iGEM 2021 — Team SDSZ_China

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# Team:SDSZ China/Notebook

![](https://static.igem.org/mediawiki/2021/e/ec/T--SDSZ_China--background-notebook-1.png)

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Project

Description

Engineering

Results

Contribution

Proof of Concept

Parts

Product

Model

Entrepreneurship

Implementation

Lab

Experiments

Notebook

Safety

HP

Human Practices

Communication

Team

Members

Collaborations

Attributions

Notebook

These are what we did during the project. Click on the dates to see more information.

May-July

What we were doing in this period of time is to search for teammates from all kinds of school, from within Beijing to other areas, from China to America...

Another thing we did was to seek for a topic. This took quite a while, from selecting from a list of topics to investigating the feasibility of the topic.

8.9-8.11

8.9

1. Preparation

- Prepare LB medium, LB plates and experiment materials. (accidently add agar into liquid medium, will redo it tomorrow)

2. Inoculation

- Inoculate MG1655 bacteria into LB plate (without antibiotics).

![](https://static.igem.org/mediawiki/2021/4/49/T--SDSZ_China--notebook-8.9-1.png)

8.10

1. LB medium Remake

- Remake two 1L LB medium (liquid) and LB plate (solid), use autoclave pans to sterilize the medium; prepare 10 LB solid plates without antibiotics and 20 more LB solid plates with antibiotics (both Amp+ & Kan+, 10 plates each); store all solid plates into 4℃ refregirator for future use.

2. Competent E.coli MG1655

- Make competent E.coli MG1655 cells using overnight cultured bacteria from yesterday (only to step 3, still need to culture it overnight and finalize the preparation tomorrow).

8.11

1. Done with Competent MG1655 cells

- Finish making competent E. Coli MG1655 cells, distribute them and store them into -80℃ freezer for future use.

8.12-8.14

8.12

1. PCR (MG1655 + PTarget)

![](https://static.igem.org/mediawiki/2021/7/79/T--SDSZ_China--notebook-8.12-1.png)

System;
  Colony PCR
    25ul 2\*Taq
    2.5ul+2.5ul F+R
    1ul Temple
    19ul ddH2O
    50ul Total
    Primer annealing temperature 55℃
    Extension 45s
  FastPfu PCR
    10ul Buffer
    4ul dNTPs
    2ul+2ul F+R
    1ul Temple
    1ul Enzyme
    30ul ddH2O
    50ul Total
    Primer annealing temperature 57℃
    Extension 75s

2. Gel Electrophoresis

1.2g Agarose
120ml 1\*TAE Buffer
10ul SYBR safe DNA Gel Stain

3. DNA Extraction

![](https://static.igem.org/mediawiki/2021/4/43/T--SDSZ_China--notebook-8.12-2.png)

Concentration (ng/ul)
  C1 9.35
  C2 46.85
  C3 16.15
  C4 19.75
  F1 9.45
  F2 7.35
  F3 12.75
  F4 12.95

4. Gibson Assembly

![](https://static.igem.org/mediawiki/2021/8/82/T--SDSZ_China--experiment-6.2.png)

![](https://static.igem.org/mediawiki/2021/4/4e/T--SDSZ_China--experiment-6.3.png)

5. Transformation & Inoculation

Transform Pcas into MG1655; Gibson-1 and Gibson-2 plasmids into Trans-5alpha.
- Transform Pcas plasmid into MG1655 competent cells (with K+ antibiotics), incubate at 30℃ for store it into 4℃ refregirator overnight; Transform Gibson plasmids into Trans-5alpha competent cells (with A+ antibiotics), incubate at 37℃ for overnight culture.

![](https://static.igem.org/mediawiki/2021/7/77/T--SDSZ_China--notebook-8.12-3.png)

8.13

Check bacteria growth, and store 2 plates of Pcas + MG1655 into 4℃ refrigerator.

1. Silicatein Transformation

- Transform Silicatein-pUC57 plasmid into LB solid plate with Amp+.

![](https://static.igem.org/mediawiki/2021/a/a6/T--SDSZ_China--notebook-8.13-1.png)

2. Silicatein Inoculation

![](https://static.igem.org/mediawiki/2021/9/9f/T--SDSZ_China--notebook-8.13-2.png)

3. Streaking plates

- For two Gibson + Trans5alpha 1 and 2 strains, make two streak plates (with A+ antibiotics) and incubate the plate into 37℃ incubator for overnight culture.
- For CsgA +Mfp5s with S010 S011 S021 strains, make three streak plates (with K+ antibiotics) and incubate them into 37℃ incubator for overnight culture.

8.14

1. Colony PCR

![](https://static.igem.org/mediawiki/2021/4/41/T--SDSZ_China--notebook-8.14-1.png)

2. Gel Electrophoresis

50ml system:
50ml 1\*TAE Buffer
0.5g Agarose
5ul Stain

Results:

![](https://static.igem.org/mediawiki/2021/3/37/T--SDSZ_China--notebook-8.14-2.png)

8.15-8.17

8.15

Got the sequencing results from yesterday: we selected sample 4, 7, 9, 10 for futhur analysis.

1. Inoculation

- Inoculate cultured bacteria (sample 4,7,9,10) from 4 Gibson-assembled pTarget-trans5alpha streak plates (see products from 8/13/2021) into 3mL of LB liquid medium (with Amp+ antibiotics), incubate it overnight on a shaker at 30°C.
- Inoculate cultured bacteria (single colony) from 2 pCas + MG1655 solid plates (see products from 8/12/2021) into 5mL of LB liquid medium (with K+ antibiotics), incubate them overnight on a shaker at 30°C.

8.16

1. pTarget Plasmid Extraction

- Making 4 PTarget plasmid extraction from yesterday's PTarget + Trans5alpha E. coli mixture (with Amp+ antibiotic). Store 4 PTarget plasmid at -20℃ freezer.

8.17

1. Making Pcas + MG1655 Competent Cells

- Transfer 0.1ml bacterial fluid (pCas) into 10ml LB (ADD K+ antibiotics!!!!)
- Cultured the strain at 30℃ for 1~2 hour, until OD600 grew to about 0.2
- Transfer 100ul 0.02% Arabinose into the strain
- Cultured the strain at 30℃ for 1.5 hour, until OD600 grew to about 0.2 - 0.25
- Immediately place it on the ice in an ice bath for 10 ~ 15 minutes to keep the bacteria in a cold environment.
- Transfer culture media into a precooled 50ml centrifuge tube, at 4℃ 4100rmp for 10min
- Add 6ml volume of cacl2-mgcl2 mixture into each tube, resuspend the cells and ice bath for 10min
- Store at -80℃

2. pT Plasmids Transformation

- Transform pTarget plasmids extracted from yesterday into 4 different Pcas + MG1655 competent cells.
- Innoculate 4 tubes of Pcas+MG1655+pTarget bacteria mixture onto 4 plates with both Amp+ Kan+ antibiotics; incubate them at 30℃ overnight without a shaker.

8.18-8.20

8.18

1. Streaking plates

- For 4 plates with 4 transformed pT plasmids, make 4 streak plates (with A+ K+ antibiotics) and incubate the plate into 30℃ incubator for overnight culture.
  - Streaking method see below image:

![](https://static.igem.org/mediawiki/2021/1/19/T--SDSZ_China--notebook-8.18-1.png)

8.19

1. Colony PCR

- Colony PCR for pCas + pTarget + MG1655 engineered bacteria (using bacteria from streaked plates from yesterday)
- Preparation & System:

![](https://static.igem.org/mediawiki/2021/5/5f/T--SDSZ_China--notebook-8.19-1.png)

- Operation:
  1. Prepare the system, including the control group (MG1655 without any engineered plasmids or enzymes)
  2. Use pippette or vortex mixer to blend the system. Then use centrifuge for 1.5minutes to make the mixture concentrate on the bottom of PCR tubes.
  3. Use PCR apparatus to duplicate templete
  4. Use electrophoresis apparatus to examine whether we knockout csgA successfully.

- Result:
  1. The results did not quite meet expectations. The control groups works fairly well, whereas the experimental groups fall to form stripes, which indicates the strain is not what we cultivate. Luckily, we find the reason: previous templete shows that the earliest known problem appeared in the process of transforming MG1655 to csgA.

![](https://static.igem.org/mediawiki/2021/3/3e/T--SDSZ_China--notebook-8.19-2.png)

2. Kill Switch Bacteria Plates

- Bacteria strains: BN063, BN065
- Method: streak the bacteria liquid mixture onto the 4 plates
- Incubate the 4 plates at 37℃ for overnight culture

3. Remake MG1655 LB Solid Plates

- Due to the unclear results from PCR of engineered bacteria, we had to redo the entire "gene knockout" process, unfortunately :(((

8.20

1. Silicatein Assay Solution Preparation

Metol-sulphite Solution
  - Add 0.6 g of anhydrous sodium sulphite to 50 mL of Milli-Q water.
  - Add 1 g of p-methylaminophenol (Metol solution).
  - Once reagents are dissolved, filter through No. 1 Whatman paper and store in a glass bottom
  - Recommended shelf life of approximately 1 month.
Oxalic Acid Solution
  - Shake 5 g of oxalic acid dihydrate in 50 mL of Milli-Q water.
  - Store in a glass bottle. Solution may be stored indefinately.

2. Remaking MG1655 Competent cells (step 1)

- Innoculate single colony from plate of MG1655 strain into 3mL of LB liquid medium (with NO antibiotics), put it into 37℃ on a shaker for overnight culture.

3. Kill Switch Innoculation

- Innoculate single colony from BN063, BN065 strains plates into 3 mL of LB liquid medium (with 30 μL K+ antibiotics)
- Incubate the liquid mixture at 37℃ on a shaker for overnight culture.

4. Silicatein Innoculation

- Innoculate single colony from selicatein-puC57 strains plates into 3mL of LB liquid medium (with 30 μL A+ antibotics)
- Incubate the liquid mixture at 37℃ on a shaker for overnight culture.

8.21-8.23

8.21

1. Kill Switch Testing

- Using 2 plates of LB solid medium (with K+ antibiotics), divide each plate into 4 sections, add 10uL of BN063 and BL065 cultured bacteria mixture into each section. (top 2 quarters without Ara, bottom 2 quarters with the addition of Ara Inducer droplet)
- Remark: Ara droplet addition: 30 uL; let Ara droplet completely dry before adding bacteria.
- Again, using 4 centrifugal tubes of LB liquid medium (1mL each), add 10uL of K+ antibiotics into each tube; add 10uL of BN063 and BL065 cultured bacteria mixture into each centrifugal tube. Then, choose 2 tubes and add 10uL of Ara Inducers into these 2 tubes.
- INCUBATE @ 37℃ OVERNIGHT CULTURE FOR ALL PLATES + TUBES.
- Why we use Ara inducer droplet?

![](https://static.igem.org/mediawiki/2021/3/38/T--SDSZ_China--experiment-11.1.png)

- Theory: When arabinose exists, AraBAD promotor(already in the bacteria) can drive the high efficiency expression of target genes which can translate into toxic protein to kill bacteria.
- Advantages:
1. Rigour
2. High inducibility
3. Low cost

8.22

1. Kill Switch Testing

Result of BN063

![](https://static.igem.org/mediawiki/2021/6/6d/T--SDSZ_China--notebook-8.22-1.png)

![](https://static.igem.org/mediawiki/2021/f/ff/T--SDSZ_China--notebook-8.22-2.png)

8.24-8.26

8.24

1. Silicatein Assay Solution Preparation

Sulfuric Acid Solution
  - Add 500 uL of Milli-Q water to 500 uL of concentrated sulfuric acid solution. (Final concentration of sulfuric acid in solution is 50%; final volume of the solution is 1mL)
  - Store in bottle.

TMOS + HCl Solution Mixture
  - Prepare a solution of 100 mM tetramethyl orthosilicate (TMOS) (1.47mL) in 1 mM HCl (3.1uL) to prepare prehydrolyzed silica particles.
  - Calculation:
    - 100mmol/L TMOS \* 152 g/mol = 15.2g/L; (15.2 g/L) / (1.03 g/mL) = 14.7 mL/L ---> 14700 uL/L ---> 1470 uL/100mL ---> 1.47mL / 100 mL
    - 1mmol/L HCl \* 36.5 g/mol = 0.0365 g/L; (0.0365 g/L) / (1.18 g/mL) = 0.0309 mL/L ---> 31uL/L ---> 3.1uL / 100mL

Acidic solution of ammonium molybdate
  - 20 g (NH4)6Mo7O24•4 H2O (0.33g) and 60 mL of concentrated Hcl [36%]
    - WILL REDUCE THE ENTIRE SYSTEM INTO 1mL INSTEAD to minimize cost.
  - Calculation:
    - 20g/(2.5 g/mL) = 8mL
    - 8mL : 60mL = 1 : 7.5 = 0.13mL : 1mL = 0.33g
    - So, if we want to use only 1mL of concentrated HCl, we need to weight out 0.33g ammonium molybdate and mix them into solution for further use.

8.25

1. Research on TEM Sample Preparation

- Need to order "Carbon Membrane Supporting Film" and "Copper Mesh" for TEM testing
  - Size: 200, 50
  - Use a centrifuge to separate calcium hydroxyphosphate
  - Sample, disperse it in absolute ethanol
  - Ultrasonic dispersion
  - Drop on the Carbon Membrane Supporting Film(on the Copper Mesh)
  - Put them into an ice box

2. Silicatein-pUC57 Plasmid Extraction

![](https://static.igem.org/mediawiki/2021/b/b4/T--SDSZ_China--notebook-8.26-1.png)

8.26

1. Redo "Gene Knock-Out"

1. Making pCas+MG1655 Competent Cells------Protocol see above (2021/8/17).
  2. Transform 4 pTarget plasmid into pCAS+MG1655 cells------Protocol see 2021/8/17.

2. Silicatein Plasmid Assembly

1. Plasmid Extraction of S010 Backbone

![](https://static.igem.org/mediawiki/2021/6/64/T--SDSZ_China--notebook-8.26-2.png)

2. Fast Pfu PCR
    10ul Buffer
    4ul dNTPs
    2ul+2ul F+R
    1ul Template (5289 bp + 1459 bp = 6748 bp)
    1ul Enzyme
    30ul ddH2O
    50ul Total
    Primer annealing temperature: 56℃
    Extension: 2 min 45 s

3. Gel Electrophoresis
    1. Add 0.3 grams of agarose, 30mL of TAE buffer and 3uL of SYBR safe DNA Gel Stain
    2. Put the gel together with the plate into the electrophoresis tank and pour 1✖️TAE buffer onto the gel surface
    3. Add 6x Loading Dye of 8 μL in each sample
    4. 50 μL of S010, silicatein-pUC57 and DNA marker(Trans2K Plus II) were added respectively (total of 5 lanes-----S1, S2, P1, P2, Marker)
    5. @ 150V, run the Gel Electrophoresis for 15min
Results:

![](https://static.igem.org/mediawiki/2021/d/d7/T--SDSZ_China--notebook-8.26-3.png)

- All of the fragements have been successfully made.
  - S1, S2 (base pair 1459 ✅)
  - P1, P2 (base pair 5289 ✅)
- Moreover, we find out that Lane S1, S2 and P1 gave the best results.

4. DNA Extraction

![](https://static.igem.org/mediawiki/2021/0/04/T--SDSZ_China--notebook-8.26-4.png)

Concentration ratio
  - Silicatein-pUC57 : S010 = 3:1

8.27-8.29

8.27

1. Gibson Assembly

![](https://static.igem.org/mediawiki/2021/9/9f/T--SDSZ_China--notebook-8.27-1.png)

2. Transformation

- Transform Gibson-3 plasmid into BL21 E.coli strain (with K+ antibiotics), incubate at 37℃ for overnight culture.

![](https://static.igem.org/mediawiki/2021/5/5b/T--SDSZ_China--notebook-8.27-2.png)

8.29

1. Streaking plate (MG1655 Gene Knockout)

- Checking bacterial growth, found out only plamid pT#4 has successfully grown bacteria.
- For that plate with transformed pT#4 plasmids, make streak plate (with A+ K+ antibiotics) and incubate the plate into 30℃ incubator for overnight culture.
- Streaking method see below image:

![](https://static.igem.org/mediawiki/2021/2/20/T--SDSZ_China--notebook-8.29-1.png)

2. Streaking plate (Si)

- For Gibson-3 (Silicatein-pUC57 & S010) + BL21, make two streak plates (with K+ antibiotics) and incubate the plate into 37℃ incubator for overnight culture.

8.30-9.3

8.30

1. Colony PCR for MG1655

- Colony PCR for pCas + pTarget + MG1655 engineered bacteria (using bacteria from streaked plates from yesterday)
- Preparation & System

![](https://static.igem.org/mediawiki/2021/2/26/T--SDSZ_China--notebook-8.30-1.png)

2. Colony PCR for Si

![](https://static.igem.org/mediawiki/2021/e/e4/T--SDSZ_China--notebook-8.30-2.png)

3. Gel Electrophoresis

50ml system
  50ml 1\*TAE Buffer
  0.5g Agarose
  5ul Stain
Results
  Failed. Nothing ran out except the control group: MG1655 without any plasmids inserted, meaning that out experiment was NOT successful. Had to debug and redo it.

8.31-9.3

1. Redo Gene Knockout for MG1655

- Day 1: Using the MG1655 LB plate, innoculate single colony into LB liquid medium for overnight culture
- Day 2: Then make MG1655 competent cells; then transform pCas plasmid into it; overnight culture
- Day 3: Then making pCas+MG1655 competent cells; then transform pT(x4) into them, overnight culture.
- Day 4: After overnight culture, select single colony of pCas+pT+MG1655 cells and do Colony PCR for testing and sequencing (to see if we finally knocked csgA gene out OR not).

2. Redo Gibson Assembly

- This time with the correct DNA concentration calculated by Nanodrop.
Concentration (ng/ul):
  - S1: 31.45
  - S2: 5.65 (disgard)
  - P1: 34.25
  - P2: 44.15

![](https://static.igem.org/mediawiki/2021/7/7c/T--SDSZ_China--notebook-8.31-1.png)

3. Transformation of Gibson-4 into BL21

![](https://static.igem.org/mediawiki/2021/8/88/T--SDSZ_China--notebook-8.31-2.png)

4. Colony PCR + Gel Electrophoresis for Gibson-4 + BL21

- Select 8 single colony from plate of Gibson-4 + BL21, then run colony PCR to test out if any of the colony can success.
- DNA Marker: 100bp Plus II DNA Ladder
- Results for 8 Colony of G-4 + BL21:

![](https://static.igem.org/mediawiki/2021/9/95/T--SDSZ_China--notebook-8.31-3.png)

![](https://static.igem.org/mediawiki/2021/7/74/T--SDSZ_China--notebook-8.31-4.png)

- Colony #4 successfully run out, meaning that this one matched with the fragment we desire. We will be using this colony to do PCR again tomorrow for both "backbone" and "fragment". And then run Gel Electrophoresis to test out if we successfully have them as the ones we designed.

- Results for backbone + fragment: (same marker as above)

![](https://static.igem.org/mediawiki/2021/0/0a/T--SDSZ_China--notebook-8.31-5.png)

- The results are positive(about 600bp)!!
- We sent both of them for sequencing, and the results for sequencing came out positive!! Now our Gibson Assembly for Silicatein + S010 strain are officially DONEEEEE!

9.4-9.6

1. Silicatein Activity Assay

- Prepare a solution of 100 mM tetramethyl orthosilicate (TMOS) in 1 mM HCl to prepare prehydrolyzed silica particles. Stir at room temperature for 15 minutes to completely hydrolyze TMOS to silica monomers.
- Prepare two reactions:

![](https://static.igem.org/mediawiki/2021/9/92/T--SDSZ_China--notebook-9.4-1.png)

- Incubate at NIST standard temperature and pressure (1 atm, 20 ºC) for 2 hours.
- Centrifuge sample at 15,000 RPM for 2 minutes to precipitate polymerized silica particles.
- Decant supernatant containing unaggregated silica particles.
- Wash 3 times with distilled H2O to remove free, hydrolyzed TMOS.
- Aliquot the unreacted hydrolyzed TMOS remaining in solution after silica aggregation.
  - Treat with 2 M NaOH for 1 hour at 80 ºC to ensure complete hydrolysis of silica particles to monomer/dimer state.
- Remove aliquots of 500 μl and add to solution of 750 uL water and 75 uL of acidic solution of ammonium molybdate (20 g (NH4)6Mo7O24•4 H2O and 60 mL of concentrated Hcl [36%]).
- Incubate at NIST standard temperature and pressure for 20 minutes. Solution should have a yellowish hue after incubation.
- Add 400 uL of reducing agent solution (see above). Solution should turn a bluish color.
- Record absorbance at 810 nm and compare to standard curve.

To make sure we have valuable comparison, in our own experiment apart from the previous protocol, we designed:
- two negative control groups----NC1, NC2;
- two experiment groups----USE ultrasonic homogenizer; NO ultrasonic homogenizer

Data:
- Qualitative Pictures:

![](https://static.igem.org/mediawiki/2021/e/ed/T--SDSZ_China--notebook-9.4-2.png)

NC1 (for NP) = Negative Control Group #1 (only TMOS + HCl)
NC2 (for P) = Negative Control Group #2 (TMOS + HCl + PBS buffer)
NP = NO ultrasound homogenization (TMOS + HCl + NaOH + silicatein extracted from 5mL of original bacterial mixture)
P = Under ultrasound homogenization (TMOS + HCl + PBS buffer + NaOH + silicatein extracted from 5mL of original bacterial mixture)

![](https://static.igem.org/mediawiki/2021/7/7f/T--SDSZ_China--notebook-9.4-3.png)

Level of colorness: NP < P < NC1 < NC2
Level of transparency: NC2 < P< NP < NC1

- Qualitative OD values:

![](https://static.igem.org/mediawiki/2021/f/f3/T--SDSZ_China--notebook-9.4-4.png)

The results show that there are sufficient amount of silicatein produced, since:
- Both NC Control are darker (NC1 remain as yellow which indicates that it needs MORE NaOH to turn blue, it contains more acids)
- Both silicatein mixture (with / without homogenization) have higher OD810nm, meaning that there are more bacteria inside the test tube (the solution is thicker / not as clear as NCs).

2. Silicatein Miniralization Products Extraction (used for TEM/SEM testing)

- We use the 80 degree Celcius dryer to try to heat-dry the silicatein protein-bacteria mixture (5mL), in hope that there will be products produced after 1~2 days of physical drying. If the results are prominent, we can move on and send the products for TEM/SEM testing.
- Results (left: control; right: silicatein after drying)

![](https://static.igem.org/mediawiki/2021/5/58/T--SDSZ_China--notebook-9.4-5.png)

![](https://static.igem.org/mediawiki/2021/f/f5/T--SDSZ_China--notebook-9.4-6.png)

3. Incubation of Gene Knockout products

- At 30 degree Celcius, overnight culture
- Failed. There was little to no bacteria grown on the plate after 2 days of culture. We reached out to a previous team who had done experiments on Gene Knockout and MG1655 bacteria strain, and asked about their results, in hoping we could find a solution. They told us that their results for knocking out MG1655 was not obvious (shown no promoninet results after and before the knockout). Thus, we decided to change our experiments setup and skip the gene knockout part, and to use the bacteria directly for our next step testing.

9.9-9.14

9.9

1. Innoculation again.

- Innoculate Silicatein, S010, S011, S012 bacteria strain from plates to LB liquid medium
- Overnight culture with 37 degree Celcius on a shaker
- They are prepared to be used for TEM/SEM testing!

![](https://static.igem.org/mediawiki/2021/b/b7/T--SDSZ_China--notebook-9.9-1.png)

2. Prepared some more LB liquid medium.

9.10

1. Add inducer IPTG + innoculated bacteria mixture

- Added 100uL of IPTG and 200uL of 4 types of bacteria mixture(Si, S010, S011, S021) from yesterday respectively, into 4 different glass bottles containing 200mL of LB liquid medium(with K+ antibiotics), put them into 37 degree Celsius incubator on a shaker for overnight culture.
  - Concentration of IPTG inducer is 5mmol/L, thus we need to use ratio 1/2000 to add inducer into LB medium to induce growth; and use 1/1000 ratio to add bacterial mixture into LB medium to make sure normal growth as well.

9.11-9.14

1. Preparing for TEM + SEM testing

- Prepare 5 pieces of “Quantifoil Carbon Film”.

![](https://static.igem.org/mediawiki/2021/5/51/T--SDSZ_China--notebook-9.11-1.png)

- TEM testing:
  - Draw 1mL of Si bacteria mixture from the original LB medium glass bottle into microcentrafigal tube. Add 10uL of bacteria mixture onto the piece of carbon film (mark as #1).
  - Dilute the original Si bacteria mixture with ratio 1:10; add another 10uL of bacteria mixture onto another piece of carbon film, repeat it twice (mark as #2, #5).
  - Repeat the step above twice, mark as #3, #4.
  - Put all 5 pieces of film into the 80 degree Celsius dryer until they are completely dry, then send them for TEM testing.
  - Pictures:

![](https://static.igem.org/mediawiki/2021/1/12/T--SDSZ_China--notebook-9.11-2.png)

![](https://static.igem.org/mediawiki/2021/4/41/T--SDSZ_China--notebook-9.11-3.png)

- Results:

![](https://static.igem.org/mediawiki/2021/0/03/T--SDSZ_China--notebook-9.11-4.png)

![](https://static.igem.org/mediawiki/2021/0/07/T--SDSZ_China--notebook-9.11-5.png)

![](https://static.igem.org/mediawiki/2021/6/6c/T--SDSZ_China--notebook-9.11-6.png)

- SDS-PAGE verification:
  - Draw 50mL of bacteria mixture from 4 different bottles from yesterday.
  - Centrifuge 4 bacteria mixture (10000rpm, 15min), decant the supernant; for all of them, add 10mL of PBS buffer and use ultrasound to homogenize the mixture (10min each).
  - Aliquot the supernants and filter the liquids.
  - Run SDS-PAGE to separate and test protein.
  - Results:

![](https://static.igem.org/mediawiki/2021/d/d0/T--SDSZ_China--notebook-9.11-7.png)

SDS-PAGE Protein Gel Electrophoresis:
1. Prepare SDS-PAGE running buffer(1X): 10mL.
2. Prepare gels and assemble the electrophoresis device.
3. Heat samples for 5 minutes at 94 degrees Celsius.
4. Cover the chamber & firmly connect both the anode and the cathode. Set the voltage on the electrophoresis power supply to a constant voltage of 120V.
5. Run the gel electrophoresis cell for 30-60 minutes.
6. After electorphoresis, remove the gel by floating it off the plate into water.
7. Stain the SDS-PAGE with Coomassie blue dyes, then destain it.
8. Observe the electrophoresis results that show up in the E-gel imager.

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# Team:SDSZ China/Engineering

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Engineering Success

The Construction of Silicatein-Contained Plasmid

Silicatein could induce silica precipitation when it is in the environment that contain the TMOS or silicic acid. As a result, in order to expose silicatein to the extracellular environment, we decided to co-express the silicatein gene with another gene which encodes a protein that is localized on the membrane. Since there are previous experiments on expressing silicatein-INP fusion protein, we design the plasmid that co-express the silicatein gene and INP gene (INP refers to ice nucleation protein, which are localized on the outer membrane). Our team successfully constructed the plasmid pET28b-INP-linker-Silicatein-7×His (Fig.1-1).

![](https://static.igem.org/mediawiki/2021/4/47/T--SDSZ_China--results_1-3.png)

Fig.1-1

Fig.1-1 is the schematic showing genetic components of the fusion protein.

We firstly found the sequences of two genes in the NCBI database, and optimized codon based on the codon bias of E.coli. After bought the silicatein from the biosynthesis company, we used PCR to extract the silciatein gene from the vector (Fig.1-2). Then, we use Gibson Assembly to transform two genes in two the BL21 strains of E.coli.

![](https://static.igem.org/mediawiki/2021/7/75/T--SDSZ_China--result_1-2.png)

Fig.1-2

Fig.1-2 represents the results of gel electrophoresis to test the fragment of silicatein extracted from previous vectors.

After the transformation, we tested whether we successfully knock in the sequences. However, two sequences are so long that they cannot be detected accurately by PCR as a whole. In order to increase the accuracy, we use PCR to detect the two connections (the connection that connect silicatein gene and vector and the connection that connect the INP gene and the vector). The results are positive.

![](https://static.igem.org/mediawiki/2021/7/7b/T--SDSZ_China--result_1-4.png)

Fig.1-3

Fig.1-3 represents gel electrophoresis of two connections.

Then, we intended to test its funciton of inducing precipitation of silica. We compared the OD values to prove its function. After placing the genetically modified E.coli in a environment that contain TMOS, HCl, and NaOH and culturing for two hours, we tested its OD value and compared it with a negative control group. The OD value of silicatein mixture is higher than the controlled group, proving the production of silica.

Furthermore, we assumed that the silicatein also presents inside the cells. As a result, we used ultrasonic comminution to break the cells and exposed the intracellular silicatein to the buffer, and compare its OD value with mixture without homogenization. The higher OD value of the mixture with the homogenezation validated our assumption, which pointed our team to a direction to improve our design, to place more silicatein on the membrane surface.

![](https://static.igem.org/mediawiki/2021/1/16/T--SDSZ_China--result_1-5.jpeg)
![](https://static.igem.org/mediawiki/2021/7/74/T--SDSZ_China--result_1-6.png)

Fig.1-4

Fig.1-5

NC1 is a negative control group #1 (only TMOS + HCl)

NC2 is a negative control group #2 (only TMOS + HCl + PBS buffer)

NP is the experimental group without ultrasound homogenization (TMOS + HCl + NaOH + silicatein extracted from 5mL of original bacterial mixture)

P is the experimental group under ultrasound homogenization(TMOS + HCl + PBS buffer + NaOH + silicatein extracted from 5mL of original bacterial mixture)

Fig.1-5 shows the OD values of four groups, as shown in Fig.1-4. In each group, the first two bars represent the OD values without centrifuge; the last bar represents the OD value after centrifuge.

TEM testing offered a more direct evidence to prove the function of silicatein. We added five drips of bacteria mixture of different concentration onto the five pieces of carbon film, and put them into the 80 degree Celsius dryer. After they are completely dry, we sent them for TEM testing. The dark particles on the surface of cells, which are the silica particles, further verified that the plasmids could induce the precipitation of silica.

![](https://static.igem.org/mediawiki/2021/3/34/T--SDSZ_China--result_1-7.jpeg)

Fig.1-6

Fig.1-6 represents the TEM result of engineered bacteria.

The Construction of Mfp5-Contained Plasmid

Same as the silicatein, only when Mfp5 is on the surface of cells can they induce the precipatation of hydroxyapatite. According to the research, we found that CsgA protein, which is one of the main components of could be used to bring the protein to the cell surface. As a result, we constructed the CsgA-Mfp5 fusion protein. As previous experiments showed that the expression of a single Mfp5 gene may be insufficient to cause a notable precipitation, our team also transformed two Mfp5 to a group of plasmids.

![](https://static.igem.org/mediawiki/2021/0/03/T--SDSZ_China--results_2-4.png)

Fig.2-1

Fig.2-1 shows gel electrophoresis of all the fragments
needed for calcium mineralization.

To further prove that construction of plasmids was successful, we also used Sanger Method to sequence the gene and compared the result with the sequence we had designed. The result also showed that the construction of the plasmids was successful, laying a solid fundation for our further reaserch.

![](https://static.igem.org/mediawiki/2021/b/bd/T--SDSZ_China--results_2-5-1.png)

Fig.2-2

Fig.2-2 shows the results of Sanger Method sequencing.

After confirming that the plasmids were as same as what we designed, we wanted to verify its function in induce the precipitation of the hydroxyapatite. We used TEM testing to prove the existence of the hydroxyapatite carbonate on the cell surface. Unfortunately, the examination was failed due to the carelessness of the reaserchers that help us, so we could only test its function in another way.

We finally used electron diffaraction to test the function of the palsmids. The electro-diffaraction diagram below showed the existence of crystal in the sample. Since there is no other crystal in the sample, it could indirectly verify the function of the Mfp5.

![](https://static.igem.org/mediawiki/2021/f/f6/T--SDSZ_China--results_2-7.png)

Fig.2-3

Fig.2-3 is the result of TEM testing.

The Construction of Kill Switch

During the research phase, we discovered two kinds of toxin which are appropriate for our experiments: kid toxin and colicin. Both of them have specificity towards E.coli, preventing them from killing other irrelevant cells. Therefore, our team planned to compare their ability in killing E.coli. As for the promoter, we used AraC due to its universality. We design two plasmids -- BN065\_pSB4K5-pBAD-colicin and BN065\_pSB4K5-pBAD-kid. However, after we successfully constructed the plasmids, the one with colicin gene showed leakage of toxin when we were incubating the bacteria, so we only used kid toxin in further research.

![](https://static.igem.org/mediawiki/2021/f/f1/T--SDSZ_China--description-Lacl-AraC-Kid_Toxin2.png)
![](https://static.igem.org/mediawiki/2021/a/aa/T--SDSZ_China--results_3-2.png)

Fig.3-1

Fig.3-2

Fig.3-1 represents the schematic of K1.

Fig.3-2 represents the schematic of K1.

We tested the function of the kill switch by performing a controlled experiment. Our team inoculated the cells in eight groups, four in liquid medium and four in solid medium.

As shown in Fig.2-3, normal E.coli, without the transformation of the plasmids, growed normally at the upper-left area of the medium; at the upper-right area, K1 cells allowed bacteria to grow normally without Arabinose; at the bottom area , K1 cells killed E.coli with the presence of Arabinose. Fig.2-4 shows that the OD value of the liquid medium that contained Arabinose was lower than that of the medium without Arabinose. These results indicate that K1 performed as effectively as expected.

![](https://static.igem.org/mediawiki/2021/7/74/T--SDSZ_China--result_3-5.png)
![](https://static.igem.org/mediawiki/2021/2/29/T--SDSZ_China--results_3-6-2.png)

Fig.3-4

Fig.3-5 shows the results of kill switch tests,
as shown in Fig.3-4.

![](https://static.igem.org/mediawiki/2021/2/2e/T--SDSZ_China--result_3-7.png)

Fig.3-3

Fig.3-3 shows the results of the experiment
testing K1 kill switch.

Fig.3-5

Fig.3-5 shows the OD value of liquid medium
with(experimental) and without(control) adding arabinose

Reference

[1] Müller, Werner EG, et al. "Bioencapsulation of living bacteria (Escherichia coli) with poly (silicate) after transformation with silicatein-α gene." Biomaterials 29.7 (2008): 771-779.

[2] Park, Jeong Chan, et al. "R5 Peptide-based Biosilicification Using Methyltrimethoxysilane." Biotechnology & Bioprocess Engineering 23.1 (2018).

[3] Wei, W. et al. An underwater surface-drying peptide inspired by a mussel adhesive protein. Adv. Funct. Mater. 26, 3496–3507 (2016).

[4] Wang, Y. et al. Living materials fabricated via gradient mineralization of light-inducible biofilms. Nature chemical biology 351-359 (2021)

[5] Dick J, Windt W D, Graef B D, et al. Bio-deposition of a hydroxyapatite layer on degraded limestone by Bacillus species[J].Biodegradation,2016(4):357-367.

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# Team:SDSZ China/Experiments

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Project

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Experiments

Protocols

These are the protocols related to the experiments in our project

Protocol for solid medium inoculation

1. Use centrifugal machine to centrifuge bacteria to the bottom (6100rpm, 3min)
2. Use pippette to discard top 3/4 LB medium supernatant, mix the bottom 1/4 (~30uL bacteria) with the rest of LB medium, drop ~40uL bacteria mixture onto the center of the solid LB plate.
3. Use glass bead spreader to spread out the bacteria mixture for optimal colony growth.
4. Incubate the sample at 37℃ for overnight culture.

Protocol for making competent E.coli cells

Day 1:
1. Inoculate the cells in 10ml LB Medium and Grow Overnight.
Day 2:
1. From the O/N culture Freshly inoculate a single colony of bacteria into 3mL of LB Medium and start growing the cells freshly with shaker at 37℃ 200rpm for 16+ hours.
2. Inoculate the mixture using 1:100 ratio (eg: 500uL innoculum into 50mL LB medium) and culture it at 37℃ 200rpm until the O.D of the culture reaches 0.3 - 0.5 at A600nm.
3. Once the O.D has reached, take the culture into two 50ml centrifuge tubes and spin down the cells (Maintain Centrifuge at 4 degrees) (2000g - 5mins)
4. Use 5mL of cold LB to resuspend the cells, add 5mL of cold Reagent (came in the box of One Step Competent Cell Preparation Kit, made of Mg2+ and Ca2+ ions), mix it and let it set in ice bath for 5 mins.
5. Resuspend the cells in 1.5mL or 0.5mL of ice Cold 0.1M CaCl2+20% Glycerol and aliqoute 100ul into each 1.5 ml tubes and store them immediately in -80℃.

Protocol for FastPfu PCR

1. Prepare PCR system on ice according to the table below(take a 50ul system as an example).

![](https://static.igem.org/mediawiki/2021/c/c6/T--SDSZ_China--notebook-3.1.png)

2. Use pippette or vortex mixer to blend the system. Then use centrifuge for 1~2 seconds to make the mixture concentrate on the bottom of PCR tubes.
3. Run PCR program according to the table below.

![](https://static.igem.org/mediawiki/2021/a/a5/T--SDSZ_China--notebook-3.2.png)

Protocol of Gel Electrophoresis

1. Place a gel mould on a horizontal table. Then place a plate (25 mL/ 50 mL/ 100 mL) in its corresponding mould. Make sure that there is no air or other impurities between the bottom of the mould and the plate and that they are closely attached. Place a comb and make sure that its bottom doesn't have any contact with the mould.
2. Weigh 0.25 g/ 0.5 g/ 1.0 g agarose and transfer it into a triangular flask. Add 25 mL/ 50 mL/ 100 mL of 1\*TAE solution and heat the flask in oven for 0.5 min~1 min. Check and blend the solution until it turns clear and even.
3. Distribute 2.5 uL/ 5 uL/ 10 uL of SYBR safe DNA Gel Stain (10000\*) and blend the solution. Then transfer the solution onto the plate carefully (to avoid bubbles). Place it under indoor temperature for 20~60 minutes.
4. After the gel is fully solidified, extract the comb to form a row of segregative holes.
5. Place the gel and the plate in the electrophoresis chamber and add 1\*TAE Buffer until it submerges the upper surface of the gel for about 2~3 mm. Move the gel and the plate carefully to get rid of bubbles in the holes.
6. Pipette samples into the holes. Make sure that samples are in proper volume to avoid overflow.
7. Taq Mix contains Loading Dye. Other samples without Gel Loading Dye should mix with it before adding them.
8. The amount needed for PCR examinations is about 3~6 ul. The amount for plasmid examination is determined by the concentration of the plasmid.
9. If samples should be retrieved, blend 50 ul of PCR mixture and Gel Loading Dye and pipette them into a larger hole.
10. DNA Marker should be added into a hole exclusively for comparison (3 ul/ 5 ul/ 10 ul).

Protocol for DNA Gel Extraction

1. Carefully excise the target DNA fragment using a clean scalpel and put it into a clean 1.5 mL microfuge tube.
2. Weight the gel slice to determine the volume of gel dissolution buffer. Assuming a quality of 0.1g Gel, add 100 uL gel dissolution buffer. Incubate the mixture at 50 degree Clesius for a few minutes until the gel has completely melted. Mix by shaking or inverting the tube every 2~3 min.
3. Add 500 uL equilibrium buffer to the adsorption column (assembly the adsorption column into a collection tube), centrifuge at 1,2000 rmp for 1 min, discard the waster liquid.
4. Cool down the gel solution to room temperature and transfer it to adsorption column, incubate at room temperature for 2 min, centrifuge at 1,2000 rmp for 30-60s, discard the waste liquid.
5. Add 600 uL of wash buffer diluted with absolute ethanol for 2 times, centrifuge at 1,2000 rmp for 30-60 s, discard the waste liquid.
6. Reusing the collection tube, centrifuge the empty column at 1,200 rmp for 2 min to dry the column matrix.
7. Place the column into a clean 1.5 mL microfuge tube, add 50-100 uL ddH2O (Sterilized, preheated to 50 degree Celsius) directly into the center of the column matrix, then incubate 2 min at 50 degree Celsius, centrifuge at 1,2000 rmp for 1 min to elute DNA.
8. The concentration of DNA fragments can be measured by Microplate reader.

Protocol for Gibson Assembly conditions

![](https://static.igem.org/mediawiki/2021/e/e6/T--SDSZ_China--experiment-6.1.png)

Protocol for Competent cell transformation

1. Mix target DNA (ligation reaction mixture, plasmids or controls) with 50 uL of competent cells, incubate for 30 min on ice.
2. Heat shock for 60s at 42 degrees Celsius, then incubate for 5 min on ice immediately. Do not shake the tube at this point.
3. Add 150 uL of LB medium without antibiotics.
4. Incubate for another 40-60 minutes at 37 degree Celsius.
5. Resuspend the cells and spread the suspension on an agar plate containing the targeted antibiotics.
6. Incubate for 16-18 hours at 37 degrees Celsius.

Protocol for streaking plates

- For two Gibson + Trans5alpha 1 and 2 strains, make two streak plates (with A+ antibiotics) and incubate the plate into 37℃ incubator for overnight culture.
- For CsgA +Mfp5s with S010 S011 S021 strains, make three streak plates (with K+ antibiotics) and incubate them into 37℃ incubator for overnight culture.
- Streaking method see below image (left one):

![](https://static.igem.org/mediawiki/2021/d/dd/T--SDSZ_China--experiment-8.1.png)

Protocol for plasmid extraction: (using TIANGEN plasmid extraction kit)

1. Culture E. coli (Trans5alpha) with plasmid (PTarget) in LB media (with Amp+ antibiotic), overnight on a shaker at 30°C.
2. Pellet 2 ml of bacterial culture in a microfuge tube by centrifuging for 1 minute at 12,000 rpm.
3. Decant the supernatant and add 150 µl of the resuspension buffer (P1 with addition of RNase A and TIANRed). In order to resuspend the pellet you may have to vortex.
4. Add 150 µl of the lysis buffer (P2), invert the tube 8 times to mix thoroughly. The solution should become clear purple and viscous.
5. Add 350 µl of the neutralization buffer (P5), invert the tube 12+ times or until a precipitate forms. The precipitate is a mixture of protein and chromosomal DNA. Centrifuge the tube for 2 minutes at 12,000 rpm. The solution should be clear yellow.
6. Transfer the solution to a spin column (CP3) and put a collection tube on the bottom of the spin column. Centrifuge the spin column with tube for 30s at 12,000 rpm. Discard the flow through.
7. Add 300 µl of the wash buffer (PWT with enathnol) and centrifuge for 30s at 12,000 rpm. Discard the flow through. Repeat this step.
8. Centrifuge for an additional 30s at 12,000 rpm to remove residual wash buffer.
9. Transfer the column to a clean microfuge tube. Add 50-100 µl of elution buffer and centrifuge for 30s at 12,000 rpm. Collect the extracted plasmid into the collection tube.

Silicatein Assay Solution Preparation

Metol-sulphite Solution
- Add 0.6 g of anhydrous sodium sulphite to 50 mL of Milli-Q water.
- Add 1 g of p-methylaminophenol (Metol solution).
- Once reagents are dissolved, filter through No. 1 Whatman paper and store in a glass bottom
- Recommended shelf life of approximately 1 month.

Oxalic Acid Solution
- Shake 5 g of oxalic acid dihydrate in 50 mL of Milli-Q water.
- Store in a glass bottle. Solution may be stored indefinately.
Sulfuric Acid Solution
- Add 500 uL of Milli-Q water to 500 uL of concentrated sulfuric acid solution. (Final concentration of sulfuric acid in solution is 50%; final volume of the solution is 1mL)
- Store in bottle.

TMOS + HCl Solution Mixture
- Prepare a solution of 100 mM tetramethyl orthosilicate (TMOS) (1.47mL) in 1 mM HCl (3.1uL) to prepare prehydrolyzed silica particles.
- Calculation:
- 100mmol/L TMOS \* 152 g/mol = 15.2g/L; (15.2 g/L) / (1.03 g/mL) = 14.7 mL/L ---> 14700 uL/L ---> 1470 uL/100mL ---> 1.47mL / 100 mL
- 1mmol/L HCl \* 36.5 g/mol = 0.0365 g/L; (0.0365 g/L) / (1.18 g/mL) = 0.0309 mL/L ---> 31uL/L ---> 3.1uL / 100mL

Acidic solution of ammonium molybdate
- 20 g (NH4)6Mo7O24•4 H2O (0.33g) and 60 mL of concentrated Hcl [36%]
- WILL REDUCE THE ENTIRE SYSTEM INTO 1mL INSTEAD to minimize cost.
- Calculation:
- 20g/(2.5 g/mL) = 8mL
- 8mL : 60mL = 1 : 7.5 = 0.13mL : 1mL = 0.33g
- So, if we want to use only 1mL of concentrated HCl, we need to weight out 0.33g ammonium molybdate and mix them into solution for further use.

Protocol for kill switch testing

- Using 2 plates of LB solid medium (with K+ antibiotics), divide each plate into 4 sections, add 10uL of BN063 and BL065 cultured bacteria mixture into each section. (top 2 quarters without Ara, bottom 2 quarters with the addition of Ara Inducer droplet)
- Remark: Ara droplet addition: 30 uL; let Ara droplet completely dry before adding bacteria.
- Again, using 4 centrifugal tubes of LB liquid medium (1mL each), add 10uL of K+ antibiotics into each tube; add 10uL of BN063 and BL065 cultured bacteria mixture into each centrifugal tube. Then, choose 2 tubes and add 10uL of Ara Inducers into these 2 tubes.
- INCUBATE @ 37℃ OVERNIGHT CULTURE FOR ALL PLATES + TUBES.

![](https://static.igem.org/mediawiki/2021/3/38/T--SDSZ_China--experiment-11.1.png)

Protocol for TEM sample testing

- Use a centrifuge to separate calcium hydroxy phosphate
- Sample, disperse it in absolute ethanol
- Ultrasonic dispersion
- Drop on the Carbon Membrane Supporting Film(on the Copper Mesh)
- Put them into an ice box

Silicatein Activity Assay

- Prepare a solution of 100 mM tetramethyl orthosilicate (TMOS) in 1 mM HCl to prepare prehydrolyzed silica particles. Stir at room temperature for 15 minutes to completely hydrolyze TMOS to silica monomers.
- Prepare two reactions:

![](https://static.igem.org/mediawiki/2021/6/62/T--SDSZ_China--experiment-13.1.png)

- Incubate at NIST standard temperature and pressure (1 atm, 20 ºC) for 2 hours.
- Centrifuge sample at 15,000 RPM for 2 minutes to precipitate polymerized silica particles.
- Decant supernatant containing unaggregated silica particles.
- Wash 3 times with distilled H2O to remove free, hydrolyzed TMOS.
- Aliquot the unreacted hydrolyzed TMOS remaining in solution after silica aggregation.
- Treat with 2 M NaOH for 1 hour at 80 ºC to ensure complete hydrolysis of silica particles to monomer/dimer state.
- Remove aliquots of 500 μl and add to solution of 750 uL water and 75 uL of acidic solution of ammonium molybdate (20 g (NH4)6Mo7O24•4 H2O and 60 mL of concentrated Hcl [36%]).
- Incubate at NIST standard temperature and pressure for 20 minutes. Solution should have a yellowish hue after incubation.
- Add 400 uL of reducing agent solution (see above). Solution should turn a bluish color.
- Record absorbance at 810 nm and compare to standard curve.

To make sure we have valuable comparison, in our own experiment apart from the previous protocol, we designed:
- two negative control groups----NC1, NC2;
- two experiment groups----USE ultrasonic homogenizer; NO ultrasonic homogenizer.

Protocol for preparing inoculated silicatein bacteria mixture with inducer IPTG.

- Inoculate Silicatein, S010, S011, S012 bacteria strain from plates to LB liquid medium
- Overnight culture with 37-degree Celsius on a shaker
- They are prepared to be used for TEM/SEM testing!

![](https://static.igem.org/mediawiki/2021/c/cb/T--SDSZ_China--experiment-14.1.png)

Protocol for TEM testing for Silicatein produced

- Draw 1mL of Si bacteria mixture from the original LB medium glass bottle into microcentrifuge tube. Add 10uL of bacteria mixture onto the piece of carbon film (mark as #1).
- Dilute the original Si bacteria mixture with ratio 1:10; add another 10uL of bacteria mixture onto another piece of carbon film, repeat it twice (mark as #2, #5).
- Repeat the step above twice, mark as #3, #4.
- Put all 5 pieces of film into the 80 degree Celsius dryer until they are completely dry, then send them for TEM testing.

Protocol for SDS-PAGE protein gel electrophoresis

1. Prepare SDS-PAGE running buffer(1X): 10mL.
2. Prepare gels and assemble the electrophoresis device.
3. Heat samples for 5 minutes at 94 degrees Celsius.
4. Cover the chamber & firmly connect both the anode and the cathode. Set the voltage on the electrophoresis power supply to a constant voltage of 120V.
5. Run the gel electrophoresis cell for 30-60 minutes.
6. After electrophoresis, remove the gel by floating it off the plate into water.
7. Stain the SDS-PAGE with Coomassie blue dyes, then de-stain it.
8. Observe the electrophoresis results that show up in the E-gel imager.

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# Team:SDSZ China/Results

![](https://static.igem.org/mediawiki/2021/0/08/T--SDSZ_China--background_results.jpeg)

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Project

Description

Engineering

Results

Contribution

Proof of Concept

Parts

Product

Model

Entrepreneurship

Implementation

Lab

Experiments

Notebook

Safety

HP

Human Practices

Communication

Team

Members

Collaborations

Attributions

![]()

Results

Overview

Our results are composed of three parts: the experiment of silicon mineralization, the experiment of kill switch, and the experiment of calcium mineralization. Each experiment includes the construction of plasmids and the tests to confirm its abilities.

Silicon Mineralization

Since silicateins can induce the precipitation of silica only when it is exposed to the TMOS, it needs to be placed on the membrane surface. Conseuqently, we planned to co-express the silicatein gene with the INP gene ( ice nucleation proteins, a kind of transmembrane proteins)(Figure 1-1), so the fusion protein can expose silicatein to the extracellular environment. We planned to use the Gibson Assembly to complete the construction and used the gel electrophoresis to test whether the constuction succeeded.

![](https://static.igem.org/mediawiki/2021/d/d3/T--SDSZ_China--results_1-1.png)
![](https://static.igem.org/mediawiki/2021/7/75/T--SDSZ_China--result_1-2.png)

Fig.1-1

Fig.1-1 shows the plasmid construction of INP-silicatein.

Fig.1-2

Fig.1-2 represents the results of gel electrophoresis to test the fragment of silicatein extracted from previous vectors.

![](https://static.igem.org/mediawiki/2021/4/47/T--SDSZ_China--results_1-3.png)

Fig.1-3

Fig.1-3 is the schematic showing genetic components
of the fusion protein.

We found the sequences of silicatein gene and INP gene in the NCBI database and optimized codon based on the codon bias of E.coli. We synthesized the INP gene by ourselves and bought the silicatein gene. After obtaining the plasmids that contain INP gene from the biosynthesis company, our team used PCR to extract the seuqence from the plasmids. We expected to see two bands in the gel electrophoresis, representing the silicatein and the vector respectively. The result (Fig.1-2) presented two binds, which proved that we extracted the silicatein successfully.

![](https://static.igem.org/mediawiki/2021/7/7b/T--SDSZ_China--result_1-4.png)

Fig.1-4

Fig.1-4 represents gel electrophoresis of two connections.

After successfully extracting the silicatein, we transformed the gene that we constructed through the Gibson Assembly. Then, we still use the gel electrophoresis to test whether we constructed the plasmids successfully. Since the gene we inserted is too long to detect as a whole, which is about 2000bp, we decided to detect sequences of two connections (one connected the silicatein with the vector and the other connected the INP with silicatein) , both of which are approximately 600bp. Conseuqently, we expected to see two binds of 600bp. From the graph above, we could see that the result was positive, proving that the plasmids are successfully constructed.

After co-culturing the E.coli with IPTG (the inducer of the silicatein gene) overnight, we wanted to test the acitivity of produced silicateins by comparing its OD values with the controlled group. Furthermore, we also wanted to prove one of our assumptions that silicateins may also exists inside the cell. Therefore, we used ultrasonic comminution to break the cells and exposed the intracelluler silicatein to the buffer, and compare its OD value with the silicatein mixture without homogenization.

![](https://static.igem.org/mediawiki/2021/1/16/T--SDSZ_China--result_1-5.jpeg)
![](https://static.igem.org/mediawiki/2021/7/74/T--SDSZ_China--result_1-6.png)

Fig.1-5

Fig.1-6

NC1 is a negative control group #1 (only TMOS + HCl)

NC2 is a negative control group #2 (only TMOS + HCl + PBS buffer)

NP is the experimental group without ultrasound homogenization (TMOS + HCl + NaOH + silicatein extracted from 5mL of original bacterial mixture)

P is the experimental group under ultrasound homogenization(TMOS + HCl + PBS buffer + NaOH + silicatein extracted from 5mL of original bacterial mixture)

Fig.1-6 shows the OD values of four groups, as shown in Fig.1-5. In each group, the first two bars represent the OD values without centrifuge; the last bar represents the OD value after centrifuge.

The above results show that both experimental groups had higher OD values, indicating that silicateins could trigger the precipitation of silica. The higher OD value of the mixture with the homogenezation also validated our assumption, which pointed our team to a direction to improve our design, to place more silicatein on the membrane surface.

![](https://static.igem.org/mediawiki/2021/3/34/T--SDSZ_China--result_1-7.jpeg)

Fig.1-7

Fig.1-7 represents the TEM result of engineered bacteria.

To test the activity of silicatein in a more direct way, we applied TEM testing as well. Our team added bacteria mixture of 5 diffenrent concentration onto the 5 pieces of carbon film and put them into the 80 degree Celsius dryer. After they are completely dry, we sent them for TEM testing. We expected to see dark particles on the membrane surface. In the Fig.1-6, we saw dark particles on the surface with diameter of 50nm, which further verified that the plasmid could induce the precipitation of silica.

Calcium Mineralization

Same as the silicatein, only when Mfp5 is on the surface of cells can they induce the precipatation of hydroxyapatite. According to the research, we found that one of several main components, CsgA protein, could be used to bring the protein to the cell surface. As a result, we constructed the CsgA-Mfp5 fusion protein (Fig.2-1). As previous experiments showed that the expression of a single Mfp5 gene may be insufficient to cause a notable precipitation, our team also transformed two Mfp5 to a group of plasmids.

![](https://static.igem.org/mediawiki/2021/9/92/T--SDSZ_China--results_2-1.png)

Fig.2-1

Fig.2-1 shows the plasmid construction of csgA-Mfp5-Mfp5.

![](https://static.igem.org/mediawiki/2021/e/e7/T--SDSZ_China--results_2-2-1.png)
![](https://static.igem.org/mediawiki/2021/b/bb/T--SDSZ_China--results_2-3-1.png)

Fig.2-2

Fig.2-3

Fig.2-2 shows the schematic of csgA-Mfp5.

Fig.2-3 shows the schematic of csgA-Mfp5-Mfp5.

The process of the construction was difficult. We failed to construct the qualified plasmids by the Gibson Assembly for several times, which baffled us for a long time until our team leader found that the sequence of CsgA was not complete. After we complemented the sequence by using the Gibson Assembly, we used the gel electrophoresis to test whether we have succuessfully constructed the plasmids. The number of bp of backbone corresponded to the bp number we had expected, and the newly inserted CsgA fragment also presented in the plasmid. Consequently, the result proved that we succeeded in constructing the plasmids.

![](https://static.igem.org/mediawiki/2021/0/03/T--SDSZ_China--results_2-4.png)

Fig.2-4

Fig.2-4 shows gel electrophoresis of all the fragments
needed for calcium mineralization.

In order to make sure that construction of plasmids was successful, we also used Sanger Method to sequence the gene and compared the result with the sequence we had designed. Fortunately, this time the experiment was proved to be a success.

![](https://static.igem.org/mediawiki/2021/b/bd/T--SDSZ_China--results_2-5-1.png)

Fig.2-5

Fig.2-5 shows the results of Sanger Method sequencing.

After confirming that the plasmids were as same as what we designed, we wanted to verify its function in induce the precipitation of the hydroxyapatite. This time, we used TEM testing to prove the existence of the hydroxyapatite. Unfortunately, the researchers that helped us to perform the TEM testing were so careless that they broke gratings and destroyed the cell. Consequently, we failed to get the image of the cell surface. We could only judged from the electro-diffaraction diagram that the crystal existed in the sample, which could indirectly verify the function of the Mfp5.

![](https://static.igem.org/mediawiki/2021/f/f6/T--SDSZ_China--results_2-7.png)
![](https://static.igem.org/mediawiki/2021/8/89/T--SDSZ_China--results_2-8.png)

Fig.2-6

Fig.2-7

Fig.2-6 is the result of TEM testing.

Fig.2-7 shows the SDS-PAGE of all functional proteins.

Kill Switch

In order to ensure the safety, our team planned to incorperate a kill switch in plasmids. We initially chose K1 and K2 as possible plasmids, of which their gene circuits are shown respectively in Fig.3-1 and Fig.3-2. Both of the K1 and K2 have AraC promoters, while in K1 the toxic protein is kid toixn, in K2 the toxic protein is colicin, both of which have high specificity to the E.coli.

![](https://static.igem.org/mediawiki/2021/f/f1/T--SDSZ_China--description-Lacl-AraC-Kid_Toxin2.png)
![](https://static.igem.org/mediawiki/2021/a/aa/T--SDSZ_China--results_3-2.png)

Fig.3-1

Fig.3-2

Fig.3-1 represents the schematic of K1.

Fig.3-2 represents the schematic of K1.

When arabinose is added to the system, it binds to AraC, which then promotes expression of toxic proteins, which are Kid Toxin in K1 and Colicin in K2, and kills the cells.

To test if these two kill switches are effective, we designed two plasmids to express the two genes, as shown in Fig.3-3 and Fig.3-4 respectively. Fig.3-3 represents the plasmid structure of K1, while Fig.3-4 represents that of K2.

![](https://static.igem.org/mediawiki/2021/8/87/T--SDSZ_China--results_3-3.png)
![](https://static.igem.org/mediawiki/2021/c/c3/T--SDSZ_China--results_3-4.png)

Fig.3-3

Fig.3-4

Fig.3-3 represents the plasmid construction of K1.

Fig.3-4 represents the plasmid construction of K2.

However, after we successfully constructed the plasmids, those carrying K2 showed leakage of toxin when we were incubating the bacteria, so we only used K1 in further research.

We wanted to know if K1 could perform as we expected. Thus, after transforming the plasmid into competent BL21 cells, we inoculated the cells in eight groups, including four in liquid medium and the other four in solid medium. The results are shown in the figures below. Arabinose was added in experimental groups. We tested and calculated the mean OD value of four liquid groups.

As shown in Fig.3-5 & Fig.3-6, in the upper-left area of the medium, normal E.coli without the transformation of the plasmids could grow normally; in the upper-right area, transformed cells could grow normally without the presence of the Arabinose; in the bottom area, the cells were killed in the Arabinose-contained environment. Fig.3-7 shows that the OD value of liquid medium after adding Arabinose is lower than that of medium without Arabinose. These results indicate that K1 performed as effectively as expected.

![](https://static.igem.org/mediawiki/2021/7/74/T--SDSZ_China--result_3-5.png)
![](https://static.igem.org/mediawiki/2021/2/29/T--SDSZ_China--results_3-6-2.png)

Fig.3-6

Fig.3-6 shows the results of kill switch tests,
as shown in Fig.3-5.

![](https://static.igem.org/mediawiki/2021/2/2e/T--SDSZ_China--result_3-7.png)

Fig.3-5

Fig.3-5 shows the results of the experiment
testing K1 kill switch.

Fig.3-7

Fig.3-7 shows the OD value of liquid medium
with(experimental) and without(control) adding arabinose

Reference

[1] Müller, Werner EG, et al. "Bioencapsulation of living bacteria (Escherichia coli) with poly (silicate) after transformation with silicatein-α gene." Biomaterials 29.7 (2008): 771-779.

[2] Poulsen, Nicole, and Nils Kroger. "Silica morphogenesis by alternative processing of silaffins in the diatom Thalassiosira pseudonana." Journal of Biological Chemistry 279.41 (2004): 42993-42999.

[3] Park, Jeong Chan, et al. "R5 Peptide-based Biosilicification Using Methyltrimethoxysilane." Biotechnology & Bioprocess Engineering 23.1 (2018).

[4] Wei, W. et al. An underwater surface-drying peptide inspired by a mussel adhesive protein. Adv. Funct. Mater. 26, 3496–3507 (2016).

[5] Wang, Y. et al. Living materials fabricated via gradient mineralization of light-inducible biofilms. Nature chemical biology 351-359 (2021)

[6] Dick J, Windt W D, Graef B D, et al. Bio-deposition of a hydroxyapatite layer on degraded limestone by Bacillus species[J].Biodegradation,2016(4):357-367.

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