iGEM 2023 — Team liuan-shanghai

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# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Notebook

### 3.4 - 4.15

* Established an IGEM team and conducted online meeting every weeks
* Discussed and chose the overall direction of the project
* Selected team leaders and organized subsequent division of labor based on the strengths of each member
* Learn theoretical knowledge related to synthetic biology together

### 4.15 - 7.13

* Conduct desk research to understand the current market demand and existing related pure natural polymer materials in the market
* Based on the current resistance of people to antibiotics, it is proposed to introduce antimicrobial peptides into materials and endow them with antibacterial properties
* Review literature to find suitable protein expression systems and appropriate antimicrobial peptides

### 7.14

* Went through the local laboratory’s safety protocol and iGEM official safety guideline
* Constructed plasmids containing CP1 gene
* Used sodium carbonate solution to steam the silk to wash away the silk gelatin protein
* Dried the separated silk protein

### 7.15

* Constructed plasmids containing CP1 gene
* Used sodium carbonate solution to steam the silk to wash away the silk gelatin protein
* Dried the separated silk protein
* Degummed silk into the ternary solution stirring and dissolution for 3h after filtration
* Dialysed the filtered solution

### 7.16

* Imported plasmids into E.coil to construct recombinant expression strains
* Unsuccessful in obtaining recombinant expression strains
* Used sodium carbonate solution to steam the silk to wash away the silk gelatin protein
* Dried the separated silk protein
* Degummed silk into the ternary solution stirring and dissolution for 3h after filtration
* Dialysed the filtered solution

### 7.17

* Designed a cell-free expression system
* Successfully expressed CP1 antimicrobial peptide
* Used sodium carbonate solution to steam the silk to wash away the silk gelatin protein
* Dried the separated silk protein
* Degummed silk into the ternary solution stirring and dissolution for 3h after filtration
* Dialysed the filtered solution

### 7.18

* SDS-PAGE and WB blot were used to verify the protein expression level
* Found that the amount of expression was too small, and the gap between the equivalent amount and the actual use was too large, so the antimicrobial peptide was abandoned

### 7.19

* Reviewed the literature and started to use M2, an antimicrobial peptide that is toxic only after enzymatic cleavage.
* Constructed a plasmid containing the M2 gene
* Conducted field research and distributed questionnaires for interviews

### 7.20

* Imported the plasmid containing the M2 gene into E. coli to construct a recombinant expression strain
* Visited a number of fruit and vegetable supermarkets to conduct interviews

### 7.21

* Added IPTG to induce the recombinant strain to express the antimicrobial peptide
* Visited a number of fruit and vegetable supermarkets to conduct interviews

### 7.22

* Lysed the bacteria
* Purified by affinity chromatography
* Used OD600 detection to check bacterial inhibitory activity
* Soaked freshly cut apple lettuce with different concentrations of silk protein and other common preservatives
* Controlled experiments were carried out
* The weight loss of fruits and vegetables was measured daily

### 7.23

* The weight loss of fruits and vegetables was measured daily
* Did microbiological analysis to determine the growth of bacteria on fruits and vegetables
* Interviewed professors from related industries
* Made a promotion video
* Drew the logo of the team
* Promoted the team through bilibili, Xiaohongshu and other channels

### 7.24

* The weight loss of fruits and vegetables was measured daily
* Did microbiological analysis to determine the growth of bacteria on fruits and vegetables
* Interviewed professors from related industries
* Made a promotion video
* Drew the logo of the team
* Promoted the team through bilibili, Xiaohongshu and other channels

### 7.25

* The weight loss of fruits and vegetables was measured daily
* Did microbiological analysis to determine the growth of bacteria on fruits and vegetables
* Made a promotion video

### 7.26 - 8.15

* made presentation video
* designed wiki pages

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# Engineering Success

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.

#### Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page)
for your team to be eligible for this criteria.

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Please see the [2023 Medals Page](https://competition.igem.org/judging/medals) for more information.

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project.

## What should this page contain?

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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).

## Inspirations

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* [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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# Experiments

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Construction of recombinant expression strains

(1)Melt the sensory cells on ice, add the plasmid into the sensory cells, flick the wall of the tube to mix, and leave it on ice for 30min.

(2)Heat-excite at 42℃ for 1min30s, incubate on ice for 1min30s.

(3)Prepare LB medium (total volume 1L): weigh 10g Tryptone, 5g Yeast Extract, 10g NaCl dissolved in ultrapure water, and then fixed to 1L, autoclaved for 20min, stored at 4℃, and add 20g Agar when spreading LB plate.

(4)Receptor cells were added into 800μL LB medium, resuscitated at 250rpm, 37℃ for 1h. After centrifugation at 3500rpm, 4min, part of the supernatant was discarded, and the plate was resuspended and coated (the plate
contained kana antibiotic), and incubated at 37℃ overnight.

(5)Pipette 5 mL of LB liquid medium into a shaker tube and add 5 μL of Kana.

(6)Use a pipette gun to pick a single strain from the strain medium

(7)The tip of the picked strain was pumped into the LB liquid medium and incubated at 250rpm and 37℃ for 8h.

Induction of expression

(1)Cultivate the strain for 20h in advance

(2)Prepare TB medium: total volume 1L, pH=7.4

Tryptone 12g; Yeast extract 24g; Glycerin(50% glycerol) 8mL; KH2PO4 2.31g; K2HPO4 12.54g.

(3)Antibiotic:medium = 1:1000

1L TB medium with 20mL of bacterial solution, add 1mL of antibiotics

Shake on shaker for 3h

(4)Add 100 μL IPTG, shake the bed for one night

Purification of protein

(1) Centrifuge at 4000rpm, 15min, 4℃ to collect the induced bacterium.

(2) Add buffer (20mM NaH2PO4, 500mM NaCl, 0.5mM Imidazole, pH=7.5) to suspend the bacterial precipitate.

(3) Use a high-pressure homogeniser to break up the organisms.

(4) Add buffer (20 mM NaH2PO4, 500 mM NaCl, 0.5 mM Imidazole, 8 M Guanidine hydrochloride, pH=7.5) at 1:1 volume ratio and mix.

(5) Centrifuge at 125000rpm, 1h20min, 4°C to collect the supernatant.

(6) The supernatant was filtered using 0.45 μm filter membrane.

(7) Use Ni column to purify the protein:

① Wash the Ni column with ddH2O;

② Equilibrate the Ni column with buffer (20 mM NaH2PO4, 500 mM NaCl, 6 M Guanidine hydrochloride, 0.5 mM Imidazole, pH=7.5);

(iii) Add the supernatant after filtration and repeat once;

④ Wash out the heteroprotein with buffer (20 mM NaH2PO4, 500 mM NaCl, 6 M Guanidine hydrochloride, 30 mM Imidazole, pH=7.5);

⑤ Elution of antimicrobial peptides with buffer (20 mM NaH2PO4, 500 mM NaCl, 6 M Guanidine hydrochloride, 1 M Imidazole, pH=7.5);

(6) Dialysis of protein solution using 3.5 kDa dialysis bag (12h)

(8) Collect protein: centrifuge at 10,000rpm, 10min, 4°C and keep the precipitate.

(9) Enzymatic digestion: take part of the precipitate and add 3mL of sterile water for suspension. Take 100μL of suspension solution, add 5μL of HRV 3C and leave it at 4℃ for 12h. freeze-dry.

(10) Add a small amount of 40% acetonitrile, 1‰ TFA, mix well, centrifuge at 21000rpm, 20min, 4℃, retain the supernatant.

(11) Further purify the protein using HPLC (flow rate 2mL/min)

(12) Remove the solution at the target peak position, rotary evaporate acetonitrile at room temperature (1h30min) and freeze-dry. The purified mature antimicrobial peptide was obtained. (Antimicrobial peptide with His tag can
be obtained in the same way as above, omitting the enzyme cutting step.)

Calibration of CFU-OD600 quantitative relationship

(1) Cultivate E. coli DH5α to logarithmic growth stage.

(2) Gradient dilution of bacterial solution to 10-9, each dilution of 100μL coated plate, each three. 37 ℃ culture 24hr, take the number of colonies between 30-300 plate count.

(3) Determine the absorbance at 600nm of each dilution concentration of bacterial solution.

(4) Construct quantitative relationship according to CFU and OD600 value.

6、OD600 value method to measure antibacterial activity:

(1) In a 96-well plate, 180 μL of E. coli DH5α bacterial solution was added, and mature antimicrobial peptide, deionised water, and 12.5 μg/μL Kana were added sequentially, and the final volume was kept consistent in each
well, and the volume shortage was made up with deionised water. The final OD600 value of the bacterial solution in each well was 0.0005, and the concentration of antimicrobial peptide was 5 mM, 1 mM, 500 μM, and 100 μM in
order. repeated twice.

(2) The 96-well plate was incubated in 37℃ incubator at 220rpm, and the OD600 value of each well was measured every 2h.

Inhibition circle method:

(1) Add deionised water to the mature antimicrobial peptide to a final concentration of 10 mM, and dilute it to 5 mM, 1 mM, 500 μM and 100 μM in a gradient;

(2) Mix E. coli DH5α bacterial fluids grown to logarithmic growth stage into LB solid medium cooled to 50-60°C (OD600 final concentration of 0.005) to obtain plates with uniform bacterial distribution;

(3) Draw a line on the plate to divide the area, stick a sterilised filter paper sheet in the centre of the area, take 10 μL of gradient diluted antimicrobial peptide solution, deionised water and 12.5 μg/μL Kana from each of
them and add them dropwise on the filter paper sheet, wait until the solution is slightly dry, and then put the plate inverted and incubate it in 37℃ incubator for 12h.

(4) Analyse the diameter of the circle of inhibition.

Combination of AMP and silk protein

a degumming

4L of water to boil, add 20 grams of sodium carbonate, add 20 grams of cocoons, cook for 30 minutes, fish out and wash with water, repeat the above steps (a total of two times)

Oven 60 ℃, drying 12 hours

Principle: silkworm cocoons contain 75% of the silk protein and 25% of the silk gelatin protein, the purpose of degumming is to remove the silk gelatin protein.

↓

b dissolve silk

with ternary solution (calcium chloride: ethanol: water molar ratio equal to 1:2:8, respectively, 58 g, 60.8 ml and 74 ml)

solution at 70 ℃, add torn to the fluffy state of degummed silk, stirring to dissolve for three hours

Filtration through 4 layers of gauze

Principle: The dissolution process destroys the original regular structure of the silk protein hydrogen bonding network, resulting in conformational changes from β-folding to random curls, the molecular chain to the
conformational existence of the random nematic and thus in the dissolved state. ↓

c dialysis

The solution is filtered, poured into a dialysis bag and dialysed for three days, reverse dialysis for 8 hours

Centrifugation, 9600 RPM, 4°C, 30 min. Take the supernatant, measure the concentration, and store it in the refrigerator at 4℃.

Purpose: to remove soluble salts from the solution

↓

d soak freshly cut apple lettuce

Apple slices:

2%wt silk protein solution (5% silk protein 100 g in 150 ml water)

2%wt silk protein solution with calcium chloride (5:1) (same conditions as above, with 1.3875 g calcium chloride)

uncoated

Morpholine fatty acid salt fruit wax

Silk protein solution plus nisin antimicrobial peptide (30mg/g)

Lettuce slices:

5%wt silk protein solution

2%wt silk protein solution with calcium chloride (5:1)

uncoated

Morpholine fatty acid salt fruit wax

silk protein solution plus nisin antimicrobial peptide (30mg/g)

↓

eTest for weight loss, photograph

Loss of weight = (mass before storage - mass after storage) ÷ mass before storage × 100 per cent

Determine the loss of water by measuring the weight loss rate, colour to see how fresh it is

↓

f infrared characterisation

Infrared spectroscopy → silk protein numerous complex amide bands

Amide I (C=O stretching vibration, C-N stretching vibration and N-H plane bending)

Amide II (N-H plane bending and C-N stretching vibration)

Amide III (C-N stretching vibration and N-H plane bending)

FTIR absorption peaks can be used to determine the relative content of different conformations.

β-folding ↑ Freshness ↑

↓

gMicrobiological analysis

PCA plate count agarose, autoclaved, poured onto plate and cooled

10 ml of decontaminated water (dd water) was added to fruits and vegetables, shaken on shaker for 10 minutes

900 µl of dd water added to individual centrifuge tubes

100 µl of stock solution added to tube 1, shaken well

100 µl of tube 1 is added to the next tube and shaken well.

Repeat until the 6th centrifuge tube (10⁶)

Generally use a solution with a dilution of 10⁴ to 10⁶

Take 50 microlitres of each and add it to 6cm medium to coat well (100mLfor9cm)

30 < colony number < 300 Available

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