iGEM 2021 — Team Yucai_SZ

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Yucai SZ/Notebook

模板！

* Team
  + [Members](https://2021.igem.org/Team%3AYucai_SZ/Team)
  + [Attributions](https://2021.igem.org/Team%3AYucai_SZ/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AYucai_SZ/Collaborations)
* Project
  + [Description](https://2021.igem.org/Team%3AYucai_SZ/Description)
  + [Implementation](https://2021.igem.org/Team%3AYucai_SZ/Implementation)
  + [Engineering success](https://2021.igem.org/Team%3AYucai_SZ/Engineering)
  + [Measurement](https://2021.igem.org/Team%3AYucai_SZ/Measurement)
  + [POC](https://2021.igem.org/Team%3AYucai_SZ/Proof_Of_Concept)
  + [Contribution](https://2021.igem.org/Team%3AYucai_SZ/Contribution)
  + [Prospect](https://2021.igem.org/Team%3AYucai_SZ/prosepect)
* [Experiment](https://2021.igem.org/Team%3AYucai_SZ/Experiments)
  + [Design](https://2021.igem.org/Team%3AYucai_SZ/design)
  + [Protocol](https://2021.igem.org/Team%3AYucai_SZ/protocol)
  + [Parts](https://2021.igem.org/Team%3AYucai_SZ/Part_Collection)
  + [Result](https://2021.igem.org/Team%3AYucai_SZ/Results)
  + [Notebook](https://2021.igem.org/Team%3AYucai_SZ/Notebook)
* Model
  + [Expression model](https://2021.igem.org/Team%3AYucai_SZ/Model)
* HP
  + [iHP](https://2021.igem.org/Team%3AYucai_SZ/iHP)
  + [Communication](https://2021.igem.org/Team%3AYucai_SZ/Science_communication)
* [Awards](https://2021.igem.org/Team%3AYucai_SZ/Awards)
* [Achievement](https://2021.igem.org/Team%3AYucai_SZ/Achievement)
* [Home](https://2021.igem.org/Team%3AYucai_SZ)

![](https://static.igem.org/mediawiki/2021/0/0c/T--Yucai_SZ--notebook.png)

### 2021.8.5

Prepare competent cells

1) Activation of bacteria: 5 mL LB was prepared the night before without antibiotics, inoculated at 1:100, and incubated overnight (less than 16 hours) at 37 ° C. At the same time, a tube of antibiotics was added to see whether bacteria grew (let bacteria revive and enter the early and middle stages of logarithmic phase). It's easier to make competent cells

2) The next morning, transfer it into 100mL medium with a density of 1:2000-500 and culture for 2h (the naked eye can see slightly cloudy, or put the palm on the bottom of the bottle, the palm lines are blurred, rather than too low density culture)

3) Prepare ice boxes and vertically insert multiple 50mL centrifugal tubes for pre-cooling. In the ultra-clean platform, the bacteria liquid was transferred to a 50m pre-cooled centrifugal tube for ice bath for 20min. Then, centrifugation was performed at 4000 RPM at 4℃ for 10 min.

4) After centrifugation, the supernatant was poured out of the ultra-clean platform, and 5mL of pre-cooled 0.1-m cacl2-MgCl2 solution was added to each tube with repeated blowing and suction with the head of a gun. The mud was re-suspended and placed in ice for 10min, followed by centrifugation at 4000 RPM at 4℃ for 10min.

5) After centrifugation, pour off the supernatant in the ultra-clean platform, add 2mL of pre-cooled 0.1m cacl2-glycerin solution to each tube, blow and suck it repeatedly with the head of a gun, re-suspend the mud, and put it in ice for 10min

6) after the ice bath, divide the competent cells into 1.5mL EP tubes, 100uL for each tube, and immediately put the tubes on ice.

7) End of packing. The competent cells were placed in a ziplocked bag, labeled and stored in a refrigerator at -80℃.

## 2021.8.6

Calculate the dosage of various drugs

Ampicillin: 0.1g/ mL 5ml (refrigerated)

IPTG: 24mg/ml=100mM 5ml (refrigerated)

M63 5× (refrigerated, not sterilized for use) : 500ml

LB culture medium and LB culture gene autoclave are faulty and need to be re-matched

## 2021.8.10

The following systems are prepared:

LB liquid medium 500ml (sterilized, refrigerated after use)

LB solid medium 250ml (sterilized), add 250U ampicillin, find the medium solidified, microwave heating to melt again, add 215U ampicillin, flip the plate again, select the best two plates reserved.

M63 1x 400ml (sterilized, purchased a pack of powder and added water to 400ml, no glucose, ampicillin added)

Glycerin 50% (stored at room temperature) sterilization (120℃, 20min)

Thawing of receptive state and plasmid (CP MFP)

3U plasmids were transferred into 5α competent cells, respectively, and coated on the above reserved LB plate. The cells were placed upside down overnight at 37℃ and cultured in a biochemical incubator

## 2021.8.11

Check the culture medium, MFP bacteria colony is not obvious, so it is reformulated
LB 250mL was prepared and AGAR 3.75g was added

Autoclave for 20min

MgSO4 1000X (0.6g 5ml)

Preparation of glucose solution 100x (2g 10ml)

Amp package added upside down on ultra-clean table (stored upside down in refrigerator)

Pick bacteria, add to 3ml LB, add to shaker culture overnight

Again, the plasmid is transferred into the receptor cell,
They were coated on the above-mentioned reserved LB plates containing Amp and placed upside down overnight at 37℃ to be cultured in a biochemical incubator

## 2021.8.12

Observe the plate in culture, and observe the blue (small) and white (large) colonies in the receptive state of CP.

Many colonies were observed on plates transferred to MFP

1L and 100mL of TAE 1x were prepared

50x tae 20ml+ddH2O980ml

50x tae 2ml+ddH2O98ml

Find the single colony suitable for picking bacteria and mark it. The labeled colonies were lightly picked on the surface of the culture medium with the tip of a small gun in the ultra-clean stage. The tip of the gun was injected into 500 µ l of LB medium containing Amp. Put them into the shaker for 3h culture (two CP [one white and one blue marked], one MFP {no label, marked as 0})

Later, five colonies (numbered as CP1-5 MFP1-5) were selected from CP (all blue colonies) and MFP respectively, and 500U LB culture medium containing Amp was injected. Incubate in a shaker for 8 hours

Take 1μL of each of the two activated bacterial solutions (CP Blue 0 MFP0), add reagents according to the table (error: unsterilized spear head was used), conduct PCR, and set the parameters as follows: 94℃ pre-denaturation for 4min, 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 55s, denaturation, annealing and extension step cycle 30 times; Finally, it was heated for 5min at 72℃ and cooled to 16 ℃.

![](https://static.igem.org/mediawiki/2021/8/8b/T--Yucai_SZ--JJu99.png)

Preparation of agarose gel solution :(1%30ml 0.3g agarose and 30ml 1xtae) turn the microwave oven to medium-high fire, boil the solution for 7 times during the process (observe before microwave when heating, take out and shake well once boiling, then put back to heat), the solution from cloudy to clear, add 3uDNA dye, shake well, and pour into the gelatinizing tank for solidification (20min).

Note: add a little more water (about 1ml) to the configuration, because boiling will cause water evaporation and loss of error
16:20

Electrophoresis: DNA Marker 5uL CP and MFP bacterial liquid PCR samples 8uL each

5μL of 2K DNA Marker (1KB DNA Ladder) and 8μL of PCR products were successively added into the upper sample Wells of the prepared agarose gel, and then electrophoresis was performed at 120V for 40min. (Negative pole - positive pole)

![](https://static.igem.org/mediawiki/2021/e/e2/T--Yucai_SZ--Junn812.png)

The exposure time of uv observation was 800ms

Then 30uL +lb3mL was inoculated at a ratio of 1:100 and cultured in a 50mL centrifuge tube. Cp0 plus IPTG inducer (100mM)15uL (final concentration should be 0.5mm), MFP no.5. Put them together in 210RPM shaker and incubate overnight at 37℃.

Bacteria-preserving CP and MFP were stored with 470uL glycerol (1:1) in a refrigerator at -20℃

## 2021.8.13

•The CP (+IPTG) of the night was taken out for observation and no obvious characterization was found. Cp1, which was later selected for insurance, had obvious blue color, while others had vague blue color

•Cp (+IPTG) was observed after long standing, and the sinking mud was white. And cp1 fungus mud is blue.

•Overnight MFP bacteria were inoculated at 1:100 on M63 medium (20mlM63 20.2umGSO4 20.4U glucose 20.7AMP 200U bacteria; Iptg101.8u) (served in petri dishes)

•Repeat the previous step to configure 3 centrifuges packed in 50ml tubes (placed in biochemical incubator without shock culture)

•Set two blank controls (without IPTG, cultured in a petri dish in a biochemical incubator)

•Develop 24-hour tomorrowwatch

In order to explore whether CP white colonies are the bacteria we need, repeat PCR of white colonies (compared with MFP) and run glue (130V 30min) [5000KB marker]

![](https://static.igem.org/mediawiki/2021/e/e7/T--Yucai_SZ--Jun813.png)

Exposure time: 1000ms

It was observed that the PCR results of CP white and CP blue were consistent, indicating different reasons to be analyzed

Cp3, 4, 0(white) and MFP1, 2 were 1:1 added with 50% glycerol and put into -20℃ refrigerator to preserve bacteria

Cp1, 2, 5 and MFP3, 4, 5 in 4℃ refrigerator (reduce activity)

## 2021.8.14

It was observed that CP0 white, CP3 and CP4 all showed blue characterization. MFP (M63) was relatively clear and had no obvious characterization

50mL 1M HCl was mixed with 2.4m HCl

With the 1 m NaOH

0.1wt/vol crystal violet solution

-0.1g crystal violet + 100mL water

## 2021.8.16

In the morning,the bacteria in the cp large test tube did not have blue characterization, while the bacteria in the small centrifuge tube had blue characterization

The petri dish of MFP is vaguely sticky.

Start double digestion. With two double digestion systems:

![](https://static.igem.org/mediawiki/2021/3/3c/T--Yucai_SZ--Junna.png)
![](https://static.igem.org/mediawiki/2021/3/3c/T--Yucai_SZ--Junna.png)

Enzymatic digestion at 37 ° for 20min on PCR instrument

Run electrophoresis 130V 30min+10min (using 5KB marker)

add loading buffer!! (After loading buffer is added, it is pumped evenly with a gun)

![](https://static.igem.org/mediawiki/2021/2/2d/T--Yucai_SZ--Jun816.png)

There should be two strips in the left column that do not meet expectations
But still cut the glue

Plastic recycling:

The colloidal CP glue weight 0.044g MFP glue weight 0.04g

Add PE sol solution (triploid product, glue 0.1g as 100U) CP134U MFP120U metal bath at 25℃ for 10min
25mlPW+ anhydrous ethanol solution

The solution was added to the CA5 adsorption column (the adsorption column was placed in the collection tube) and stood at room temperature for 2min

Centrifuged at 12000rpm for 45s, the waste liquid was dumped and the adsorption column was put into the collection tube

Add 600uPW+ anhydrous ethanol, centrifuge at 12000rpm for 45s to drain the waste liquid, and put the adsorption column into the collection tube (twice)

Centrifuge at 12000rpm for 2min, and dry

Change the centrifugal tube, add buffer TB50ul to the adsorption film, place at room temperature for 2min, centrifuge at 12000rpm for 2min, collect DNA solution

Experimental results: The plasmid containing MFP gene ran gel electrophoresis results only one strip
To verify whether the results do not meet expectations due to experimental operation errors

Double digestion was carried out again, and the total system was 50U according to the instructions
Enzymatic digestion at 37 ° for 20min on PCR instrument

![](https://static.igem.org/mediawiki/2021/1/19/T--Yucai_SZ--JJu1.png)![](https://static.igem.org/mediawiki/2021/f/f2/T--Yucai_SZ--JJu2.png)

The result is the same as last time...

Analysis may be the enzyme inactivation or plasmid restriction site problems (dangerous (# 'O'))
The cut glue is stored in the refrigerator at 4℃

Contacted Dr. Wang Yanyi to ask about MFP biofilm, and decided to take a petri dish for crystal violet staining tomorrow

![](https://static.igem.org/mediawiki/2021/6/65/T--Yucai_SZ--ihpw2.png)

### 2021.8.17

Recycle the glue again like yesterday's steps (stored at 4℃ yesterday)

Plastic recycling:

The colloidal CP glue weight 0.044g MFP glue weight 0.04g

Add PE sol solution (triploid product, glue 0.1g as 100U) CP134U MFP120U metal bath at 25℃ for 10min
25mlPW+ anhydrous ethanol solution

The solution was added to the CA5 adsorption column (the adsorption column was placed in the collection tube) and stood at room temperature for 2min

Centrifuged at 12000rpm for 45s, the waste liquid was dumped and the adsorption column was put into the collection tube

Add 600uPW+ anhydrous ethanol, centrifuge at 12000rpm for 45s to drain the waste liquid, and put the adsorption column into the collection tube (twice)

Centrifuge at 12000rpm for 2min, and dry

Change the centrifuge tube, add buffer TB50ul to the adsorption film, place at room temperature for 2min, centrifuge at 12000rpm for 2min, collect DNA solution [The blue mark on the side wall of the centrifuge tube is 2, CP6ng /uL mFP7.6ng /uL, store in the refrigerator at -20℃]

1.Single digestion verification (verify whether the enzyme is inactivated)
With 2 enzyme digestion systems:

![](https://static.igem.org/mediawiki/2021/5/57/T--Yucai_SZ--JJu3.png)

(Add 2μL 6×loading buffer when running gel)

 ![](https://static.igem.org/mediawiki/2021/9/99/T--Yucai_SZ--JJu4.png)

(Add 2μL 6×loading buffer when running gel)

Enzymatic digestion at 37 ° for 20min on PCR instrument

![](https://static.igem.org/mediawiki/2021/7/78/T--Yucai_SZ--JJu5.png)

Note: Don't forget to add plasmids ！！！！！！！！！！ (; '⌒ `)
2.Crystal violet staining test

Remove supernatant and rinse 3 times with 3mlddH2O

+CV solution 10mL staining for 15min

When the dye +ddH2O was poured out for several times, the bottom layer of the medium could not be rinsing, indicating that mucin was formed and mineralization could begin

![](https://static.igem.org/mediawiki/2021/6/64/T--Yucai_SZ--Jun1.png)![](https://static.igem.org/mediawiki/2021/e/e4/T--Yucai_SZ--Ju11.png)![](https://static.igem.org/mediawiki/2021/1/19/T--Yucai_SZ--Ju12.png)

3:00 PM SBF mineralization

One petri dish with and without IPTG was reserved as the control, and the rest of the supernatant was poured out and added with about 11mL SBF solution. Culture in biochemical incubator at 37℃.

Run electrophoresis 130V 30min+10min（5kb marker）

![](https://static.igem.org/mediawiki/2021/9/9c/T--Yucai_SZ--Ju13.png)

### 2021.8.18

In the morning

Single enzyme 1 h

Run glue

![](https://static.igem.org/mediawiki/2021/0/05/T--Yucai_SZ--Ju14.png)

In the afternoon
The system with double digestion is as follows

![](https://static.igem.org/mediawiki/2021/4/4e/T--Yucai_SZ--JJu6.png)![](https://static.igem.org/mediawiki/2021/2/2f/T--Yucai_SZ--JJu7.png)

run

![](https://static.igem.org/mediawiki/2021/6/64/T--Yucai_SZ--Ju15.png)

Plasmid plasmid CP MFP 50ul+5mlLB (Amp included)
Cp1 300U +3mlLB (including Amp)

Shaker culture at 37℃ overnight

Double enzyme cutting glue refrigerator preservation [Date marked]

### 2021.8.19

Plasmid extraction (two tubes of CP and MFP, each with 1ml bacteria)
PWT+ 50mL anhydrous ethanol was configured

RNaseA and TIANRed were added to P1

1ml bacterial solution 12000rpm 1min, and the supernatant was sucked out

Add 150uP1 and re-suspend the fungus mud

Add 150uP2 and gently flip it up and down 7 times to change color to clear purple

After adding 350uP5, mix it up and down quickly for 12-20 times, turn yellow and flocculent precipitation appears at 12000rpm for 2min

Transfer the suction supernatant to adsorption column CP3 (adsorption column into the collection tube) 12000rpm 30s

Dump the waste liquid, add 300uPWT 30s to the adsorption column, and dump the waste liquid
12000rpm 1min (remove the residual bleaching solution)

The adsorption column was placed in a clean collection tube, and eluting buffer TB (CP1, CP, 2 a senior student added 60U MFP1, MFP2WR added 50U) was dropped to the center of the adsorption film for 12000rpm 30s
Drop adsorption column

Plasmid concentration was measured
Cp1 (34.9ng/ul), CP2 (33.2ng/ul), MFP1 (59.4ng/ul), MFP2 (78.6ng/ul)

### 2021.8.20

In the morning
MFP PCR system is as follows

![](https://static.igem.org/mediawiki/2021/3/3c/T--Yucai_SZ--JJu8.png)

pcr

![](https://static.igem.org/mediawiki/2021/6/69/T--Yucai_SZ--Junn8201.png)

In the afternoon
Recycle according to instructions

MFP single enzyme digestion (X enzyme)/ double enzyme digestion system

![](https://static.igem.org/mediawiki/2021/2/24/T--Yucai_SZ--JJu9.png)![](https://static.igem.org/mediawiki/2021/3/3a/T--Yucai_SZ--JJu10.png)

Enzyme digestion at 37 ° for 1h and inactivation at 80 ° for 20min

Run rubber 40 min ~ ~

The DNA of morning PCR had no restriction sites

Therefore, the energy p can be exchanged to re-pcr the primers of the restriction site

50 ul distribution system

![](https://static.igem.org/mediawiki/2021/5/50/T--Yucai_SZ--JJu11.png)

PCR was followed by gel running (one part), purification of the other part and preservation at -20℃
Cut cp again

![](https://static.igem.org/mediawiki/2021/c/cc/T--Yucai_SZ--Jun8202.png)![](https://static.igem.org/mediawiki/2021/6/6a/T--Yucai_SZ--JJu12.png)

recycle

### 2021.8.21

Two Gibson systems are prepared

![](https://static.igem.org/mediawiki/2021/0/0e/T--Yucai_SZ--Ju18.png)

The PCR apparatus was incubated at 50 ℃ for 30min

Two tubes of DH 5α receptor cells were co-transformed

### 2021.8.23

![]()

With a PCR system (fluorescent protein)

![](https://static.igem.org/mediawiki/2021/a/ae/T--Yucai_SZ--JJu13.png)

PCR data extension time was changed to 75s

With a double digestion system (blue light)

![](https://static.igem.org/mediawiki/2021/9/9b/T--Yucai_SZ--JJu14.png)

Enzyme digestion was performed at 37℃ in water bath and then transferred to a centrifugal tube for inactivation at 80℃ for 20min in metal bath

PCR products and enzyme digestion products were stored in the refrigerator at -20℃

The bacteria selected no. 22 were inoculated at 1:100 (30U bacteria, 3mlLB) and shaken overnight

### 2021.8.24

In SBF
Purified yesterday's PCR product
Enzyme MFP

![](https://static.igem.org/mediawiki/2021/1/11/T--Yucai_SZ--JJu15.png)

run

![](https://static.igem.org/mediawiki/2021/8/8e/T--Yucai_SZ--Ju19.png)

The PCR MFP concentration was 50.8ng/ul

The cp concentration of glue recovery was 6.2ng/ul

The concentration of CP was 23.1ng/ul

The T4 connection system is equipped according to the instruction

![](https://static.igem.org/mediawiki/2021/7/71/T--Yucai_SZ--JJu16.png)
![](https://static.igem.org/mediawiki/2021/8/84/T--Yucai_SZ--JJu17.png)

Store overnight at -20°C

Chloramphenicol 25mg/ml 4ml (0.1g chloramphenicol 4ml anhydrous ethanol) was divided into 4 small centrifuge tubes

[Note: Chloramphenicol is configured with anhydrous ethanol]

Chloramphenicol 1:1000 LB was given

Pour on the plate

### 2021.8.25

T7 and GFP plasmids were extracted

PCR run

![](https://static.igem.org/mediawiki/2021/6/63/T--Yucai_SZ--Ju20.png)

87/5000

Crystal violet staining 8.22 plate of MFP and cp

The staining effect of both plates is not good

In the afternoon

Purify the morning plasmid

Mfp SBF pour water and dry at 60°C for 24h

Gibson: T7, GFP and Blue light plasmid

![](https://static.igem.org/mediawiki/2021/0/07/T--Yucai_SZ--JJu18.png)

T4 connection cp+ MFP, P3K3
Coconversion

Colony PCR (Gibson-linked CP + MFP)

![](https://static.igem.org/mediawiki/2021/b/ba/T--Yucai_SZ--JJu19.png)

Wrong downstream primer!!

### 2021.8.26

Pick out 4 BL+T7 strains

3 CP + MFP strains connected by T4

BL+GFP four, P3K3 three

### 2021.8.27

The bacteria selected yesterday were inoculated with 30U bacteria 3mlLB at 1:100

Take the remaining 200u bacteria 1:1 and add 50% glycerol retaining bacteria into the same ziplocked bag at -20℃ and write down the strains

### 2021.8.28

New primers are in. Re-pcr 11 tube（enzyme cp+mfp1、2、3、4 T4 cp+mfp1、2、3）

![](https://static.igem.org/mediawiki/2021/2/2c/T--Yucai_SZ--JJu20.png)

Extension time changed to 45s and other unchanged
run（marker 胶1、2、3、4 enzyme1、2、3、4 T4 1、2、3）

![](https://static.igem.org/mediawiki/2021/6/69/T--Yucai_SZ--Ju21.png)

Enzyme 3 T4 1, 2 and 3 with obvious bands were selected for plasmid extraction

extract the plasmid enzyme3 T4 1、2、3 T7+BL1、2、3、4 P3K3 1、2、3

![](https://static.igem.org/mediawiki/2021/f/f8/T--Yucai_SZ--JJu21.png )

Put the date in the fridge at -20℃

### 2021.8.29

A double anti LB plate was poured into 5 plates and the remaining LB(no antibiotics) was stored at room temperature

BL+T7 and CP + MFP co-transformation (BL21 receptor cells have T7 enzyme, can not be used, using the 5α sensible state (without T7)

### 2021.8.30

Recoconversion

### 2021.8.31

Pick the bacteria and number 1① 1② 1③ 2① 2② 2③ 3① 3② 3③ 4① 4② 4③

Wrap the culture in aluminum foil

### 2021.9.2

Bacteria 2② were inoculated at 1:100 for 1.5h at 7:30:100 and inoculated on M63 medium 20mlM63

20.2umGSO4 20.4U glucose 20.7AMP and Cm 200U; Iptg101.8u) blue light irradiation culture

### 2021.9.6

20mlM63

 20.2uMgSO4

 20.4uGlucose

20.7Amp

20.7uCm

 200uBacteria;

IPTG101.8u
drew 2 pictures with CP

### 2021.9.7

drew another 2 pictures with CP
got another M63

### 2021.9.8

CV dyeing

![](https://static.igem.org/mediawiki/2021/d/d6/T--Yucai_SZ--Ju22.png)

### 2021.9.12

In order to improve the CP element, enzyme digestion is required

![](https://static.igem.org/mediawiki/2021/9/9b/T--Yucai_SZ--JJu22.png)
![](https://static.igem.org/mediawiki/2021/5/57/T--Yucai_SZ--JJu23.png)
![](https://static.igem.org/mediawiki/2021/3/34/T--Yucai_SZ--Jun912.png)

Above is the PCR product of CP

![](https://static.igem.org/mediawiki/2021/6/65/T--Yucai_SZ--JJu24.png)![](https://static.igem.org/mediawiki/2021/c/c3/T--Yucai_SZ--JJu25.png)![](https://static.igem.org/mediawiki/2021/4/4b/T--Yucai_SZ--Ju24.png)

recycle

### 2021.9.13

Change cp to GFP

The fragment was recovered by ligase T4

![](https://static.igem.org/mediawiki/2021/8/89/T--Yucai_SZ--JJu26.png)

### 2021.9.14

The strain was cultured overnight in liquid LB medium.

### 2021.9.15

The bacteria solution was added into the fresh LB medium (containing IPTG) at 1:200 and cultured at 220 RPM at 37 ℃ for 8 hours.

The 200 µ l culture was transferred to a 96-well plate and the 485/510 fluorescence and OD600 were measured using a microplate reader. The ratio of fluorescence value to OD600 was used to indicate the expression level of sfGFP (vertical axis in the figure below).

![](https://static.igem.org/mediawiki/2021/0/09/T--Yucai_SZ--Jun555.png)

### 2021.9.27

Changed the sensor system

The cotransferees were connected to two 600U LB (including Amp cm) tubes at 1:100. One tube wrapped in tin foil, one tube not wrapped. Put them together on the shaker.

Then connect 4 M63 20mL disks, of which Amp CM glucose and MgSO4 were added 20U each in two disks and Amp glucose and MgSO4 were added 20U each in the other two disks

Two plates of CP bacteria were transferred at 1:100, respectively

One of the plates of cotransferrable bacteria was wrapped in cut-out paper and illuminated with blue light in a biochemical incubator

### 2021.9.30

M63 was dumped and added to 10mlSBF for mineralization

The two tubes were centrifuged 12000rpm for 1min to observe and compare

![](https://static.igem.org/mediawiki/2021/1/15/T--Yucai_SZ--Juna.png)

The left side is not hiding from light and the right side is completely hiding from light

### 2021.10.2

Replace the SBF

### 2021.10.4

Replace the SBF

### 2021.10.6

Replace the SBF

### 2021.10.8

Drain the SBF and dry it

![](https://static.igem.org/mediawiki/2021/b/b2/T--Yucai_SZ--Junb.png)

Cotransferrable bacteria are on the left and CP bacteria are on the right

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Yucai SZ/Engineering

模板！

* Team
  + [Members](https://2021.igem.org/Team%3AYucai_SZ/Team)
  + [Attributions](https://2021.igem.org/Team%3AYucai_SZ/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AYucai_SZ/Collaborations)
* Project
  + [Description](https://2021.igem.org/Team%3AYucai_SZ/Description)
  + [Implementation](https://2021.igem.org/Team%3AYucai_SZ/Implementation)
  + [Engineering success](https://2021.igem.org/Team%3AYucai_SZ/Engineering)
  + [Measurement](https://2021.igem.org/Team%3AYucai_SZ/Measurement)
  + [POC](https://2021.igem.org/Team%3AYucai_SZ/Proof_Of_Concept)
  + [Contribution](https://2021.igem.org/Team%3AYucai_SZ/Contribution)
  + [Prospect](https://2021.igem.org/Team%3AYucai_SZ/prosepect)
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![](https://static.igem.org/mediawiki/2021/e/e3/T--Yucai_SZ--engineering_successed.png)

## 1 CsgA-Mfp3S-pep fusion protein

![](https://static.igem.org/mediawiki/2021/e/ef/T--Yucai_SZ--mfp3s.png
)

**Fig.1**CsgA-Mfp3S-pep sequence

## 1.1 Introduction

CsgA is a bacteriophage hair protein of Escherichia coli that assembles extracellularly into long hairs; Mfp3S is a mussel foot filament mucin; and pep can adsorb ions from the culture environment, which results in mineralization.We fused CsgA with Mfp3S-pep to form a new part.

The Mfp protein is the protein we selected from the [1] paper. The mussel foot protein type is considered to be a key protein for mussel adhesion in humid environments; it contains about 13 mol% DOPA and consists of about 80 repeats of a decapeptide shared sequence. Attempts have been made to produce recombinant mfp proteins in a variety of expression systems, including E. coli and yeast.

Here, inspired by functionally graded composites in nature, we developed active composites with precise spatial patterning and stepwise gradient control of mineral density by using light-induced biofilms and biomimetic mineralization in E. coli. We show that biofilm structural proteins in E. coli fused with mussel foot protein (Mfp) analogs confer engineered biofilms to promote HA mineralization and interfacial binding. When blue light induced strains are used to grow functional biofilms with controlled spatial and biomass densities, living mineralized materials with pattern and gradient characteristics can be generated following a benign biomimetic HA mineralization process.

In search of a good method to restore murals, we turned our attention to this gene.

Yucai\_SZ iGEM2021 plans to express this fusion protein module in engineered bacteria capable of fulfilling three requirements to design biofilms for pro-selective mineralization. First, the fusion protein will be tolerated by the secretion machinery of the curl system; second, it will have little effect on biofilm growth and will not disrupt the intrinsic self-assembly of adhesive fibers; and third, it will initiate HA mineralization while exhibiting strong interfacial adhesion to the mineralized HA layer. Previous studies have shown that Mfps exhibit strong adhesion to various surfaces and that they contain negatively and positively charged residues that are believed to promote HA mineralization.28 Among them are Mefp530 and Mfp3S peptide (Mfsp3S-pep; an Mfp3S analogue protein)27 which can initiate HA mineralization and promote interfacial adhesion as a potential protein module. A CsgA-Mfp fusion protein (aTc)-inducible system containing CsgA (the major protein component of the E. coli biofilm) and the C-terminal Mfp family of fusion domains was constructed under the modulation of anhydrous tetracycline.

![](https://static.igem.org/mediawiki/2021/7/71/T--Yucai_SZ--egr.png)

**Fig.2**CsgA and Mfp3S-pep[1]

csgA-Mfp3S and CsgA-Mfp3S-pep can be secreted from engineered cells . However, faster growth rates and higher biomass production were observed for the CsgA-Mfp3S-pep-secreting strain, whose biofilm biomass was similar to that of the CsgA control strain. The CsgA-Mfp3S-pep fusion protein was selected in the paper as a representative of the HA mineralization functional module. Results from in vitro and in vivo experiments performed in the thesis to compare CsgA-Mfp3S-pep and CsgA nanofibers under mineralization conditions in 1.5-fold simulated body fluids (SBF) indicate that while HA mineral deposition occurs in both systems, CsgA-Mfp3S-pep samples show a faster mineralization rate. Furthermore, examination of in vivo mineralization by TEM and selected area electron diffraction (SAED) confirmed that biofilms expressing CsgA-Mfp3S-pep triggered denser mineral formation with more pronounced crystalline features after 5 days of mineralization. Thus, these results highlight the role of Mfp3S-pep fusion protein in promoting HA mineral formation and crystallization. Quartz crystal microbalance (QCM) tests were performed to compare the interfacial binding of CsgA and CsgA-Mfp3S-pep to HA surfaces. The results showed that CsgA-Mfp3S-pep protein monomer and nanofibers adsorbed faster and stronger to the HA surface compared to their CsgA counterparts.

In this way we hope to use this to develop an efficient and low-cost method for restoration of murals.

## 1.2 Assay design

According to the findings of the above paper, fusing and expressing these three proteins allows the host to immobilize on a solid surface to form a biofilm and adsorb calcium ions to form a mineralized layer. To test the function of this protein, we placed the protein downstream of the T7 promoter, constructed an expression system on pSB1A3, transformed it into BL21(DE3) host bacteria, and performed induction tests.

## 1.3 Test results

![](https://static.igem.org/mediawiki/2021/b/b8/T--Yucai_SZ--rs.png)

**Fig.3**Graph of crystalline violet staining results

Its staining results proved the effect of having biofilm formation i.e. CsgA with Mfp3S. Mineralization was performed on the other unstained biofilms. The SBF was changed every two days for 7 days of mineralization. samples from day 0 and day 7 were sent to SEM for examination and the results were as follows.

![](https://static.igem.org/mediawiki/2021/9/99/T--Yucai_SZ--csga.png)

**Fig.4**CsgA-Mfp3S-pep bacteria (Left) and amilCP bacteria (Right) cultured in SBF

![](https://static.igem.org/mediawiki/2021/9/93/T--YucaI_SZ--r3.png)
![](https://static.igem.org/mediawiki/2021/e/e1/T--YucaI_SZ--r4.png)

**Fig.5** SEM image showing the surface morphology of the biofilm (i.e., unmineralized). Scale bars: 10μm (left)3μm (right)

![](https://static.igem.org/mediawiki/2021/6/6d/T--YucaI_SZ--r5.png)![](https://static.igem.org/mediawiki/2021/c/c2/T--YucaI_SZ--r6.png)

**Fig.6** SEM image showing the surface morphology of the mineralized composite (mineralization 7d). Scale bars: 10μm (left)3μm (right)

Figure 3 shows day 0 and Figure 4 shows day 7. It can be observed that the samples on day 0 are still in the biofilm indicating state (i.e. unmineralized) the samples on day 7 show the surface morphology of mineralized composites (i.e. mineralized) the pep sequence can function as expected.
Taken together, the test results demonstrated the film-forming and mineralizing effects of CsgA-Mfp3S-pep fusion protein.

## 2.p70-VVD-VVD-T7R-179

![](https://static.igem.org/mediawiki/2021/e/e7/T--Yucai_SZ--bl.png)

**Fig.7** Sensory system p70-VVD-VVD-T7R-179 sequence

## 2.1 Introduction

This is a similar design to Part:BBa\_K3628024 but split the T7RNAP between aa179 and aa180. The split T7RNAP has little activity in dark environment. When the two segments were exposed to blue light, they will bind together due to the photodimerization of VVD domain and exhibit full RNA polymerase activity. So it can be used for constructing a photo sensor system with T7 promoter.

## 2.2 Assay design

We inserted this part into a pSB4C5 backbone, and transformed it into the Escherichia coli DH5α host cell which had harbored a pSB1A3 plasmid containing a pT7-amilCP transcriptional unit. Then test it in tubes.

Inoculate a colony into LB medium and culture overnight in dark (200 rpm, 37 ℃).

Overnight cultures are inoculated 1:100 into two tubes with fresh LB medium. When cultured in shaking table, one tube is exposed to 480 nm blue light, and the other one is covered by tin foil, to protect it from light
After 12 hours of shaking culture, 500 μL culture were sampled and transferred into 1.5 mL EP tube.
Centrifugate the sample tubes at 10 000 g for 30 seconds and observe the colors of bacterial precipitations.

## 2.3 Test results

![](https://static.igem.org/mediawiki/2021/6/69/T--YucaI_SZ--r%3F.png)

**Fig.8**(Upper) Light culture (Lower) Light-proof culture

The experiment demonstrates that the photoreceptor system can work as we expect.

[1]Wang Y, An B, Xue B, et al. Living materials fabricated via gradient mineralization of light-inducible biofilms[J]. Nature Chemical Biology, 2021, 17(3): 351-359.

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# Team:Yucai SZ/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:Yucai SZ/Results

模板！

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![](https://static.igem.org/mediawiki/2021/c/c3/T--Yucai_SZ--result.png)

## System construction - molecular experiment results

The plasmid containing the CsgA-Mfp3S-pep fusion protein gene was first synthesized,transferred into the BL21 (DE3) receptor state, and subjected to colony PCR. the resulting.DNA electrophoresis bands are shown below.

![](https://static.igem.org/mediawiki/2021/e/e2/T--Yucai_SZ--Junn812.png
)

**Fig.1** CsgA-Mfp3S-pep fusion protein bacteriumColony PCR DNA electrophoresis band diagram

The successfully transferred bacteria were divided into two groups, one with IPTG and one without IPTG, and the induction of expression was started in M63 broth medium at an OD600 of about 1.
Four days later, a dish with IPTG and a blank control dish of amilcp bacteria were stained with crystalline violet, and the staining results are shown below

![](https://static.igem.org/mediawiki/2021/e/e2/T--Yucai_SZ--Junn812.png
)

**Fig.2**amilCP bacteria Colony PCR DNA electrophoresis band diagram

Its band of about 800bp represents a successful transfer.
PCR was performed on the CsgA-Mfp-pep fragment, and its PCR product was purified by DNA product and then double digested, and its DNA electrophoresis bands were as follows

![](https://static.igem.org/mediawiki/2021/6/69/T--Yucai_SZ--Junn8201.png)

**Fig.3** CsgA-Mfp3s-pep fragment PCR DNA band map

The band is about 750bp, which is the target fragment we want.
The amilCP plasmid was double digested and the DNA electrophoresis bands were as follows

![](https://static.igem.org/mediawiki/2021/4/41/T--YucaI_SZ--r10.png)

**Fig.4** amilCP plasmid double digestion DNA band map

The products were subjected to DNA purification product recovery and the recovered fragments were ligated with T4 ligase, respectively. The resulting plasmids were transferred into DH5α receptor state and then subjected to colony PCR with the following DNA electrophoresis bands.

![](https://static.igem.org/mediawiki/2021/7/78/T--YucaI_SZ--r11.png
)

**Fig.5**Synthetic Plasmid Bacteria Colony PCR DNA strip map

The band size of about 1500bp represents successful transfer.

## System test results

## I. Induction system testing

The bacteria transferred into amilCP plasmid were divided into two groups, one with IPTG and one without IPTG while expression was induced at an OD600 of about 1.
The induction results were as follows

![](https://static.igem.org/mediawiki/2021/7/7f/T--YucaI_SZ--r8.png
)

**Fig.6**amilCP graph of induction expression results

The bacterium in the right tube in the figure has a clear blue phenotype compared to the bacterium in the left tube, which proves that the induction system can function as expected.

## II. Results of CsgA-Mfp3S-pep fusion protein expression

The bacteria transferred into CsgA-Mfp3S-pep plasmid were divided into two groups, one with IPTG and one without IPTG while expression was induced in M63 broth medium starting at an OD600 of about 1.
Three days later, a dish with IPTG and a blank control dish of amilCP bacteria were stained with crystal violet, and the staining results are as follows

![](https://static.igem.org/mediawiki/2021/b/b8/T--Yucai_SZ--rs.png)

**Fig.7** Crystalline violet staining results

Their staining results demonstrated the presence of biofilm formation and the effect of CsgA with Mfp3S. Mineralization of the other unstained biofilms was performed. The SBF was changed every two days for 7 days of mineralization. samples from day 0 and day 7 were sent for scanning electron microscopy and the results were as follows.

![](https://static.igem.org/mediawiki/2021/8/8c/T--Yucai_SZ--aaa0.png)

**Fig.8** SEM image showing the surface morphology of the biofilm (i.e., unmineralized). Scale bars: 10μm (left)3μm (right)

![](https://static.igem.org/mediawiki/2021/2/2e/T--Yucai_SZ--aaa1.jpeg)

**Fig.9**SEM image showing the surface morphology of the mineralized composite (mineralization 7d). Scale bars: 10μm (left)3μm (right)

Figure 7 shows day 0 and Figure 8 shows day 7. It can be observed that the samples on day 0 are still in the biofilm indicated state (i.e. unmineralized) the samples on day 7 show the surface morphology of mineralized composites (i.e. mineralized) showing the success of CsgA-Mfp3S-pep fusion protein expression and mineralization.

## III.the light-sensitive system test results

The plasmid containing the photosensitive system gene was first synthesized and transferred into the DH-5α receptor state together with the CsgA-Mfp3S-pep-amilCP plasmid. The bacteria were inoculated 1:100 in two groups of fresh LB, one group completely wrapped in tinfoil to avoid light and the other group was illuminated using a blue LED light. The results after two days of simultaneous incubation and centrifugation at 12,000 rpm for 1 min were as follows

![](https://static.igem.org/mediawiki/2021/3/39/T--Yucai_SZ--ggxt.png
)

**Fig.10**(Left) Light culture (Right) Sheltered culture

It can be clearly observed that the bacteria on the left side of the light culture appear a distinct blue phenotype compared to the bacteria on the right side of the light-proof culture. The experimental results show that the light-sensitive system can function as expected.

## III. Co-transfer to expression

The photoreceptor system-T7RNAP plasmid was transferred into DH-5α receptor state along with CsgA-Mfp3S-pep-amilCP recombinant plasmid. The bacterium was inoculated 1:100 in two plates of M63 broth medium, and one plate was irradiated using a light-proof cloth wrapped around the Petri dish and the other plate was irradiated using a blue LED light, and mineralized after three days of induction culture. The mineralization results were as follows

![](https://static.igem.org/mediawiki/2021/c/c1/T--YucaI_SZ--r14.png
)

**Fig.11** (Left) Mineralization results of blue light irradiation culture (Right) Mineralization results of light-proof culture

The results demonstrate the success of amilcp-CsgA-Mfp3S-pep in combination with the blue light sensing system, the success of our experiments and the feasibility of our project.

## IV. PT7-PhlO test results

To reduce the amount of leaked expressions. We were inspired to apply it in our project. Referring to the structure of BL21(DE3)+pET28 protein expression system, we wanted to make both T7 promoter and T7RNAP under the control of PhlF to reduce the leakage activity, so we added PhlO sequence after the T7 promoter. We attached BBa\_K3254024 elements downstream of both the original T7 promoter and the modified T7 promoter with PhlO binding site to characterize the strength of the promoter.

![](https://static.igem.org/mediawiki/2021/4/40/T--YucaI_SZ--d7p5r15.png
)

**Fig.12**Diagram of PhlO device

We attached each of these two test devices to the pSB4C5 backbone and transformed them into an E. coli that constitutively expresses T7RNAP.1 The experimental results showed that this modification did not significantly affect the activity of the T7 promoter (compare the blue column in the figure below).

Subsequently, we transferred to this system the BBa\_K2525007 element attached to the pSB1K3 backbone. We inserted the strain into liquid LB medium and incubated it overnight. Bacterial broth 1:200 was inserted into fresh LB medium (containing IPTG) and incubated at 37 degrees C, 220 rpm for 8 hours. 200 µl of culture was taken into 96-well plates and 485/510 fluorescence and OD600 were measured using an enzyme marker. sfGFP expression was expressed as the ratio of fluorescence value to OD600.

![](https://static.igem.org/mediawiki/2021/e/e1/T--Yucai_SZ--pt7.png
)

**Fig.13**PhlO and PhlF assay results

We plotted the results in the above figure. The experimental results showed that PhlF significantly inhibited the activity of pT7-phlO by more than 70-fold.

1. Zong, Y., Zhang, H. M., Lyu, C., Ji, X., Hou, J., Guo, X., Ouyang, Q., and Lou, C. (2017) Insulated transcriptional elements enable precise design of genetic circuits, Nature communications 8, 52.