In the high pressure steam sterilization pot 121 degrees Celsius, 30 min.
2. Take 50ul of competent cells BL21(DE3) melted in ice bath, add 1ul of target DNA, mix gently, and place in ice bath for 30min.
3. 42 degrees Celsius of water bath heat shock 90 seconds, and then quickly transferring pipe to the ice bath for 2 minutes, the process is not to shake the centrifugal tube.To join in each of the centrifuge tube 500 ul sterile SOC or LB culture medium, after blending at 37 degrees Celsius, 200 RPM cultivate 1 h.
4. Take 150 ul has transformed cells to the corresponding antibiotics LB AGAR culture medium, add 1/1000 of antibiotics. Put the plate culture medium into the shaker at 37 degrees Celsius and cultivate it overnight.

##### Induced expression of L593 and hCOL3

Experiment time:July 20, 2021
Experimental staff: Jin Shuming, Yifan Zhu, Yunfeng Qu
1. The plump available bacteria were selected from the overnight plate culture medium and cultured in the liquid medium at 37 ℃ until OD600 reached 0.6. the expression of triglyceride was induced by adding 1 mM isopropyl β-D-glucosinolates. The cells were divided into 18 degrees Celsius and 30 degrees Celsius, and the blank group was cultured in the shaker for 8 hours.
2. Preservation of glycerol test tube: add a small amount of bacterial liquid to glycerin (- 80 ℃) to facilitate subsequent use.

##### Cell-breaking extraction of protein and Electrophoretic Verification of SDS protein

Experiment time:July 21, 2021
Experimental staff: Shuming Jin, Xiao Xiao, Sijia Huang，Yunfeng Qu
1. 120 r/min, the centrifugal to the bottom of the precipitation, appeared a tube in qing dynasty, a total of 7 pipe, each join PBS(1\*),
2. First blow wash in the centrifugal tube 500 multips to add 2.5 ml.After broken cell(Is better to clearing), Put in the ice water bath.
3. To break the cell machine use: open the machine power supply.Wipe the machine within the needles.Take appropriate to the size of the glass plate, add some ice cubes and water, the foam board，Water should not be foam board, will stay centrifuge tube into the broken cells.First place needles at the bottom of the centrifuge tube, but should not be in contact with the wall.Fixed. Adjust the centrifuge tube location make needle impending downward.First test, such as sharp sound and as can be.Formally began to break the cell.
3. After the cell breaking is completed, the liquid in the centrifuge tube is centrifuged and the precipitation is washed three times.
4. Sample preparation for electrophoresis: 50ul sample = 40ul (uniform precipitation and PBS) + 10ul (blue), boiling 10min
5. According to the description PAGE gel preparation: first select suitable thickness of the glass, this experiment choose 1.0 mm. Overlay the two pieces of glass in the correct way, clamping on the transparent carrier. According to the instruction manual configuration is required for the lower gel liquid, mixing, between pipetting gun in the glass. After filling the deionized water was added into the lower rubber surface level, the water filling. Place a period of time to clear water and gel layer.Pour water net. According to the instruction manual configuration suitable upper gel.The upper gel filled with can.Taking the thickness of the adapter insert top comb.Let stand until set.
6. Sample in the glue hole, Maker (two-color pre-dyed wide molecular weight protein) 5ul, nourishment 10ul, blank / control 5ul, start running glue, first 80v, set 30min (usually less than 30min) until the sample is pressed into a line, then set the current to 20mA, about 1h to the marginal bottom layer about 1.2cm voltage for electrophoresis
7. Decolorize the glue and observe the results.

##### Homologous recombination of L593 plasmid

Experiment time:July 25-26, 2021
Experimental staff: Xiao Xiao, Yunfeng Qu
Improvement of the first round experiments: examine the L593 sequence and find that L593 could not be expressed normally due to the existence of this hydrophobic region (we guess it was a signal peptide): MKTVTIITIIVVIIVVILII MVLSKSCVMKTVTIITIIVVIIVVILIIMVLSKSCV. We decided to use homologous recombination to remove part of the sequence.
1. In the software snapgene, the primers used for homologous recombination are designed and sent to the company for synthesis. The newly bought primers are centrifuged (13000rpm 3.1\*100ul for 2min) and slowly opened. After adding the sterilized distilled water (see instructions, 3.1\*100ul), shake well, dissolve, and place the primers at room temperature.
2. PCR.
20ul system (9.3ul ddH20, 0.25ul upstream primers, 0.25ul downstream primers, 0.2ul template plasmid, 10ul dNTP, 1ul DNA polymerase).
PCR operation.
95 ℃ pre-denaturation for 30s.
95 ℃ denaturation for 15s.
62 ℃annealing for 15s.
72 ℃ extension (6327bp) 6min.
30 cycles.
72 ℃ complete extension of 5min
3. Homologous connection was carried out according to the instructions of the homologous kit and verified by DNA electrophoresis.
4. Heat transduction (Transduction of plasmid into bacteria）
pET16b-L593 AmpR antibiotics
[1] LB solid medium(0.5% 300ml sodium chloride. Yeast 0.5% peptone 1% agar 1.5%),In the high pressure steam sterilization pot 121 degrees Celsius, 30 min.
[2]Take 50ul of competent cells BL21(DE3) melted in ice bath, add 1ul of target DNA, mix gently, and place in ice bath for 30min.
[3]42 degrees Celsius of water bath heat shock 90 seconds, and then quickly transferring pipe to the ice bath for 2 minutes, the process is not to shake the centrifugal tube.To join in each of the centrifuge tube 500 ul sterile SOC or LB culture medium, after blending at 37 degrees Celsius, 200 RPM cultivate 1 h.
[4]Take 150 ul has transformed cells to the corresponding antibiotics LB AGAR culture medium, add 1/1000 of antibiotics. Put the plate culture medium into the shaker at 37 degrees Celsius and cultivate it overnight.
5. The fuller colonies were selected and sent to the company for sequencing to verify whether part of the sequence was successfully removed.

##### Synthesis of hydroxylated collagen by enzymatic modification in vitro

Experiment time:August 1-3, 2021
Experimental staff: Shuming Jin, Sijia Huang, Yifan Zhu
1. The modified pET28a-hCOL3 and pET16b-L593 were expressed in E. coli BL21 (the specific steps have been written above)
2. 5 ug L593 prolyl hydroxylase were added to acceptor peptides at 0.5mg/ml in 50 mM Tris-HCl, pH 7.4, 100 μM FeSO4, 1 mM ascorbate, 100 μM DTT, 60 μM 2-oxoglutarate and 100 nCi of 2-oxo[14C]glutarate in a total volume of 100 μl and incubated at 37 °C for 45 min.

##### Hydroxylated Collagen purification

Experiment time:August 4-5, 2021
Experimental staff: Shuming Jin, Yunfeng Qu, Xiao Xiao
1.Expand cultivating of hydroxylated Collagen: in vitro cultured species Numbers for: 30-228 - k species seeds, vaccination to 75 MLLB medium, quantity of 1%, 30 degrees and 18 degrees of inducing expression.
2.Enriching bacteria: 120x100r/min,1min, collecting Escherichia coli after induction and expression for 14h in 1min.
3.Heavy suspended bacteria: to use current now matches the PH = 7.2 Tris HCl buffer, heavy suspended bacteria 20 ml in 50 ml centrifuge tube. 4 s broken cell: set parameters, warming, cooling 6 s, broken cell 15 min, put in ice water.
4.Centrifugal, discard the clear liquid 120X100r/min
5.Protein that let stand for 10 min, the supernatant fluid with E1, 5 ml elution 3 times, E2, 5 ml elution 3 times, E3, 5 ml elution 4 times, discard 1 times E3 elution liquid, collecting liquid, 2, 3, 4 times marked 30-2;30-3;30-4(On behalf of 30 degrees).18-2;18-3;18-4 (on behalf of 18 degrees), a total of 6 pipe so that the follow-up measure protein content and electrophoresis detection.
6.E1:10mM imidazole，50mM Tris-HCl（PH=7.2），100mM NaCl
E2:30mM imidazole，50mM Tris-HCl（PH=7.2），100mM NaCl
E3:350mM imidazole，50mM Tris-HCl（PH=7.2），100mM NaCl

##### Measuring purified hydroxylated collagen concentration

Experiment time:August 6-7, 2021
Experimental staff: Shuming Jin, Yunfeng Qu, Yifan Zhu
1. Using coomassie brilliant blue + bovine serum albumin (BSA) comparison method. (OD=596)
The preparation of standard solution:

![](https://static.igem.org/mediawiki/2021/0/09/T--BUCT-China--notebook2.png)

2. Configuration of the sample under test.30-2;30-3;30-4; 18-2;18-3;18-4 each 10ul. Add 990 ul purified water, 5 ml1XG250, standard solution and sample under test are diluted 10 times after measuring the absorbance.
Standard solution diluted 10 times after the absorbance measurement results are as follows.

![](https://static.igem.org/mediawiki/2021/8/88/T--BUCT-China--notebook3.png)

3. To make a figure as follows, absorbance and the concentration equation is: y = 0.0544 + 0.0011 x,
4. To measure the sample diluted 10 times absorbance measurement results are as follows, and turn it into the equations of the appeal of concentration.

![](https://static.igem.org/mediawiki/2021/4/4b/T--BUCT-China--notebook4.png)

![](https://static.igem.org/mediawiki/2021/a/aa/T--BUCT-China--notebook5.png)

5. The total elution concentration is 3 times the sum of elution concentration for their temperature.Due to the sample preparation when diluted 10 times diluted 10 times again, And three eluent volume of 15 ml, strain system for 75 ml, so the results are as follows:
Take the example of 30 degrees: 30, the total concentration was 0.003385+0.02514+0.006159+0.01209. Bacterial protein production quantity/(g/L):0.012058\*15\*10\*10/75=0.241168. Conclusion: in terms of expressing protein content as an index, and the expression of 30 degrees condition is superior to 18 degrees.

### IV.Experiment Record-----Cell culture

##### August 13

Extraction of primary muscle satellite cells from chicken
All according to the experimental design.
A few caveats:
1. When taking a muscle sample, try to select the muscle close to the bone.
2. We took a lot of muscle samples, so we carried them out in two times. The remaining part of muscle was soaked in DMEM-F12 medium and placed in an incubator. After 24 hours, the muscle tissue was taken out and cells were further extracted.

##### August 14

Cell extraction was observed & primary muscle satellite cells were extracted from the tissue of the previous day
1. Observe the cells extracted yesterday

![](https://static.igem.org/mediawiki/2021/6/61/T--BUCT-China--notebook6.png)

Looking at the cells isolated the day before, it was found that some of the muscle satellite cells had been extracted.
The red dots indicate muscle satellite cells and the green dots indicate tissue fragments, dead cells, and non-adherable red blood cells, which were cultured for 24 hours.

2. Continue to extract primary cells
This time, muscle tissue was digested in two ways: one was digested directly with collagenase, and the other continued to use a mixture of enzymes

##### August 15

Observation of muscle satellite cells
1. The muscle satellite cells extracted on 13 August were presented

![](https://static.igem.org/mediawiki/2021/a/a5/T--BUCT-China--notebook7.png)

After fluid exchange, most of the tissue debris, dead cells and non-adherent red blood cells were removed, and the muscle cell morphology was more obvious.

2.Cells extracted on August 14 were presented

Collagenase digestion: few muscle cells

![](https://static.igem.org/mediawiki/2021/d/da/T--BUCT-China--notebook8.png)

Mixed enzyme solution digestion: As last time, the situation is optimistic.

For more detail of experiment of scaffold and cell culture, please go to the following links:
[2021.igem.org/Team:BUCT-China/Design[2021.igem.org/Team:BUCT-China/Proof\_Of\_Concept](https://2021.igem.org/Team%3ABUCT-China/Proof_Of_Concept)](https://2021.igem.org/Team%3ABUCT-China/Design)

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IGEM

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BUCT

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TEAM

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# Team:BUCT-China/Engineering

[![](https://static.igem.org/mediawiki/2021/f/fa/T--BUCT-China--Toplogo.png)](https://2021.igem.org/Team%3ABUCT-China)

* [Home](https://2021.igem.org/Team%3ABUCT-China)
* Team
  + [Members](https://2021.igem.org/Team%3ABUCT-China/Team)
  + [Attributions](https://2021.igem.org/Team%3ABUCT-China/Attributions)
* Project
  + [Description](https://2021.igem.org/Team%3ABUCT-China/Description)
  + [Design](https://2021.igem.org/Team%3ABUCT-China/Design)
  + [Engineering](https://2021.igem.org/Team%3ABUCT-China/Engineering)
  + [Notebook](https://2021.igem.org/Team%3ABUCT-China/Notebook)
  + [Proof of Concept](https://2021.igem.org/Team%3ABUCT-China/Proof_Of_Concept)
  + [Implementation](https://2021.igem.org/Team%3ABUCT-China/Implementation)
  + [Contribution](https://2021.igem.org/Team%3ABUCT-China/Contribution)
* [Model](https://2021.igem.org/Team%3ABUCT-China/Model)
* Human Practices
  + [Integrated Human Practices](https://2021.igem.org/Team%3ABUCT-China/Human_Practices)
  + [Scientific Education](https://2021.igem.org/Team%3ABUCT-China/Communication)
  + [Collaborations](https://2021.igem.org/Team%3ABUCT-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ABUCT-China/Partnership)
* Parts
  + [Part1](http://parts.igem.org/Part%3ABBa_K3742000)
  + [Part2](http://parts.igem.org/Part%3ABBa_K3742001)
  + [Part3](http://parts.igem.org/Part%3ABBa_K3742002)

* [Step1](#h1)
* [Step2](#h2)
* [Step3](#h3)
* [Step4](#h4)
* [Reference](#h5)

## Engineering Success

Due to the coronavirus in 2021, we don’t have sufficient time to enter the laboratory to complete the experiment. We completed the experimental design of this project in the early stage and completed part of the experiments in the limited time. We hope to verify the feasibility of our experimental design through part of the experimental content, and do a good foundation for our follow-up work. This page contains the results of our experiments regarding to first section.

We have described our project design ideas (including project section I and section II, design ideas, 4 steps in section I , etc.) in the design page of Wiki：
[https://2021.igem.org/Team:BUCT-China/Design](https://2021.igem.org/Team%3ABUCT-China/Design)

![](https://static.igem.org/mediawiki/2021/2/26/T--BUCT-China--EG0.png)

### Step1

We take advantage of the fatty acid synthesis pathway of *E.coli* to synthesize myristoleic acid from glucose, we construct an artificial metabolic pathway that can desaturate the ACP-fatty acid by Δ9 desaturase(TsAab2) and finish the fatty acid synthesis to produce 14C fatty acid by thioesterase (UcFadD).

![](https://static.igem.org/mediawiki/2021/3/31/T--BUCT-China--EG11.png)

T: Tsfab2 desaturase gene (promoter ARA)
U: Ucfadd thioesterase gene (promoter LAC)

Plasmid pet28a-tsfab2-ucfadd was designed. The T (desaturase gene) gene fragment was connected with arabinose promoter C to obtain C promoter Ara. Then, the U (thioesterase gene) fragment was connected to the t-weight long fragment through T4, and the T-U recombinant plasmid was constructed as the target plasmid.

For the pET28a-TsAab2-UcFadD we want to build, we first construct plasmid of pET28a-TsAab2 with C (Ara promoter). We synthesize two DNA primers which sequences are pairing with 5’ of based on sequencing results and gel picture comparing with marker, we can prove that the plasmid is successfully constructed, and plasmid size is 7943bp. The plasmid was successfully built and can be used in further construction of pET28a-TsAab2-UcFadD.

![](https://static.igem.org/mediawiki/2021/a/a5/T--BUCT-China--cha-3.jpg)

After enzyme digestion (NheI, XhoI and XbaI, XhoI) and ligation, we have successfully transfered the pET28a-TsAab2-UcFadD into *E.coli*. And the bacteria has formed single colony on Kanamycin LB medium, however we haven’t do the sequencing to verify the plasmid yet due to the limited time.

![](https://static.igem.org/mediawiki/2021/9/9e/T--BUCT-China--EG12.png)

### Step2

In this part, we designed and constructed a plasmid containing FadL gene and P450 BM3 gene sequences. After the plasmid was introduced into the FadD gene-deficient E. coli cells in the laboratory, the cells achieved increased absorption of exogenous long-chain fatty acids (LCFA). Then, hydroxylation reaction was carried out to make the host cells produce more hydroxyl fatty acids.

![](https://static.igem.org/mediawiki/2021/0/06/T--BUCT-China--EG13.png)

Fig1. Expected results of the experiment

Our objective was to hydroxylate LCFA which had already entered into the membrane. Thus, the presence of the FadD gene would hinder the experiment. By consulting the relevant literature, we found that Jin H and his team have done two experiments to prove that knockout of FadD gene and overexpression of Fadl in Escherichia coli can increase the production of hydroxylate in Escherichia coli. First, they knocked out the FadD gene in E. coli and introduced the P450 enzyme gene into the bacteria to convert palmitic acid into omega-hydroxypalmitic acid. The results of the experiments demonstrate that the production of ω-hydroxypalmitic acid is increased in the FadD-deleted, P450 overexpressed mutants. The experimental results are shown in fig. 3 (a). [1]

Later, they overexpressed Fadl gene on the basis of FadD gene deletion and P450 expression, and found that the production of ω-hydroxypalmitic acid was greatly increased. The experimental results are shown in fig. 3 (B). Because LCFA accumulates in the periplasm through FadL membrane protein transport and is consumed in the cytoplasm through P450 enzyme, the diffusion of LCFA is driven and will not be participated in β-Oxidation. As a result, the yield of hydroxylation products increase. [1]

![](https://static.igem.org/mediawiki/2021/b/bc/T--BUCT-China--EG14.png)

Fig3. Experimental results by Jin H and his team[1]

![](https://static.igem.org/mediawiki/2021/2/23/T--BUCT-China--EG15.png)

After the completion of the plasmid design, we contacted the company to synthesize the fadL gene at the MCS I site of pCOLADuet-1, and linked it to the P450 gene by homologous recombination. At the same time, we extracted the plasmids which were successfully transferred into the cells and verified the vector part and P450 part by PCR. As shown in fig. 5.

![](https://static.igem.org/mediawiki/2021/8/85/T--BUCT-China--EG16.png)

Fig.5 The PCR result.
The first bright band is DNA Marker.
 The second bright band is the vector part which has 4862 bp.
 The third bright band is P450 which has 3150 bp.

### Step3

We mainly want to express two proteins, CoA ligase and acyltransferase. The function of the two proteins as follows (EC for CoA ligase; WS2 for acyltransferase) At the same time, in our design, it contained a T7 promoter of EC between the two segments (EC / WS2)

![](https://static.igem.org/mediawiki/2021/1/1b/T--BUCT-China--EG17.png)

In order to verify the functions of EC and WS2, we constructed the plasmid pETDuet (WS2/EC). In the verification experiment, we used hydroxydodecanoic acid and nonanoic acid as substrates. We expressed pETDuet (WS2/EC) into E. coli BL21 (DE3). The functions of EC and WS2 are shown below. During the polymerization process, EC acts as a CoA ligase to remove H in CoA-SH and -OH in the carboxyl group of hydroxy fatty acid through dehydration and condensation, and connect CoA and hydroxy fatty acid at the same time; WS2 As an acyltransferase, the acyl group in the hydroxy fatty acid CoA replaces the H in hydroxyl group of the hydroxy fatty acid, and CoA-SH is generated at the same time.

![](https://static.igem.org/mediawiki/2021/c/c4/T--BUCT-China--EGG1.png)

##### Experimental results and analysis

##### 1.SDS protein electrophoresis

![](https://static.igem.org/mediawiki/2021/5/54/T--BUCT-China--EG1.png)

Molecular weight of target protein
EC 62 kDa
WS2 52.5 kDa
ACS2 61.9 kDa
Through SDS protein electrophoresis, we can confirm that our genes EC and WS2 are expressed, and the *E.coli* has the enzymes we need by comparing with marker. Which allowe us to conduct further experiments.

##### 2.Results of GC-MS with Hydroxydodecanoic acid and nonanoic acid as substrates

After the engineering bacteria were successfully constructed, we added an exogenous substrate and added 3~4% DMSO to increase the solubility of the substrate hydroxy fatty acid and the permeability of the cell membrane. After the induction of fermentation with the IPTG inducer, the cells were broken and used Trichloromethane extracts the fermentation broth and prepares samples for gas phase detection. Set 1 group of control substrates nonanoic acid and hydroxydodecanoic acid (brown line) and 3 groups of experimental group substrates are 1. hydroxydodecanoic acid and isopropanol (blue line) 2. Nonanoic acid and hydroxydodecanoic acid (Purple line) 3. Nonanoic acid and isopropanol (black line).

According to the GC-MS spectrum, in addition to experimental group 3, the other two groups of experimental group 1 hydroxylauric acid and isopropanol (blue line) and experimental group 2 nonanoic acid and hydroxylauric acid (purple line) pass and control Dimeric hydroxy dodecanoic acid was found in the comparison of group nonanoic acid and hydroxydodecanoic acid (brown line), indicating that the polymerization pathway of engineered bacteria is feasible to polymerize hydroxydodecanoic acid; in addition, experimental group 2 nonanoic acid and hydroxydodecanoic acid ( Purple line) The product Nonanoic acid hydroxydodecanoate appears, which proves that the hydroxyl group of the hydroxy fatty acid can be used as the catalytic site of WS2 acyltransferase, and the carboxyl group at the other end does not affect the catalytic reaction of the acyltransferase. Our research has proved that the polymerization process of EC coenzyme A ligase and WS2 acyltransferase can play a role in two identical or different monomers and form at least two degree of polymerization. Due to the limitation of the experimental time and the experimental equipment caused by the epidemic, we have not been able to provide more evidence of terpolymers and above polymers. The vaporization temperature of the gas-phase analyzer we use is 380℃, which is such a temperature. It may not be possible to vaporize the higher poly hydroxy fatty acids and enter the analytical instrument, so we did not get the relevant data. In the next experiment, we plan to further increase the intracellular substrate concentration during fermentation and use a gas phase instrument with a higher vaporization temperature for product detection.

![](https://static.igem.org/mediawiki/2021/6/63/T--BUCT-China--EG2.png)

### Step4

In this step, we want to synthesis functional collagen. The first gene sequence is the gene encoding prolyl hydroxylase enzyme from aquatic giant viruses. The second gene sequence is a fragment of human collagen type III COL3A1cDNA(hCOL3).We planned to use bacterially active prolyl hydroxylase enzymes from the giant virus mimivirus to modify collagen encoded by hCOL3 to produce hydroxylated collagen, which can provide a better cell culture

##### 1.pET16b-L593 and pET28a-hCOL3 > plasmids

![](https://static.igem.org/mediawiki/2021/a/ad/T--BUCT-China--EG3.png)

We found that some aquatic giant viruses, belonging to Phycodnaviridae and Mimiviridae, harbor genes encoding prolyl hydroxylase enzyme[2]. These viral hydroxylases are soluble and active when expressed in E. coli, thus opening new possibilities for the production of hydroxylated collagen in bacterial expression systems[3].We planed to use bacterially active prolyl hydroxylase enzymes from the giant virus mimivirus to produce hydroxylated collagen. For figure, it is the pET16b-L593 vector that we synthesized uses pET-16b as the expression vector and lacks His-Tag.

Primers and restriction Enzyme cutting sites:
5’-CCATGGATGAAAACTGTAACAATAATCACG-3’
5’-CGAACGTAAGTTCAGCTAAGGATCC-3’

Nucleic acid sequence

![](https://static.igem.org/mediawiki/2021/0/0f/T--BUCT-China--EG4.png)

Tips:
(1) The red part is T7 promoter:
(2) The green part is Prolyl 4-hydroxylase [Acanthamoeba polyphaga mimivirus] gene (NCBI Reference Sequence: YP\_003987108.1）
(3) The blue part is T7 terminator.

![](https://static.igem.org/mediawiki/2021/3/30/T--BUCT-China--EG5.png)

We chose to use a fragment of human collagen type III COL3A1cDNA, encompassing 1206 bp and lacking propeptide-encoding regions, was custom synthesized using codons optimized for bacterial expression and including NcoI and BamHI sites at 5’- and 3’-ends. The pET28a expression vector was first digested with NcoI-BamHI, which eliminates the His-tag at the 5N-terminal site. The resulting hCOL3 segment was inserted as a NcoI-BamHI fragment into pET28a, yielding pET28a-hCOL3 with His-tag.

Primers and restriction Enzyme cutting sites:
NcoI 5’-CCATGGATGTACGACAGTTATG-3’
BamHI 5’-CCACCATCACCACTAAGGATCC-3’

Nucleic acid sequence:

![](https://static.igem.org/mediawiki/2021/7/7c/T--BUCT-China--EG6.png)

Tips:
(1) The red part is T7 promoter:
(2) The blue part is synthetic construct for human collagen gene, type III, alpha 1 (COL3A1 gene) fragment(hCOL3)(GenBank: HG779440.1);
(3) The yellow part is 6xHis tag gene sequence;
(4) The purple part is T7 terminator.

Protein sequence:

![](https://static.igem.org/mediawiki/2021/6/69/T--BUCT-China--EG7.png)

Tips:
(1) The green part is synthetic construct for human collagen, type III.
(2) The red part is 6xHis tag.

##### 2. Experimental results and analysis (1)SDS protein electrophoresis of expression of L593

We put pET16b-L593 into E. coli (BL21) and induced expression by adding IPTG at 18 °C and 30 °C.Through SDS protein electrophoresis, and comparing with marker, we can confirm the expression of L593.

![](https://static.igem.org/mediawiki/2021/2/2d/T--BUCT-China--EG8.png)

##### 2.SDS protein electrophoresis of purified hydroxylated collagen and content determination of hydroxylated collagen

The expressed hCOL3 protein and L593 enzyme were carried out enzyme modification reaction a suitable and vitro environment to obtain hydroxylated collagen. Because it has His-tag, it can be purified. The following picture shows SDS protein electrophoresis of purified hydroxylated collagen and content determination of hydroxylated collagen.

(Temperature-The times of elution product)
![](https://static.igem.org/mediawiki/2021/4/41/T--BUCT-China--EG9.png)

Through SDS protein electrophoresis, and comparing with marker, we can confirm the expression and purification of hydroxylated collagen.

![](https://static.igem.org/mediawiki/2021/5/57/T--BUCT-China--EG10.png)

### Reference

[1] Bae Jin H; Park Beom Gi; Jung Eunok; Lee Pyung-Gang; Kim Byung-Gee. fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity[J].Applied Microbiology and BiotechnologyVolume 98, Issue 21. 2014. PP 8917-25
[2]Eriksson M, Myllyharju J, Tu H, Hellman M, Kivirikko KI (1999) Evidence for 4-
hydroxyproline in viral proteins. Characterization of a viral prolyl 4-hydroxylase and its
peptide substrates. J Biol Chem 274(32):22131-4
[3]Luther KB, Hulsmeier AJ, Schegg B, Deuber SA, Raoult D, Hennet T (2011) Mimivirus
collagen is modified by bifunctional lysyl hydroxylase and glycosyltransferase enzyme. J
Biol Chem 286(51):43701-9

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# Team:BUCT-China/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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# Team:BUCT-China/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)