iGEM 2020 — Team WHU-China

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# Team:WHU-China/Notebook

Attributions

![](https://static.igem.org/mediawiki/2020/c/c4/T--WHU-China--logomaybe.png)

Wet Lab>Notebook

![](https://static.igem.org/mediawiki/2020/f/f0/T--WHU-China--notebook.jpg)

[Protocols](#Protocol)

[Notebook for Quenching Module](#no1)

[Notebook for Sensing Module](#no2)

[Notebook for Chemotaxis](#no3)

[Notebook for TA systems](#no4)

[Notebook for Cell-free quorum sensing](#no5)

Protocols

[Plasmid purification](https://static.igem.org/mediawiki/2020/2/25/T--WHU-China--note1.pdf)

[Restriction endonuclease digestion](https://static.igem.org/mediawiki/2020/c/c0/T--WHU-China--note2.pdf)

[PCR](https://static.igem.org/mediawiki/2020/e/e5/T--WHU-China--note3.pdf)

[DNA purification](https://static.igem.org/mediawiki/2020/c/c1/T--WHU-China--note4.pdf)

[Cell-free protein synthesis](https://static.igem.org/mediawiki/2020/0/04/T--WHU-China--note5.pdf)

[Suspension cell line subculture](https://static.igem.org/mediawiki/2020/5/54/T--WHU-China--note6.pdf)

[Differentiation induction of HL-60](https://static.igem.org/mediawiki/2020/e/e0/T--WHU-China--note7.pdf)

[Chemotaxis assay: preliminary experiments](https://static.igem.org/mediawiki/2020/9/97/T--WHU-China--note8.pdf)

[Cell cryopreservation](https://static.igem.org/mediawiki/2020/b/b9/T--WHU-China--note9.pdf)

[Preparation and cryopreservation of RNA samples from dHL-60 cells](https://static.igem.org/mediawiki/2020/f/f6/T--WHU-China--note10.pdf)

[Chemotaxis assay of THP1](https://2020.igem.org/File%3AT--WHU-China--protocol_11_12.pdf)

[RNA extraction from dHL-60 and HL-60](https://static.igem.org/mediawiki/2020/0/01/T--WHU-China--note13.pdf)

[Flow cytometry](https://static.igem.org/mediawiki/2020/3/36/T--WHU-China--14.pdf)

Notebook for Quenching Module

31/8/2020

Picked 3 single colonies of recovered DH5α strains and transferred them into 3 test tubes with LB liquid medium.

2/9/2020

Added 50μL of DH5α into a new test tube with 5mL LB liquid medium and then cultured in a shaker for 3h(OD600=0.941).

Prepared competent bacteria according to the protocol of One Step Competent cell Preparation Kit, Beyotime (D0301) and transferred GFP, rhlR, AiiA and sensor parts from previous iGEM kits respectively into the competent bacteria (100μL/each), then cultured under 37℃ overnight. Failed to grow single colonies.

5/9/2020

3μL sensor part from previous iGEM kit was transferred into 100μL newly purchased DH5α strains according to the instruction of the kit, then spread them on the plates with chloramphenicol under 37℃ overnight.

The absorption peak of E0040 (GFP) and sensor parts solution were both 270nm after measuring.

25/9/2020

Dissolved the AiiC gene dry powder from IDT to 10ng/μL and the primers to 100μM. Used PCR to get the sequence containing AiiC and Gibson overlap sequence, Ta was set to 48℃.
Failed to get the target band.

26/9/2020

Used EcoRI and PstI to digest AiiC and PSB1A3 (from previous iGEM kit).

Incubated under 37℃ overnight, then inactivated the enzymes under 80℃ for 5 min.

27/9/2020

Purified AiiC and PSB1C3 according to the protocol of TIANquick Midi Purification Kit, Tiangen (DP204), then used agarose gel electrophoresis to observe the result of purification. Only can see PSB1A3 band.

Used agarose gel electrophoresis to observe the original AiiC gene solution. Got the target band.

Used the Rapid DNA Ligation Kit to ligate AiiC and PSB1A3.

After ligation, transferred them into competent bacteria according to the kit and spread it on a plate with ampicillin.

29/9/2020

Used colony PCR to check the AiiC in the 3 strains inoculated on 28/9.

2/10/2020

Extracted the plasmids containing AiiA from TOP10 (ordered from Genscript) according to the instruction of TIANquick Midi Purification Kit, Tiangen (DP204).

Used agarose gel electrophoresis to check the plasmids containing AiiA. Observe one band which is much larger than the target band.

Used colony PCR to get pvdq from PAO1.

Transferred plasmids containing AiiA into prepared competent BL21, then cultured on LB plates with kanamycin under 37℃ overnight.

3/10/2020

Used agarose gel electrophoresis to check the result of pvdq colony PCR. Failed to get target band.

Haoyu Zheng helped us pick single colonies containing AiiA into liquid LB medium.

4/10/2020

Used colony PCR to get pvdq from PAO1. Prepared 8 PCR tubes of reaction systems in total for different Ta from 50-56℃.

Used agarose gel electrophoresis to check the results of pvdq colony PCR. Failed to get target band.

5/10/2020

Used colony PCR to get pvdq from PAO1. Prepared 3 PCR tubes of reaction systems in total for different Ta from 51.5-56.4℃.

Used agarose gel electrophoresis to check the result of pvdq colony PCR. Failed to get target band.

Took 1mL of AiiA-pET28a strains from each of the 3 tubes, then amplified and cultured in LB liquid medium (about 120 mL with 0.6 mL kanamycin) overnight under 37℃ and 200 rpm.

6/10/2020

Added 5mL bacteria solution from each conical flask prepared on 5/10 into 120 mL LB liquid medium, then cultured under 200 rpm for 1h (OD600=0.456). Added 60μl IPTG (1M) each bottle and incubate under 28℃ (negative control: added 10ml bacteria solution without adding IPTG into 50 ml EP tube).

Used colony PCR to check the AiiA in the three flasks of the bacteria solutions. Used agarose gel electrophoresis to check the result of AiiA colony PCR. Only got one band over 12kb. Then stopped induced expression and picked 3 original TOP10 single colonies and transferred them into LB liquid medium (5mL LB with 25μL kanamycin) respectively, cultured overnight under 37℃,200 rpm.

Used EcoRI and PstI to digest 17 μL AiiC (about 10 ng/μL) and 8 μL PSB1C3 (about 25 ng/μL). Prepared 2 reaction systems of AiiC and 1 of PSB1C3.

Incubated under 37℃ overnight, then inactivated the enzymes under 65℃ for 25min.

7/10/2020

Extracted the plasmids containing AiiA from TOP10 according to the instruction of TIANquick Midi Purification Kit, Tiangen (DP204).

Used agarose gel electrophoresis to check the products of digestion of PSB1C3, the result of AiiC digestion, plasmids containing AiiA. Got one band over 12kb from plasmids extracted from TOP10.

Used colony PCR to check AiiC (3 reaction systems) from previous bacteria solutions which may contain AiiC ligated with pSB1A3(do the same thing as before, but change annealing temperature this time).

Used agarose gel electrophoresis to check the result of AiiC colony PCR. Failed to get target band.

Used EcoRI and PstI to digest 17 μL 12kb plasmids from TOP10.

Incubated under 37℃ for 2h, then used agarose gel electrophoresis to check the result of digestion. Got 2 bands of 6-8kb.

Purified AiiC and PSB1C3 according to the instruction of the DNA Purification Kit.

8/10/2020

Transferred 100μL BL21 into 5mL LB liquid medium and then cultured in a shaking table for until OD600 reached 0.510.

Used PCR to get the sequence containing AiiC and Gibson overlap sequence (3 reaction systems for 56-65.6℃).

Prepared competent BL21 (OD600=0.510).

Used the Rapid DNA Ligation Kit to ligate AiiC and PSB1C3, then transferred the product into competent BL21, then cultured on LB plates overnight under 37℃.

Used agarose gel electrophoresis to check the result of PCR. Failed to get the target band.

9/10/2020

Used EcoRI and PstI to digest plasmids from company to check AiiA. 37℃ water bath for 2h.

Used agarose gel electrophoresis to check the result of digestion. The negative control had a 12kb band while the enzyme-digested product had a 1kb band and a 7kb band.

Picked 6 single colonies from the 3 plates cultured on 8/10 (the colonies had grown into lawn), then transferred into 6 tubes of LB liquid medium (6mL LB with chloramphenicol).

Prepared competent BL21 (OD600=0.34).

Used the Rapid DNA Ligation Kit to ligate AiiC and PSB1K3, PSB1C3. Transferred the production into competent BL21, then cultured on LB plates overnight under 37℃.

10/10/2020

Picked 3 single colonies (1 containing AiiC-PSB1C3 and 2 containing AiiC-PSB1K3) and transferred them respectively into 3 test tubes of 6mL LB liquid medium (containing 50ng/μL antibiotics(Cm and Kan separately)), cultured overnight under 37℃, 200rpm.

11/10/2020

Built a cell-free system according to the protocol. After incubation in 37℃ for 1h, added 100μL cell-free system into 400μL nickel column.

Used SDS PAGE to check the expression of AiiA in the cell-free system.

Took each 1.5 mL bacteria solution from LB liquid medium cultured on 10/10 (2 tubes of PSB1K3-AiiC and 1 tube of PSB1C3-AiiC), extracted the plasmids (PSB1K3-AiiC and PSB1C3-AiiC) according to the instruction of TIANprep Mini Plasmid Kit (final volume: 100μL each).

Used EcoRI and PstI to digest PSB1K3-AiiC and PSB1C3-AiiC.

Used agarose gel electrophoresis (1%) to check PSB1C3-AiiC plasmids, PSB1K3-AiiC plasmids and the result of digestion. Got target band of PSB1C3-AiiC plasmids and only one band of the enzyme-digested products of PSB1C3-AiiC (about 2000bp).

12/10/2020

Used agarose gel electrophoresis (0.6%) to check the result of digestion again. Got only one band of the enzyme-digested products of PSB1C3-AiiC.

Transferred 60μL BL21 solution into 5mL LB liquid medium, cultured under 37℃, 200rpm until OD600 reached 0.305, then prepared competent BL21. Transferred AiiA plasmids in to the competence bacteria, then plated them on the plates with corresponding antibiotics overnight under 37℃.

Transferred bacteria containing PSB1C3-AiiC into 120 mL LB liquid medium (containing 200μL chloramphenicol) .

Checked the results of SDS-PAGE done on 11/10. Only find blurry or no target band. Conducted SDS-PAGE again. But still no target bands.

13/10/2020

Prepared competent BL21.

Transferred plasmids containing AiiA into the competent BL21.

Added 5 mL bacteria (containing PSB1C3-AiiC) solution into 120 LB liquid medium (containing 200 μL chloramphenicol), cultured under 37℃, 200rpm for 2h. Then added 60 μL IPTG into the culture, cultured overnight under 28 ℃.

14/10/2020

Picked 4 single colonies (pET28a-AiiA) cultured on 12/10, then added them respectively into 4 tubes of 5mL LB liquid medium (containing 25μL kanamycin).

Extracted pET28a-AiiA plasmids according to the instruction of TIANprep Mini Plasmid Kit, Tiangen (DP103), then used EcoRI and PstI to digest the product.

Used agarose gel electrophoresis (1%) to check the result of digestion. Both the original solutions of plasmids and solutions of plasmids with enzyme digestion had no bands.

Checked the result of PSB1C3-AiiC induced expression.

Notebook for Sensing Module

SIAT shared their stock of nissle1917 strain. Stored at 4°C after receipt.

23/8/2020

Inoculated three single colonies of nissle1917 into 5ml LB liquid medium and cultured overnight in a 30°C shaker.

24/8/2020

Took one of nissle1917 culture to streak on the LB plate, cultured overnight at 30°C. Other stored at 4°C.

25/8/2020

The cultivation plate of nissle1917 was sent to Tongji.

30/8/2020

Took 50 μl glycerol stock of DH5α and spread on the LB plate, cultured overnight at 30°C

Picked three single colonies of BL21 from the LB plate stored in the laboratory, inoculated them into 5ml LB liquid medium and cultured overnight in a 30°C shaker.

31/8/2020

Prepared the solution of ampicillin (50mg/ml), kanamycin (10mg/ml) and chloramphenicol (34mg/ml, ethanol), filtered and sterilized through 0.22 micron syringe filters.

1/9/2020

Prepared competent cells of DH5α according to the protocol of One Step Competent cell Preparation Kit, Beyotime (D0301).

Used 10 μl ddH2O to dissolve the DNA of BBa\_I746361, BBa\_B0010, pSB4A5 and BBa\_J23100 in the dry powder plate.

Transformed the target gene and spread on the LB plate containing the corresponding antibiotic, cultured overnight at 37°C.

2/9/2020

No transformed colonies on the plate, ordered from GenScript instead.

8/9/2020

Picked three single colonies of BL21 from the LB plate stored in the laboratory, inoculated them into 5ml LB liquid medium and cultured overnight in a 37°C shaker.

15/9/2020

Colony PCR to obtain CDS of pqsR gene according to the protocol of PCR Kit with Taq, Beyotime (D7232).

16/9/2020

Detected PCR products by TAE agarose gel electrophoresis (1%, 120V, 30min) and found no target band.

18/9/2020

Repeated the colony PCR but reduced Ta to 52°C.

19/9/2020

Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found no target band.

24/9/2020

Repeated the colony PCR but used primers with fewer overhangs and extended pre-denaturation time to 5min.

Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.

Gel extracted according to the protocol of TIANgel Midi Purification Kit, Tiangen (DP209).

Used 10 μl ddH2O to dissolve the DNA of TEVprotease and TEVprotease+histag.

Took 4 μl of TEVprotease, TEVprotease+histag and pSB1A3 linearized vector, digested them with EcoRI and PstI overnight at 37°C according to the protocol of EcoRI, Beyotime (D6329) and PstI, Beyotime (D6565).

25/9/2020

Detected gel extracted products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.

Detected digested products of TEVprotease, TEVprotease+histag and pSB1A3 by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.

Extracted digested products of TEVprotease, TEVprotease+histag and pSB1A3.

Repeated the PCR but used extracted products of pqsR CDS as template and used the first pair of primers to add biobricks prefix and suffix.

26/9/2020

Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found no target band.

Streaked on the LB plate containing kanamycin to resuscitate glycerol stock of TOP10 with plasmids which inserted BBa\_B0010 and expression devices of phiR73 delta and GFP, cultured overnight at 30°C.

27/9/2020

Ligated digested products of TEVprotease, TEVprotease+histag and pSB1A3 separately according to the protocol of Rapid DNA Ligation Kit, Beyotime (D7002).

Transformed the ligated products and spread on the LB plate containing ampicillin, cultured overnight at 30°C.

28/9/2020

Normal recombinants appeared on the plates, picked three single colonies, inoculated them into 5ml LB liquid medium containing ampicillin and cultured overnight in a 30°C shaker.

29/9/2020

Used recommended standard vector sequencing primers to build colony PCR system to check the recombinant plasmid.

Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.

Repeated the PCR of pqsR CDS using longer primers but extended pre-denaturation time to 5min.

30/9/2020

Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found no target band.

2/10/2020

Took 12 μl of pSB1A3 and pSB1C3 linearized vector, plasmid inserted BBa\_B0010 and GFP expression device, digested them with EcoRI and PstI overnight at 37°C according to the protocol of EcoRI, Beyotime (D6329) and PstI, Beyotime (D6565).

Took 12 μl of plasmid inserted TEVprotease, plasmid inserted BBa\_B0010 and GFP expression device, digested them with XbaI and PstI overnight at 37°C according to the protocol of XbaI, Beyotime (D6713) and PstI, Beyotime (D6565).

Repeated the PCR of pqsR CDS but change primers.

3/10/2020

Detected digested products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.

Gel extracted according to the protocol of TIANgel Midi Purification Kit Tiangen (DP209).

Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.

Repeated the PCR of pqsR CDS but used the first pair of primers.

4/10/2020

Detected PCR products by 1% agarose gel electrophoresis (TAE, 120V, 30min) and found target band.

Gel extracted according to the protocol of TIANgel Midi Purification Kit Tiangen (DP209).

5/10/2020

Ligated digested products of GFP expression device and pSB1A3 according to the protocol of Rapid DNA Ligation Kit, Beyotime (D7002).

Transformed the ligated products and spread on the LB plate containing ampicillin, cultured overnight at 30°C.

7/10/2020

No transformed colonies on the plate.

Repeated but failed again.

Notebook for Chemotaxis

2020/9/4

Subculture of HL-60

1. The HL-60 cell line (culture bottle) was obtained from the Preservation Center（CCTCC）

2. Transfer 2 / 3 cell suspension to another two culture bottles, three culture bottles together (37 ℃, 5% carbon dioxide), the total amount of cell suspension in each bottle is 6ml.

Medium: RPMI-1640 (10% FBS)

2020/9/5

Subculture of HL-60

1. The cell density was more than 90% in three HL-60 culture bottles under inverted microscope.

2. In three culture bottles, two parts of 3 ml cell suspension were taken from each bottle and injected into 10 cm culture dish respectively, and 7 ml RPMI-1640 (10% FBS) was added to each dish. No. 1-6.

3. Under the inverted microscope, the cell density decreased significantly, about 20%.

4. 37 ℃, 5% carbon dioxide incubator.

5. Order 500 ml RPMI-1640.

2020/9/6

HL-60 culture

1. The culture dish was observed in the morning, and the cell density was not high, so it was not passaged. Put them back into the incubator for cultivation.

2020/9/7

Medium preparation & HL-60 subculture

A. Medium preparation

RPMI-1640: 45 ml, FBS: 5 ml, Total volume: 50 ml

Multiple tubes were prepared and packed in 50 ml centrifuge tubes.

B. Subculture of HL-60

1. The density of No. 1-6 culture dishes was more than 90%.

2. After blowing each dish, take 5 ml cell suspension and add it into a new culture dish numbered X-1 (x is the original number), and add 5 ml medium to each dish.

3. The cell density of the old and new culture dishes decreased compared with before, but the cell density of the old one was significantly higher than that of the new one. It is speculated that the reason is that the air mixing is not even enough. Put it into the incubator for cultivation.

2020/9/8

Culture of HL-60 by changing medium

1. Take out 6-7 ml cell suspension from each plate of 12 plates, add 6-7 ml medium, and put them back into the incubator for culture

2020/9/9

HL-60 was cultured by changing medium and the acquisition of THP1

1. HL-60 liquid change.

2. The THP1 cell line was obtained from the Preservation Center and cultured in the incubator.

2020/9/10

HL-60 changing medium and THP1 subculture

1. When HL-60 was cultured in different medium, it was found that there were small particles around the cells, which may be the fragments left by cell death, but the risk of contamination was not ruled out.

25/9/2020

2. THP1 subculture: the cell suspension (about 9 ml) in the culture bottle was divided into 3 times, 3 ml suspension was taken each time to a new 10 cm cell culture dish, and 7 ml RPMI-1640 (10% FBS) was added to each culture flask, and then cultured in the incubator, labeled as plates 1, 2 and 3.

2020/9/11

HL-60 changing medium and subculture of THP1

1. After changing the solution of HL-60, it was found that the particles still existed and the cell morphology was slightly irregular.

2. THP1 subculture: take 2.5 ml cell suspension from each dish into a new dish, and add 7.5 ml medium to each dish, labeled as 1-1, 1-2, 1-3, 1-4, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-3, 3-4. Put it into the incubator for cultivation.

2020/9/12

Differentiation induction of HL-60

A. Preparation of cell suspension

1. Select the culture dish 1, 2, 3, 2-1, 4-1, 6-1 of HL-60 cell line, and draw 50 ml cell suspension (packed in two 50 ml centrifuge tubes, 25 ml in each tube).

2. 1 ml cell suspension was taken out from each centrifuge tube and counted by cell counting plate and trypan blue exclusion method to estimate cell density.

3. Take 10 ml suspension from each centrifuge tube to two 10 ml centrifuge tubes, and take 1 ml cell suspension from each tube to two new 10 ml centrifuge tubes, and add 9 ml culture medium.

4. Repeat operation 3 to make the final cell concentration reach about 5.5e6/ml.

B. Preparation of differentiation inducible medium

RPMI-16401: 45 ml, FBS: 15 ml, DMSO: 1.9 ml

Total volume: 150 ml

C. Establishment of differentiation inducible culture system

1. Take out 1 ml diluted cell suspension and inject it into a new 10 cm culture dish, and add 6.5 ml medium with differentiation induction.

2. Repeat operation 1 until all cell suspensions are used up. 20 culture plates, labeled 1-20, are obtained and cultured in the incubator.

2020/9/13-2020/9/16

Replacement of HL-60 and THP1

2020/9/17

Chemotaxis of CXCL1 to THP1, a preliminary experiment

A. Preparation of concentration gradient of CXCL1 solution

B. Cell suspension preparation

C. Transwell Migration Assay

Sampling sequence: the first row: Control1, control2, control3, 10nM-1, 10nM-2, 10nM-3

The second row: 1nM-1, 1nM-2, 1nM-3, 0.1nM-1, 0.1nM-2, 0.1nM-3

2. Put into the incubator (37 ℃, 5% carbon dioxide) for 15 h (overnight)

D. Result count

Results: it was found that the number of cells in each counting room was almost less than 10, and the data was not statistically valid. It was considered to select a higher concentration of chemokines, a longer culture time and more cells in the formal experiment, and use flow cytometry to count cells.

2020/9/21-2020/9/24

Changing medium of HL-60 and THP1

1. It was found that all THP1 cell suspensions except one dish were turbid and pinkish white. The growth of THP1 cells was poor by microscopic examination, which was determined as fungal contamination.

2. Cryopreserved cells: the only one dish of THP1 in good condition

2020/9/27

HL-60 cells were cultured and passaged

1. Fungal contamination was also found in HL-60 cells

2. Cryopreserved cells: HL-60 in good condition

3. Order a new batch of HL-60 and THP1 cells from the Preservation Centre

2020/10/1

Passage culture of THP1 cells

1. THP1 cell line, about 7 ml cell suspension, was obtained from the Preservation Center

2. Subculture: 3 ml and 4 ml cell suspensions were respectively injected into two 10 cm culture dishes, and each dish was supplemented with medium to 15 ml

3. Prepare 40 ml medium with antibiotics

RPMI-1640: 36 ml, FBS: 4 ml, Double antibody: 0.4 ml

2020/10/2

Passage culture of THP1 cells

1. Observation found that one dish of cells in poor condition, suspected of contamination, so abandoned

2. Another dish of cells was introduced into 5 new culture dishes (2 ml cell suspension was left in each dish, and the medium was added to 17 ml)

2020/10/3

Observation of THP1 cell culture

1. No contamination was found, but the cell growth was slow, so the solution was not changed.

2020/10/4

Observation of THP1 cell culture

1. No pollution was found, but a large number of cells agglomerated in the shape of mulberry, and the agglomerations decreased significantly after blowing and mixing.

2020/10/6

Observation of THP1 cell culture

1. Individual clusters of THP1 became larger, and the cell density was still not high after blowing and dispersing

2020/10/7

Observation of THP1 cell culture & acquisition of Escherichia coli K-12 MG1655

1. The density of THP1 increased by visual inspection, but it was not enough for Transwell migration experiment

2. One strain of E. coli K-12 MG1655 was obtained from Zhixiong Xie's laboratory and cultured overnight in 37 ℃ incubator

2020/10/8

Exchange of THP1 cells & cryopreservation of Escherichia coli K-12 MG1655

1. It was found that the density of THP1 in one dish was enough for Transwell migration experiment

2. All THP1 culture dishes were changed

3. The Escherichia coli K-12 MG1655 plate was stored in 4 ℃ refrigerator

2020/10/9

Chemotaxis of THP1 by chemokines (formal experiment) 1

A. Preparation of chemokine mother liquor

Volume of sterile water: 6.33 μl (CXCL1, cxcl2, cxcl3), 5.95 μl (CXCL8), 5.75 μl (CCL2), 6.41 μl (CCL3), 5.62 μl (CCL8).

The mother liquor of 1E5 nmol / L of seven chemokines was obtained

B. Cell suspension was prepared

C. Transwell Migration Assay

Sampling order:

First board: first row: 1Control 1, 1Control 2, 1Control 3, cxcl1-1, cxcl1-2, cxcl1-3

The second row: 1Control 3, 1Control 4, 1Control 5, cxcl2-1, cxcl2-2, cxcl2-3

Starting time: 10:12

The second board: the first row: 2control 1, 2control 2, 2control 3, cxcl3-1, cxcl3-2, cxcl3-3

The second row: 2control 4, 2control 5, 2control 6, cxcl8-1, cxcl8-2, cxcl8-3

Starting time: 10:42

The third board: the first row: 3control 1, 3control 2, 3control 3, ccl2-1, ccl2-2, ccl2-3

The second row: ccl3-1, ccl3-2, ccl3-3, ccl8-1, ccl8-2, ccl8-3

Starting time: 10:24

2. Put it into the incubator for overnight cultivation

dHL-60 and HL-60 RNA were extracted

Failure, reason: did not shake well after adding isopropanol

2020/10/10-2020/10/11

Chemotaxis of THP1 by chemokines (formal experiment) 2

Count the number of cells in the lower chambers by flow cytometry to calculate the chemotactic index

The number of cells was recorded with SSc as the horizontal axis and FSC as the longitudinal axis.

Notebook for TA systems

2020/10/13

Liquid culture of Escherichia coli K-12 MG1655

1. Select a single colony of E. coli K-12 MG1655 into the test tube containing 5 ml LB medium. 37 ℃, 200 rpm overnight

2020/10/14

MazE gene colony PCR, pET-28a plasmid extraction

A. Colony PCR of MazE gene

1. The experimental group and negative control group were set up. The formula and operation were shown in colony PCR protocol

2.1% agarose gel electrophoresis, see agarose gel electrophoresis protocol

The order of sampling: marker, MazE experimental group, MazE negative control group

Results: in the experimental group, there were obvious target bands between 200 bp and 300 bp, and there were obvious primer dimer bands in both groups

B. Extraction of pET-28a plasmid

A total of 100 μl plasmids were extracted from two tubes of bacterial liquid (1.5 ml each tube) with TIANprep Mini Plasmid Kit, Tiangen (DP103)and then refrigerated in 4 ℃

2020/10/15

Colony PCR of PemI and HipB genes

1. The bacterial solution used on October 14 continued to be cultured overnight, and colony PCR was carried out with the bacterial solution. The two genes were respectively set up in the experimental group and the control group, and the PCR conditions were the same as those in 2020/10/14

Sample order: Marker, P2, P1, P-negative, H, H-negative

Results: there was no target band and there was a very obvious primer dimer band

Analysis: the primer design is not ideal, the annealing temperature is not suitable, and the concentration of bacterial solution is too high

2. The bacterial liquid was stored at 4 ℃

2020/10/16

Colony PCR of PemI and HipB genes

1. 10.15 refrigerated bacterial solution was used for colony PCR. PemI was divided into P1 (annealing temperature 61 ℃), P2 (annealing temperature 57 ℃) and negative control, and HipB was divided into H (annealing temperature 57 ℃) and negative control. The other PCR conditions were the same as 10.14

Results: there was no target band, and there were obvious primer dimer bands in all groups, but it was weaker than that of 10.15

2. Another E. coli K-12 MG1655 colonies were cultured overnight at 37 ℃ at 200 rpm in a tube containing 5 ml LB medium

2020/10/17

PemI and HipB colony PCR & pET-28a identification by electrophoresis

1. 10.16 inoculated bacterial solution was used for colony PCR. The annealing temperature of PemI group was 61 ℃ and that of HipB group was 57 ℃. The other PCR conditions were the same as those of 10.14

2.10.14 the two tubes of pET-28a were identified by electrophoresis, and a band between 3KB and 4KB was obtained, which was supposed to be the super helix pET-28a

2020/10/18

PemI and HipB electrophoresis, MazF colony PCR and pET-28a single restriction digest

1. PemI and HipB electrophoresis is conducted, and no target bands was identified

2. MazF colony PCR, annealing temperature was 46℃, other conditions are the same as 10.15

3. pET-28a single restriction digest by NotI enzyme for 12 hours

2020/10/19

MazE PCR and MazF, PemK, HipA colony PCR

1. MazE PCR was conducted again with another pair of primers to add the homologous arm at the 3’ end. The annealing temperature was 58.0℃ and the extension time was 1 min 20 s. Other PCR conditions are the same as 10.15

2. MazF, PemK and HipA colony PCR were conducted. The annealing temperature for MazF was 60.9℃, for PemK was 53℃ and for HipA was 53.9℃

Result: No target bands were detected.

Notebook for Cell-free quorum sensing

9.11 preparation of culture media (LB, LA, 2x YTPG)

9.15 3A Assembly of plasmids (transcription factors overexpression-T, las activator-L, rhl activator-R) and transformation

9.16 single colonies observed in the Ampr plates

9.17 colony PCR, failed to see any right band

9.18 ligation of remaining digested fragments, colony PCR (failed), and another round of digestion

9.19 single colonies observed in the Ampr plates

9.20 colony PCR, failed to see any right band; ligation of digested fragments, transformation

9.22 preparation of culture media (LB, LA); failed to detect any colony

9.24 PCR amplification of fragments using primer pairs synthesized by Sangon Biotech, failed

9.26 massive digestion of plasmids and fragments, failed to detect any band of fragments in the agarose gel

9.29 PCR amplification of fragments using redesigned primer pairs, successful; ligation using Hieff Clone® Plus Multi One Step Cloning Kit and transformation

9.30 colony PCR, positive for colonies T1, T2, T5, C1-C6, negative for colonies L and R

10.1 plasmid extraction of C; PCR amplification of fragments, ligation and transformation of plasmids L and R

10.2 plasmid extraction of T1 and T5; colony PCR, positive for colonies L6, L7 and R4

10.3 plasmid extraction of T5, L6, L7 and R4

10.10 plasmid F (fluorescence reporter) construction using previously described seamless assembly method

10.11 colony PCR, positive for F2 and F7; preparation of mother solution for CFPS premix

10.12 plasmid extraction of F2, F7, L6, R4; cell-free assays using commercial kit E. coli S30 Extract System for Circular DNA (Promega)

10.13 plasmid extraction of C, R4, F2

10.15 transformation of plasmid T to E. coli BL21 (DE3); preparation of S30 buffer

10.16 recombinant BL21 (DE3) single colonies picked for inoculation; preparation of CFPS premix

10.17 E. coli lysates preparation using sonication

10.18 cell-free assays using self-made E. coli lysates pre-enriched with transcription factors (LasR, RhlR); SDS-PAGE

10.19 plasmid samples (T, L, R, F, C) and pSB1A3 sequencing primers sent to Sangon Biotech; later verification of inserted genes by sequencing

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# Team:WHU-China/Engineering

engineering

![](https://static.igem.org/mediawiki/2020/c/c4/T--WHU-China--logomaybe.png)

Wet Lab>Engineering

![](https://static.igem.org/mediawiki/2020/e/e8/T--WHU-China--logoengineering.jpg)

[Overview](#title1)

[DBTL cycle of Quenching module](#title2)

[DBTL cycle of Sensing module](#title3)

[DBTL cycle of

  cell-free quorum sensing](#title4)

[DBTL cycle of Hardware](#title5)

Overview

The Design-Build-Test-Learn (DBTL) cycle has always been regarded as one of the major ideas of iGEM
and synthetic biology. Due to the limited time of performing experiments this year (we didn’t return
school until September actually), not much useful data was acquired from laboratory. However, we
still endeavored to learn from what we obtained for improving our methodologies, and meanwhile paid
extra efforts to dry lab work for demonstrating our designs. No matter how tough the situation was,
the idea of engineering and DBTL was always kept in our minds, and finally we did generate expected
results to a certain degree. Together with the results, four DBTL cycles are described in detail
below.

DBTL cycle of Quenching module

Quorum quenching enzymes have been placed at the core position of our design. As there are two
classes of them (AHL lactonases and AHL acylases) and they have various substrate specificity
(prefer short-chain or long-chain AHLs or both), together with the fact that P.aeruginosa has two
quorum sensing systems composed of one short-chain and one long-chain AHL, we really need to select
the appropriate enzyme or enzyme combination to obtain better AHL degradation effects. After looking
through plenty of literatures for a long period, we learned that the products of AHL degradation
catalyzed by AHL acylases are more stable so the reaction is usually irreversible while the reaction
catalyzed by AHL lactonases is reversible. So we initially planned to choose several AHL acylases as
the candidates. However as most of the acylases prefer to degrade long-chain AHLs and there is not
much research on those that can cover a broad spectrum, we still chose one lactonase as the
candidate[1]. Finally, we decided to use three broad-spectrum acylases (AiiC[2], AiiO[3] and
PvdQ[4]) and one well-known broad-spectrum lactonase (AiiA) to conduct further experiments. Besides,
in order to utilize the experimental data more efficiently, we also designed a quorum dynamics model
to help us. By connecting the Km value we got from the experiment with the model, we can rapidly
decide the best enzyme(s) to use. For more details, you can see the [model](https://2020.igem.org/Team%3AWHU-China/Model) part of our wiki.
We designed four devices of these enzyme genes. But when we asked company to synthesize these genes,
the failed to synthesize pvdQ and aiiO genes for some reason. And we failed to get pvdQ gene from
P.aeruginosa directly by colony PCR. Therefore we only built the two devices in Figure 1. But again
the process was hard as the aiiC was so large that it was difficult to be ligated with a plasmid. So
we got many false positive colonies. And we couldn’t use colony PCR to check whether we built the
vector successfully as doing PCR on such a large gene was tough as well(only enzyme digestion
verification can be used). So although confirmed by the enzyme digestion test, the vector really
should be tested further. As for aiiA, which was successfully synthesized and put on pET28a(+)
plasmid by the company, it was much easier to test the proper ligation of it.

![](https://static.igem.org/mediawiki/2020/9/9c/T--WHU-China--eng2.png)
![](https://static.igem.org/mediawiki/2020/5/50/T--WHU-China--eng3.png)

Fihure 1: the maps of pSB1C3+aiiC and pET28a (+)+aiiA.

Next step we wanted to test the functions of the two devices. We firstly used the commercial
cell-free kit (Promega: E. coli S30 Extract System for Circular DNA) to express the AiiA. After
purification and SDS-PAGE, we failed to see the target band (Figure 2).

![](https://static.igem.org/mediawiki/2020/a/a2/T--WHU-China--eng4.png)

Figure 2: SDS-PAGE to test AiiA after purification of the cell-free reaction system. L1: control
group, without purification. L2-L5: purified and eluted by different concentrations of imidazole.

From the experiment we learned that the cell-free kit may not be used to express a large amount of
proteins. So we then tried to transform the pSB1C3+aiiC into E.coli BL21(DE3), and used IPTG to
induce the expression of the enzymes. Next, the standard AHL solutions and reaction with crude cell
extract were tested by the method described in the [measurement](https://2020.igem.org/Team%3AWHU-China/Measurement) (used crude cell extract
instead of purified enzymes) part of our wiki. And the HPLC results were as follows (Figure 3).

(A)

![](https://static.igem.org/mediawiki/2020/6/68/T--WHU-China--eng5.png)

(B)

![](https://static.igem.org/mediawiki/2020/2/2c/T--WHU-China--eng6.png)

Figure 3: HPLC analysis of the standard solutions of C4-HSL(A) and 3-oxo-C12-HSL(B) (1mg/ml). A: the
two peaks for C4-HSL occur at about 6 min and 8.4 min. B: the peak for 3-oxo-C12-HSL occurs at about
4.3 min.

These results showed that we successfully proved the availability of our method. As for the
measurement of crude cell extract reacting with AHLs, the results were shown in Figure4. From these
data (compared with the results of standard solutions above, certain amount not shown), we observed
that the area of one of the C4-HSL peaks didn’t change much while the other peak reduced
significantly after reacting with the crude cell extract (Figure4 A-B). And in the other reaction,
the area of the 3-oxo-C12-HSL peak reduced to nearly the half (Figure4 C-D). So we assumed that the
enzymes may be expressed by the cells and they could degrade AHLs with both short and long chains.
However, as the height and area of all the peaks of our samples were too small, due to the small
amount of AHLs we added to the reaction system maybe, the errors may be too large. So the data we
got may not be convincing and we couldn’t tell whether the enzymes were successfully expressed.
However, the preliminary experiments on both the standard solutions and our samples at least prove
the possibility of using HPLC to quantify the AHLs which corresponds to the methods mentioned in
other literatures. Still further experiments are needed to decide the amount of AHLs used in this
experiment.

(A)

![](https://static.igem.org/mediawiki/2020/6/69/T--WHU-China--eng7.png)

(B)

![](https://static.igem.org/mediawiki/2020/5/5e/T--WHU-China--eng8.png)

(C)

![](https://static.igem.org/mediawiki/2020/f/f3/T--WHU-China--eng9.png)

(D)

![](https://static.igem.org/mediawiki/2020/a/a8/T--WHU-China--eng10.png)

Figure 4: HPLC analysis of the crude cell extract reacting with AHLs. A: control group of C4-HSL
reaction system: inactivated crude cell extract (heating at 95℃ for 5 minutes) mixed with C4-HSL. B:
experimental group of C4-HSL reaction system: crude cell extract mixed with C4-HSL. C: control group of 3-oxo-C12-HSL reaction system: inactivated crude cell extract
(heating at 95℃ for 5 minutes) mixed with 3-oxo-C12-HSL. D: experimental group of 3-oxo-C12-HSL
reaction system: crude cell extract mixed with 3-oxo-C12-HSL.

Moreover, the relatively unsuccessful attempt of enzyme expression made us learn a lot. First, long
genes and high GC content genes are not easy to get either from synthesis or colony PCR. Second, the
traditional enzyme digestion and ligation method of molecular cloning is not that efficient. So in
the next season, we may use Gibson assembly to do the molecular cloning and try to avoid cloning
long genes. In addition, the first DBTL cycle made us think more---as we really wanted to find
enzymes with broad specificity towards substrates, are there any ways to directly create one which
will largely save our time? After doing another period of research, we put forward an idea that we
could find the ancestral enzyme of the existing enzymes. Because ancestral enzymes usually went
through less evolution and their substrate specificity was very broad. Although they may be less
catalytically efficient, we could use directed evolution on the enzymes (introduce several mutations
that are essential to their catalytical efficiency) to obtain both efficient and broad-spectrum
ones. With the help of our mathematic model, we successfully found several ancestral enzymes of our
candidates（Figure 5）. Due to the limited time, we only tested them in silico. But after
optimization, the sequences we got can be used directly next season, which will greatly reduce the
time of selecting candidate enzymes from such large amount of existing quenching enzymes. And the
enzymes we created may be even more efficient than the existing broad-spectrum enzymes! For more
details, you can see the [model](https://2020.igem.org/Team%3AWHU-China/Model) part in our
wiki.

![](https://static.igem.org/mediawiki/2020/2/21/T--WHU-China--eng11.png)

Figure 5: phylogenetic tree of PvdQ protein, generated by iTOL.

Above all, through the first DBTL cycle and initiating a second cycle, we are trying to tackle the
enzyme selecting problem we meet, which we believe is approaching success!

DBTL cycle of Sensing module

Chemokines are the crucial effectors of the sensing module in our design, since we want to express
them in our engineered bacteria to recruit immune cells, mainly monocytes and neutrophils to clear
pathogens. We have chosen 7 chemokines (CXCL1, CXCL2, CXCL3, CXCL8, CCL2, CCL3 and CCL8) as our
parts collection of chemokines, which are all known with chemotaxis effects on monocytes and
neutrophils and have no reported glycosylated modification. Chemokines from CXCL family are known to
have better chemotaxis effect on neutrophils and CCL family is known to have a preference to attract
monocytes [6]. To evaluate the chemotaxis effects of the 7 chemokines and select out 2 to 4
chemokines which are suitable for our project, cytological experiments were conducted. In these
experiments, we successfully cultured THP1 cell line as our model of monocytes and carry out the
Transwell migration assay and flow cytometry to evaluate their chemotaxis effect on THP1 cells.
Finally, we selected out CCL2 as the best chemokine part to attract monocytes at our given chemokine
concentration (10 nmol/l)[Figure 6 and Figure 7].

![](https://static.igem.org/mediawiki/2020/b/b6/T--WHU-China--eng12.png)

Figure 6. Transwell migration assay result of CCL2 treatment, shown by cell number.(n=3)

![](https://static.igem.org/mediawiki/2020/5/5c/T--WHU-China--81.png)

Figure 7. Chemotactic index(CI) of 7 chemokines to THP1.(n=3)

In our plan, we will furtherly use differentiated HL-60 cells as the model of neutrophils and
evaluate the chemotaxis effect of these 7 chemokines to select out most suitable 1 to 2 chemokines
to attract neutrophils. Gradients of concentration are considered to be set as well, so that the
chemotaxis effect of all chemokines can be analyzed at different concentration levels and the most
suitable range of chemokine concentration can be identified. Nevertheless, the preference of
chemokines to one of the immune cells restricts their use, since both monocytes and neutrophils are
important in acute inflammatory reactions. Two chemokines may need to be produced simultaneously.
Through searching information, we found that chemokines may interact with each other and strengthen
or weaken their effectiveness [7]. Thus combined Transwell migration assay of 2 chemokines should be
carried out to test their interaction effect and select the best combination. Later, the selected
chemokine parts will be expressed in our engineered bacteria. Finally, purified products expressed
and secreted by our engineered bacteria will be examined by Transwell migration assay again to
validate their functional efficiency. However, because of time limitation caused by COVID-19, we did
not carry out these experiments, which are planned to be done next year.
Besides, in order to maintain the secretion system with sufficient sensitivity while not causing
serious immune factor storms due to leaked expression, we decided to add a secretion signal peptide
to the N-terminus of the cytokine, and add a TEV protease cleavage site between that and the
chemokine itself. In this way, the fusion chemokine can be cleaved by the co-expressed TEV protease
to remove its secretion signal peptide and inhibit its secretion. As you might expect, this system
has the risk of causing the expressed chemokines to accumulate in bacteria chassis, so we added the
ssrA rapid degradation tag to the C-terminal of the cytokine to antagonize its side effects. In our
plan, this part is an important part of safety. GFP will replace chemokines for expression and
secretion in E. coli to verify whether the cleavable secretion signal peptide and ssrA tag work as
expected compared to the form without them. Unfortunately, the PCR reaction of adding a homology arm
to GFP for recombination failed to obtain the target band. This error prevented our urgent
experiment from proceeding as expected to quantify the expression of intracellular and extracellular
proteins, so it was difficult for us to determine whether the system we designed works
effectively.
Although our experiments are not complete, we did other work to help prove and refine our design.
Our model work of inflammation storm helps us to evaluate the risk to causing inflammation storm
with in vivo chemokine treatment and may help direct dose and time of in vivo chemokine treatment.
Our hardware design of an organoid based on micro-fluidic chip will help us to validate and evaluate
the effectiveness of in vivo chemokine treatment by our engineered bacteria without using any living
organisms. Furthermore, based on the data we got from Transwell test, we have created a new part
collection composed of several chemokines to share with other teams for future usage. You can see
the [parts](https://2020.igem.org/Team%3AWHU-China/Parts) section in our wiki for more
details!

DBTL cycle of cell-free quorum sensing

The envisioned workflow of cell-free quorum sensing includes: (i) in vitro prototyping of quorum
sensing networks; (ii) in silico screening of quorum sensing inhibitors and in vitro rapid and
high-throughput testing; and (iii) in vivo testing for validation. In phase I, we planned to
complete in vitro prototyping of quorum sensing networks but failed. However, the idea of
design-build-test-learn cycle was still implemented throughout our cell-free experiments, and we
wanted to share our experiences.
To reconstitute the basic logic of las and rhl systems of P. aeruginosa in cell-free systems, four
plasmids were successfully constructed with pSB1A3 as the backbone (Figure 8), which include: (i)
Plasmid-T (K2965011+LasR; K2965011+RhlR) will overexpress LasR and RhlR in E. coli BL21(DE3) after
IPTG induction, and the bacteria will then be lysed to prepare cell-free systems; (ii) Plasmid-L
(J23100+LasI; K649000+LasI) will express LasI to synthesize OdDHL in cell-free systems, and achieve
a positive feedback by its second device; (iii) Plasmid-R (K649000+RhlI; R0071+RhlI) will express
RhlI to synthesize BHL in cell-free systems under the control of Plasmid-L, and achieve a positive
feedback by its second device; (iv) Plasmid-F (K649000+GFPmut3b; R0071+mCherry) will express
GFPmut3b and mCherry under the control of in vitro established las and rhl systems, and the green or
red fluorescence will be measured by a microplate reader to quantify the activated level of these
two systems. Another plasmid (C) was constructed (J23100+GFPmut3b) as a negative control in
subsequent drug screening process, for validating the function of potential inhibitory molecules.

![](https://static.igem.org/mediawiki/2020/5/50/T--WHU-China--eng1.png)

Figure 8: Four recombinant plasmids constructed to reconstitute the basic logic of las and rhl
systems.

Initially, we attempted to make a proof-of-concept of cell-free quorum sensing by a commercial
cell-free kit (Promega: E. coli S30 Extract System for Circular DNA), with four plasmids
co-expressed in one pot. However, the attempts failed and no fluorescence was detected by the
microplate reader. Considering the regular method of expressing one plasmid per cell-free reaction,
we deemed that the imbalance of plasmid expression and energy consumption led to the failure.
Therefore, we turned back to the original plan, as preparing lysates with pre-expressed LasR and
RhlR to express the rest three plasmids in a chronological and hierarchical order. Besides, we
suspected the addition of SAM (an important co-factor and a substrate of acyl homoserine lactones)
might sustain the biosynthesis of OdDHL and BHL, whereby helping to establish the systems.
Subsequently, sixteen 20-μL cell-free reactions with different reaction conditions were established
and incubated in 30℃ for 4 hours, and several reactions ‘turned’ green! We got crazy for quite a
while, but later discovered blue light didn’t excite the expected green fluorescence, and the colour
was more likely the colour of diluted E. coli lysates (Figure 9), totally different from the
beautiful results in a research article [11].

![](https://static.igem.org/mediawiki/2020/c/c1/T--WHU-China--eng14.png)
![](https://static.igem.org/mediawiki/2020/c/c8/T--WHU-China--eng15.png)

Figure 9: No fluorescence observed in cell-free quorum sensing systems under white light (left) and
blue light (right).

Then a SDS-PAGE assay was performed to answer our questions. It’s strange that the protein bands
during IPTG inducing process (0, 0.5 hour, 1 hour, 1.5 hour, 2 hour) were very light, thus cannot
serve as the expected negative controls. However, considering that the molecular weight of LasR was
predicted as 26.62 kDa and RhlR 27.58 kDa, these two proteins seemed not to be pre-overexpressed in
the lysates (Figure 10). Several factors might contribute to the failed overexpression. Firstly, to
obtain efficient cell extracts, the protocols [9,10] varied in the appropriate incubation time after
IPTG induction, while a protocol described ‘If enzyme overexpression is the objective, incubate in
30℃ shaker for 4 h, or ~8 doublings, postinduction for protein expression; If cell-free protein
synthesis is the objective, incubate in 30℃ shaker to reach OD600 of 3’ [10]. However, in our
design, we need to both make use of pre-expressed proteins and implement cell-free protein synthesis
in the lysates, which means we should re-evaluate the inducing time and conduct a set of preliminary
experiments. Also, the quality of IPTG might be a problem, as a senior in the laboratory mentioned
this based on his experience on the bag of IPTG we’re using.

![](https://static.igem.org/mediawiki/2020/7/7f/T--WHU-China--eng16.png)

Figure 10: SDS-PAGE shows LasR and RhlR seem not to be pre-overexpressed in the lysates.

Unfortunately, there has been no time left for us to prepare cell-free systems for another round of
DBTL process. Hopefully, after debugging these factors, we will prototype las and rhl systems in
vitro, and facilitate massive screening of potential quorum sensing inhibitors whereafter.

DBTL cycle of Hardware

Our ultimate goal is to construct engineered probiotics that will face Gram negative pathogens
directly in lower respiratory tracts, being capable to quench their quorum sensing and expel them by
recruiting immune cells. This firstly demands the survival of our probiotics in lower respiratory
tracts: they should grow well and should not be excluded or poisoned by the presence of G- pathogens
(e. g. Pseudomonas aeruginosa). So, how viable our probiotics are when they are exposed to G-
respiratory pathogens has become a problem. To solve this problem raised from our design, a
co-culture device was built (the structure is shown below in Figure 11; further information is
available on Hardware page), aiming to find out whether our probiotics will be poisoned by G-
pathogens in a lower-respiratory-tract-like environment.

![](https://static.igem.org/mediawiki/2020/1/11/T--WHU-China--hardware112.png)

Figure 11: the co-cultivation device and its components.

Control element:
1-3: Three-way valve; 4-9: Double pass valve; 10-13: Pressure-Controlled needle (including
pressure-controlled needle piston);
Sampling and ventilation elements:
14: Pressure regulating ventilation pipe; 15, 18: Sterile sampling tube; 16, 19: Bacteria sampling
tube (consisting of rubber pipe, disposable bacterial filter, double-pass valve, joint and
countersunk head of rubber pipe); 17, 20: Sampling mouth of bacterial liquid in the left culture
bottle (consisting of rubber pipe, double-pass valve, joint and countersunk head of rubber pipe)
Other components:
21, 22: Culture bottle; 23, 24: Multi-use bacterial filters; 25: Completely closed bulkhead; 26:
Connecting bridge

This system is basically composed of a culture of probiotics (E. coli Nissle 1917 (EcN)) and a
culture of G- pathogens (Pseudomonas aeruginosa PAO1), which are separated by two sieves that allow
controlled medium exchange but prevent cells to pass. EcN and P. aeruginosa PAO1 were cultured to
log phase and added separately into this device, which was kept at 37℃ (similar to lower respiratory
tract environment). Medium from P. aeruginosa PAO1 incubator was ejected into the EcN incubator to
test that whether EcN would be affected by P. aeruginosa PAO1 metabolites.

![](https://static.igem.org/mediawiki/2020/0/02/T--WHU-China--proof1.png)

Figure 12: OD600 value versus time in co-cultivation device (experiment) and conical flask
(control).

To confirm that the growth of our probiotics will not be interfered by the metabolites of
Pseudomonas aeruginosa and to test the basic function of our hardware, we designed a simple
experiment: First, we added 2ml bacteria solutions (which was cultured overnight) to 120ml fresh LB
medium (two flasks for EcN and one for P. aeruginosa PAO1) and cultured them in a shaker (37℃,
200rpm) for 2 hours to obtain an initial bacteria density. And then we poured one flask of EcN and
P. aeruginosa PAO1 in the left and right bottle separately. And we used the hardware to mix the
medium (without mixing the bacteria!) of the two bottles and took 2ml samples per 45 minutes to
measure the OD600 value in a spectrophotometer as the quantification of bacteria density since then.
The same procedure was also used on the other flask of our probiotics which was not mixed with the
medium of P. aeruginosa PAO1as the control group. And we used Matlab to draw the growth curve of the
bacteria in the three bottles. The mixing point was regarded as the 0 point at x axis. .
From Figure 12 we can see that the growth curve of our probiotics in the hardware is similar to that
in the control group. Although maybe due to the medium we used, the density of P. aeruginosa PAO1 is
not so high, both the pathogens and probiotics are still growing. This partially proves that our
probiotics can be cultured with P. aeruginosa PAO1 normally, which provides evidence for the
possibility of using our probiotics as chassis to fight against the P. aeruginosa PAO1 in our lungs.
Besides, the function of our hardware is verified again..
In this DBTL cycle, based on the first arising problem of the practicability of our project in
applicational situations—tolerance of EcN to pathogen metabolites, we designed a non-contact
co-cultivation device and tested the wild type EcN growth curve under a condition of limited cell
contact and controllable media exchange. The result revealed that it is possible for EcN to work in
the anticipated environment. Due to the limited time, further experiments, e. g. testing the
tolerance of fully engineered EcN to pathogen metabolites are required. Nevertheless, these works
still laid the first foundations of realization of our project in the next season.

Reference

1.Susanne Fetzner. Quorum quenching enzymes[J]. Journal of Biotechnology, 2015, 201:2-14.
2.Manuel R, Diggle S P, Stephan H, et al. Quorum quenching activity in Anabaena sp. PCC 7120:
identification of AiiC, a novel AHL-acylase.[J]. Fems Microbiology Letters, 2010(1):73-80.
3. Czajkowski R, Krzyzanowska D, Karczewska J, et al. Inactivation of AHLs by Ochrobactrum sp. A44
depends on the activity of a novel class of AHL acylase[J]. Environmental Microbiology Reports,
2011, 3(1):59-68.
4. Bokhove Marcel, Jimenez et al. The quorum-quenching N-acyl homoserine lactone acylase PvdQ is an
Ntn-hydrolase with an unusual substrate-binding pocket.[J]. Proceedings of the National Academy of
Sciences of the United States of America, 2010.
5. Yi-Hu, Dong, Gusti et al. Identification of Quorum-Quenching N-Acyl Homoserine Lactonases from
Bacillus Species.[J]. Applied & Environmental Microbiology, 2002.
6. Kenneth Murphy. Janeway’s Immunobiology[M]. New York: Garland Science, 2011. 99-103
7. von Hundelshausen P, Agten SM, et al., Chemokine interactome mapping enables tailored
intervention in acute and chronic inflammation. Sci Transl Med. 2017 Apr 5;9(384):eaah6650.
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Nuzzi PA, Lokuta MA, Huttenlocher A. Analysis of neutrophil chemotaxis. Methods Mol Biol.
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system by systematic titration of transcription and translation components. Biochemical Engineering
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Advances, 4 (2018).
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397, 1-21, doi:10.1515/hsz-2015-0158 (2016).

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# Team:WHU-China/Experiments

# Experiments

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

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

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:WHU-China/Results

Results

![](https://static.igem.org/mediawiki/2020/c/c4/T--WHU-China--logomaybe.png)

Wet Lab>Results

![](https://static.igem.org/mediawiki/2020/9/99/T--WHU-China--results.png)

[Overview](#title1)

[Plasmids construction
  in Cell-Free Quorum Sensing](#title2)

[Quenching Module](#title3)

[Sensing Module](#title4)

Overview

Goal

Cell-Free Quorum Sensing
1. To reconstitute the basic logic of las and rhl systems in E. coli lysates
2. To screen quorum sensing inhibitors from a library of natural products

Quenching Module
AHLase Expression
1. To express lactonase AiiA and acylase AiiC, PvdQ and AiiO in E. coli BL21(DE3)
2. To test their degradation efficiency

Sensing Module
TEV protease expression
1. To express TEV protease with and without His-tag
Primary Chemokine selection
1. To test the efficiency of inducing cell migration of each chemokine
2. To calculate chemotaxis index of each chemokine and choose appropriate chemokines
Toxin-Antitoxin Systems
1. To clone toxin and anti-toxin pairs from E. coli K-12 MG1655
2. To test their efficiency of preventing HGT and choose a best TA system

Achievement

Cell-Free Quorum Sensing
1. Four plasmids to reconstruct quorum sensing systems were constructed
2. A plasmid for negative control in the drug screening process was constructed

Quenching Module
AHLase Expression
1. AiiC and AiiA genes were constructed, transferred and expressed in E. coli BL21(DE3), but further
data was required; PvdQ and AiiO were unavailable

Sensing Module
TEV protease expression
1. TEV protease with and without His-tag was successfully constructed (confirmed by colony PCR), but
there was no expression attempt
Primary Chemokine selection
1. Successfully obtained the migrated number of THP1 cells induced by each chemokine (shown in
Figure 6 to Figure 12)
2. Determine the optimal chemokine for THP1 (at a concentration of 10nm/L)
Toxin-Antitoxin Systems
1. Successfully constructed MazE and MazF (confirmed by colony PCR), while the attempts to clone
PemI, PemK, HipB, and HipA failed

Plasmids
construction in Cell-Free Quorum Sensing

All the plasmids in Cell-Free Quorum Sensing need two devices to be inserted, and the expected
efficiency is low. After nearly two weeks of unsuccessful digestion and ligation, Hieff Clone® Plus
Multi One Step Cloning Kit was suggested by our advisor as an alternative. With Sangon Biotech
sending primers for us, we used PrimeSTAR® GXL DNA Polymerase to add homologous arms flanking the
genes synthesized by IDT and GenScript (Figure 1). Using Hieff Clone® Plus Multi One Step Cloning
Kit, the ligation (50℃, 20 minutes) was performed, as well as later transformation.

![](https://static.igem.org/mediawiki/2020/f/f6/T--WHU-China--res1.png)
![](https://static.igem.org/mediawiki/2020/9/9b/T--WHU-China--res2.png)

Figure 1. PCR amplification by PrimeSTAR® GXL DNA Polymerase

Successfully, several right bands were observed by colony PCR (Figure 2), and corresponding single
colonies were inoculated to 10 mL LB media and incubated overnight (37℃, 220 rpm).

![](https://static.igem.org/mediawiki/2020/a/a7/T--WHU-China--res3.png)
![](https://static.igem.org/mediawiki/2020/f/ff/T--WHU-China--res4.png)

Figure 2. Colony PCR for correct single colonies

Plasmids were extracted from overnight cultures, and quantified by Thermo Scientific™ NanoDrop™ One.
Later the five plasmids were digested by EcoRI and SpeI, and right but dim bands were observed. For
final validation, all five plasmids and pSB1A3 sequencing primers (BBa\_G00100 and BBa\_G00101) were
sent to Sangon Biotech for gene sequencing.

In summary, five plasmids with confirmed inserted sequences were constructed, and further
experiments based on these five plasmids will be implemented after this season.

Quenching Module

Expression of AHLases

The main goal of our quenching module is inserting the genes encoding quenching enzymes into
expression vectors and transferring them into host bacteria to express the enzymes. With the help of
GenScript, we got the AiiA gene synthesized and inserted into pET-28a (+) directly. And to verify
the correct ligation of the gene, we used EcoR1 and Pst1 to digest the plasmids and got the expected
result, which was also the same as the company’s (Figure 1). We noticed that the original plasmid
band (over 12kb) is much larger than expected(6-7kb), but it may be due to the complex conformation
of the pET-28a(+)(which we asked our instructor for explanation). And as there are two bands in the
lane of digestion products (one of which is dim) of correct size, we can basically conclude that the
expression vector is successfully constructed. And we have successfully transformed it into E. coli
BL21(DE3) (not shown). But this vector still needs to be further confirmed by colony PCR and gene
sequencing. And next season, it can be used directly to get the enzymes we want.

![](https://static.igem.org/mediawiki/2020/a/ad/T--WHU-China--res5.png)

Figure 3. Enzyme digestion for correct plasmids (pET-28a(+)-AiiA. L1: plasmids got from company. L2:
digestion products of plasmids synthesized by company.

For the gene fragment encoding AiiC we got from IDT, we digested it and the pSB1C3 vector from iGEM
kit with EcoR1 and Pst1(37°C, overnight). And then we used Rapid DNA Ligation Kit, Beyotime (D7002)
to ligate the gene with the pSB1C3 vector. After the transformation of it into E. coli BL21(DE3), we
extracted the plasmids and digested them again with the same enzymes to check if the result was
positive. And the electrophoresis showed the target band of the recombinant plasmid but only one
trailing band in the lane of the digestion products (Figure 2). So, we proposed that it may be due
to the unsuccessful separation of the AiiC gene and the vector fragment after digestion. Thus, it
also needs colony PCR and gene sequencing for further verification.

![](https://static.igem.org/mediawiki/2020/2/27/T--WHU-China--res6.png)

Figure 2: Enzyme digestion for correct single colonies carrying pSB1C3+AiiC. L1: plasmids extracted
from BL21(DE3) colonies. L2: enzyme digestion products of the plasmids extracted.

However, synthesis of pvdQ was failed due to its high GC content and failure occurred on AiiO as
well for unknown reason. And we failed to obtain PvdQ from Pseudomonas aeruginosa PAO1 directly by
colony PCR. So, unfortunately, we can’t conduct experiments with PvdQ and AiiO this year. And we may
continue to experiment with them in next season but may change them for other genes that are easier
to get.

Sensing Module

TEV protease expression system with
and without His-tag

The TEV protease with His-tag is planned to be used in the subsequent detection of the effectiveness
of the secretion restriction device, and the TEV protease without His-tag is used to conveniently
remove from the system after cleaving the N-terminal of the chemokine. The TEV protease gene was
digested with EcoR1 and Pst1 and then ligated to pSB1A3 with the same operation. The colony PCR
result of the positive colonies after the ligation product is transformed shows that the connection
is successful. Due to the failure of the related parts transformation, neither of the two TEV
proteases tried to be expressed in the bacteria.

![](https://static.igem.org/mediawiki/2020/4/4b/T--WHU-China--res7.png)

Figure 4. Colony PCR confirmed the success of the connection

Chemokines primary selection

In our design, we want to use chemokines to recruit inflammatory cells, mainly neutrophils and
monocytes to clear the pathogens. We had chosen 7 chemokines, CXCL1, CXCL2, CXCL3, CXCL8, CCL2, CCL3
and CCL8, which may be suitable for our project. We planned to select 2 to 4 chemokines with high
chemotaxis ability at appropriate concentrations from these chemokines to express in our engineered
bacteria. We used differentiated HL-60 cell line as the model of neutrophils and THP1 cell line as
the model of monocytes. To evaluate the chemotaxis of the 7 chemokines to THP1 cells, transwell
migration assay was conducted, each condition with 3 parallel experiments and each transwell board
with at least 3 parallel controls. The migrated THP1 cells in the lower chamber were counted by
flow-cytometry. The primary results are shown in the form of scatter diagram [Figure 5].

![](https://static.igem.org/mediawiki/2020/8/8b/T--WHU-China--res8.png)

Figure 5. An example of primary cell counting result from flow-cytometry, in which CCL2 was
examined. The red marked dots in the red frame are records of THP1 cells in the lower chamber. The
number of these dots indicates the number of THP1 cells in the lower chamber.

Results shown by cell number

The number of cells in each lower chamber was then collected according to these scatter diagrams and
analyzed. Analyzed results are shown in Figure 6 to Figure 12. The results suggested that at the
concentration of 10 nmol/l, 3 chemokines from CCL family have the potential to attract THP1 cells,
especially CCL2, which shows statistical significance(p < 0.05). However, 4 chemokines from CXCL
family do not show a potential to attract THP1 cells and they even have a tendency to inhibit
the cells’ migration at the concentration of 10 nmol/l. Moreover, the data of several chemokines
like CCL3, CXCL8 and CXCL3 shows great standard error, which indicates their unstable
performance of chemotaxis at the concentration of 10 nmol/l.

![](https://static.igem.org/mediawiki/2020/4/40/T--WHU-China--11.png)

Figure 6. Chemotaxis of CCL2 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)

![](https://static.igem.org/mediawiki/2020/2/28/T--WHU-China--21.png)

Figure 7. Chemotaxis of CCL3 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)

![](https://static.igem.org/mediawiki/2020/a/ae/T--WHU-China--31.png)

Figure 8. Chemotaxis of CCL8 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)

![](https://static.igem.org/mediawiki/2020/3/35/T--WHU-China--41.png)

Figure 9. Chemotaxis of CXCL1 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)

![](https://static.igem.org/mediawiki/2020/8/8c/T--WHU-China--51.png)

Figure 10. Chemotaxis of CXCL2 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)

![](https://static.igem.org/mediawiki/2020/5/5b/T--WHU-China--61.png)

Figure 11. Chemotaxis of CXCL3 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)

![](https://static.igem.org/mediawiki/2020/c/c5/T--WHU-China--71.png)

Figure 12: Chemotaxis of CXCL7 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)

Results shown by CI

The chemotactic index (CI) of each chemokine was calculated and displayed in Figure 13. It is obvious that CI of CCL2 was the highest, and CCL3 comes the second and CCL8 the third.

![](https://static.igem.org/mediawiki/2020/5/5c/T--WHU-China--81.png)

Figure 13. Chemotaxis of 7 chemokines to THP1, shown by chemotactic index. Chemokine concentration is 10 nmol/l.(n=3)

According to the results all above, CCL2 was the best chemokine to attract THP1 cells, because CCL2 has the highest CI and its enhancement of cell migration has statistic significance. Although the CI of CCL3 is just a little lower than that of CCL2, its performance of chemotaxis is unstable and shows no statistic significance compared with the control. Therefore, the result suggests CCL2 to be the one we should select to use.
         Because of time limitation and failure on Hl-60 cell culture, the transwell migration assay on differentiated HL-60 cells was not conducted. Moreover, the concentrations of all chemokines are equal to 10 nmol/l. The results when the concentration differs remains unknown. Next year, further experiments may include these tests.

PCR of toxin-antitoxin
systems

Toxin-antitoxin systems are used to prevent horizontal gene transfer in our design. We planned to put two pairs of toxin and antitoxin gene separately on two different plasmids. To achieve that, on each plasmid the toxin comes from a system while the antitoxin comes from another. In order to get the toxin and antitoxin genes separately, we decide to use colony PCR. Here shows our colony PCR results of MazE and MazF gene from Escherichia coli K-12 MG1655 [Figure 14 and Figure 15]. The bright band in front of 2oobp band was identified as primer dimers for defective design of primers.

![](https://static.igem.org/mediawiki/2020/6/60/T--WHU-China--res82.png)

Figure 14. Agarose gel electrophoresis results of MazE colony PCR. The size of MazE CDS is 264bp.

![](https://static.igem.org/mediawiki/2020/d/dd/T--WHU-China--res84.png)

Figure 15. Agarose gel electrophoresis result of MazF colony PCR. The size of MazF CDS is about 333bp.

We also extracted the pET-28a, preparing to use it as the carrier of our toxin and antitoxin genes[Figure 16]. The bands of pET-28a extracted is in front of 4000bp, smaller than the size of itself(5.4kb). This may be caused by the supercoiled conformation of the extracted pET-28a.

![](https://static.igem.org/mediawiki/2020/f/fa/T--WHU-China--res83.png)

Figure 16. Agarose gel electrophoresis of extracted pET-28a. The size of pET-28a is about 5.4kb.

The colony PCR of PemI, PemK, HipB and HipA all failed. This may be caused by unsuitable annealing temperature or poor design of primers. Solving this problem requires more experiments. However, because of time and conditional limitation, we neither made more trials nor did any further molecular cloning experiments. These further works are planned to be conducted next year.

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