iGEM 2021 — Team NU_Kazakhstan

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# Team:NU Kazakhstan/Notebook

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- [Background](/Team%3ANU_Kazakhstan/Background)
- [Description](/Team%3ANU_Kazakhstan/Description)
- [Design](/Team%3ANU_Kazakhstan/Design)
- [Safety](/Team%3ANU_Kazakhstan/Safety)
- [Contribution](/Team%3ANU_Kazakhstan/Contribution)
- [Awards](/Team%3ANU_Kazakhstan/Awards)
- [Sponsors](/Team%3ANU_Kazakhstan/Sponsors)

- [Notebook](/Team%3ANU_Kazakhstan/Notebook)
- [Experiments](/Team%3ANU_Kazakhstan/Experiments)
- [Proof of Concept](/Team%3ANU_Kazakhstan/Proof_Of_Concept)
- [Results](/Team%3ANU_Kazakhstan/Results)
- [Proposed Implementation](/Team%3ANU_Kazakhstan/Implementation)

- [Engineering Success](/Team%3ANU_Kazakhstan/Engineering)
- [Parts](/Team%3ANU_Kazakhstan/Parts)
- [Model](/Team%3ANU_Kazakhstan/Model)

- [Integrated Human Practices](/Team%3ANU_Kazakhstan/Human_Practices)
- [Science Communication](/Team%3ANU_Kazakhstan/Communication)

- [Members](/Team%3ANU_Kazakhstan/Team)
- [Attributions](/Team%3ANU_Kazakhstan/Attributions)
- [Collaborations](/Team%3ANU_Kazakhstan/Collaborations)
- [Sharing Experience](/Team%3ANU_Kazakhstan/Sharing)

- [Sustainability](/Team%3ANU_Kazakhstan/Sustainable)
- [Education](/Team%3ANU_Kazakhstan/Education)
- [Inclusivity](/Team%3ANU_Kazakhstan/Inclusivity)

## June

## Week 1

### Day 1 - 17.06.2021

1. 1ml of cell suspension was obtained and mixed with 1ml of DI water in a falcon tube. The tube was centrifuged at 4700 rpm for 1 hour.
2. The supernatant was discarded. The pellet with bacteria was mixed with a TE buffer (10mM Tris-HCl, pH 8.0, 0.1mM EDTA) of volume 1ml. The mixture was thoroughly mixed and centrifuged at 4700 rpm for 20 min.
3. With the clear division of pellet and supernatant, the supernatant was discarded leaving 300ml of the solution. The mixture was gently resuspended and divided into new 1.5ml reaction tubes.
4. One 1.5ml reaction tube with the cell suspension was treated with 1ml lysis buffer (50mM Tris, 1% SDS, pH 8.0) (BioRAD). While the second tube was mixed with lysis solution from BioRAD Aurum Plasmid Minikit of volume 1ml. Both tubes were incubated in a thermomixer at 37°C for 1 hour without applying rotations during incubation.
5. The tubes with cell lysates were kept in the fridge (4°C) overnight.

### Day 2 - 18.06.2021

1. 1ml of cell suspension was obtained and mixed with 1ml of DI water in a falcon tube. The tube was centrifuged at 4700 rpm for 1 hour.
2. The supernatant was discarded. The pellet with bacteria was mixed with TE buffer (10mM Tris-HCl, pH 8.0, 0.1mM EDTA) of volume 1ml. The mixture was thoroughly mixed and centrifuged at 4700 rpm for 20 min.
3. With the clear division of pellet and supernatant, the supernatant was discarded leaving 300ml of the solution. The mixture was gently resuspended and divided into new 1.5ml reaction tubes.
4. One 1.5ml reaction tube with the cell suspension was treated with 1ml lysis buffer (50mM Tris, 1% SDS, pH 8.0) (BioRAD). While the second tube was mixed with lysis solution from BioRAD Aurum Plasmid Minikit of volume 1ml. Both tubes were incubated in a thermomixer at 37°C for 1 hour without applying rotations during incubation.
5. The tubes with cell lysates were kept in the fridge (4°C) overnight.

Table 1. The tubes with cell lysates

|  | tube 1 | tube 2 | tube 3 | tube 4 |
| Volume in a tube | 0.4ml | 0.5ml | 0.6ml | 0.4ml |
| 0.1v Sodium acetate | 40μl | 50μl | 60μl | 40μl |
| 1v absolute EtOH | 400μl | 50μl | 60μl | 40μl |

1. Tubes were kept in the -20°C fridge for 3 days

## Week 2

### Day 1 - 21.06.2021

1. The tubes were centrifuged at 10 000 rpm for 15 minutes.
2. The supernatant was descarded
3. 200 microliters of 70% ethanol was added to each tube and were centrifuged for 15 minutes at 10 000 rpm (x3)
4. The ethanol solution was decanted and tubes containing pellets were left to air dry.
5. The dried tubes were treated with ultrapure water in volume 100 microliters.

Nanodrop measurements

1. The ultrapure water was used to blank the nanodrop 8000 spectrophotometer (Thermo FIsher Scientific)
2. Four samples were measured at the same time, and the measurements were recorded in Table 2

Table 2. Nanodrop measurements of four samples

| Tube | Concentration | Buffer / Solution | 260 / 280 |
| 1 | 50.44 ng/ul | LS | 1.55 |
| 2 | 30.14 ng/ul | LS | 1.54 |
| 3 | 58.52 ng/ul | LB | 1.85 |
| 4 | 150.1 ng/ul | LB | 1.60 |
| 5 | 290 ng/ul | LB and LS | 1.93 |

![Notebook Figure 1](https://static.igem.org/mediawiki/2021/f/f6/T--NU_Kazakhstan--Notebook_Figure_1.jpg)

Figure 1. Nanodrop measurements of four samples

![Notebook Figure 2](https://static.igem.org/mediawiki/2021/e/e7/T--NU_Kazakhstan--Notebook_Figure_9.jpg)

Figure 2. The DNA extraction results

1. The DNA extraction was repeated again and results are in Table 3

### Day 2 - 22.06.2021

1. After the tubes were incubated for 24 hours at 37 **°**C, the tubes were treated with lysis solution and lysis buffer, and mixed. The solution was divided into 4 tubes.
2. The tubes were allowed to stay for 5 minutes and were centrifuged at 10'000 rpm for 15 minutes.
3. The upper aqueous layer was separated and loaded into new 1.5 mL tubes (1 tube).
4. The tube with DNA solution then was separated into 2 tubes.
5. Each tube was treated with 0.1 volume of sodium acetate 3M solution and 1 volume of absolute ethanol.
6. The tubes were allowed to precipitate at -20 **°**C for 24 hours.

### Day 3 - 24.06.2021

Table 3. Results of washing of DNA and nanodrop measurements

| Tube | Concentration | 260/280 |
| 1 | 290 ng/uL | 1.93 |
| 2 | 64.22 ng/uL | 1.81 |

## Week 3

### Day 1 - 29.06.2021

1. LB agar was prepared
2. 3 g of Luria Bertani powder was weighed and added to the bottle 500 mL.
3. 200 mL pg di water was added into the bottle and mixed until powder was dissolved.
4. 3 g of Agar powder was added into the bottle, mixed.
5. The solution was autoclaved for 15 minutes at 121**°**C.

### Day 2 - 30.06.2021

1. Turned UV in the hood for 30 min.
2. Autoclaved LB agar.
3. Prepared 6 petri dishes by painy 20 ml of LB agar onto them.
4. While plates were solidified, prepared Nutnent Broth by adding 10 grams of NB both to the 400 ml of distilled water.
5. Using sterile loops and L-spreaders prepared 3 steak plates and 3 spread plates by inoculating E. coli-DHsd.
6. Closed each petri dish with parafilm and gathered them.
7. Put them into an incubator for 48 hours at 37**°**C.
8. Using left LB agar prepared 2 slant agars of 20 ml in cellstar tubes (Falcon (centrifuge bottles) bottles).
9. Put slant tube sand NB into the autoclave for ~ hour.

## July

## Week 1

### Day 1 - 01.07.2021

1. Get out the petri dishes (cell cultures) from the incubator.
2. From one of the streak plates isolated a single colony and inoculated prenanly prepared LB agar slants.
3. Put them into an incubator for 24 hours at 37**°**C.

![Notebook Figure 3](https://static.igem.org/mediawiki/2021/8/8d/T--NU_Kazakhstan--Notebook_Figure_76.jpg)

Figure 3. *E.Coli* spread plate 30/06/2021

![Notebook Figure 4](https://static.igem.org/mediawiki/2021/d/de/T--NU_Kazakhstan--Notebook_Figure_6.jpg)

Figure 4. *E.Coli* spread plate 30/06/2021

![Notebook Figure 5](https://static.igem.org/mediawiki/2021/f/fd/T--NU_Kazakhstan--Notebook_Figure_21.jpg)

Figure 5. *E.Coli* spread plate 30/06/2021

![Notebook Figure 6](https://static.igem.org/mediawiki/2021/a/ab/T--NU_Kazakhstan--Notebook_Figure_38.jpg)

Figure 6. *E.Coli* streak plate 30/06/2021

![Notebook Figure 7](https://static.igem.org/mediawiki/2021/2/26/T--NU_Kazakhstan--Notebook_Figure_54.jpg)

Figure 7. *E.Coli* streak plate 30/06/2021

![Notebook Figure 8](https://static.igem.org/mediawiki/2021/c/ca/T--NU_Kazakhstan--Notebook_Figure_70.jpg)

Figure 8. *E.Coli* streak plate 30/06/2021

1. One single colony was taken from the streak plate cultures with *E.coli*and inoculated into centrifuge tubes (30 mL) containing Nutrient Broth.
2. The same was done with the second tube containing NB in 30 mL.
3. LB agar in volume of 200 mL was prepared and autoclaved.

## Week 2

### Day 1 - 05.07.2021

1. Brainstormed protocols for further lab procedures
2. Made a list of reagents that will be required

![Notebook Figure 9](https://static.igem.org/mediawiki/2021/f/f4/T--NU_Kazakhstan--Notebook_Figure_78.jpg)

Figure 9. Quick-Load Purple 1 kb DNA Ladder

![Notebook Figure 10](https://static.igem.org/mediawiki/2021/9/9c/T--NU_Kazakhstan--Notebook_Figure_4.jpg)

Figure 10. Gel Loading Dye Purple (6X)

![Notebook Figure 11](https://static.igem.org/mediawiki/2021/3/3b/T--NU_Kazakhstan--Notebook_Figure_40.jpg)

Figure 11. Gel Loading Dye Purple (6X)

![Notebook Figure 12](https://static.igem.org/mediawiki/2021/c/ce/T--NU_Kazakhstan--Notebook_Figure_79.jpg)

Figure 12. Quick-Load Purple 1 kb DNA Ladder

![Notebook Figure 13](https://static.igem.org/mediawiki/2021/a/af/T--NU_Kazakhstan--Notebook_Figure_15.jpg)

Figure 13. Loading dye 5X

### Day 2 - 06.07.2021

1. Prepared 100 mL 1M CaCl2 -> stored in a small refrigerator.
2. 10% of SDS (1 g SDS + 10 mL DI).
3. 2N NaOH 50 mL (4 g NaOH + 50 mL DI).
4. Nutrient Agar 200 mL, then autoclaved.
5. LB broth 200 mL, then autoclaved.

### Day 3 - 07.07.2021

1. Planning of the transformation experiment
2. Prepared LB agar by adding LB agar (7.4 g) into 200 ml of distilled water
3. Autoclaved LB agar and 25 ml of distilled water
4. Microwaved Nutrient Agar
5. Prepared 2 agar slants and 1 steak plant using E.coli plates from 30/06/22 and 20 microliters nutrient agar.
6. Inoculated the E.coli into LB broth prepared 06/07/2021
7. Incubated 2 agar slants, 1 streak plate and E.coli broth for 24 hours of 37**°**C.
8. Get 10 ml of autoclaved water and added 0.5 g of kanamycin to it --> prepared kanamycin solutions 50 mg/ ml
9. Poured 0.2 mL = 200 uL of kanamycin solution into 200 ml of LB agar (07/07/2021 preparation)
10. Poured 20 ml of selective media (LB agar with kanamycin) into one petri dish
11. 2 petri dishes with selective medium were prepared

P.S. smth was wrong with Nutrient agar (06/07/2021 preparation) **:(**

### Day 4 - 08.07.2021

1. Measured OD ~ 0.646
2. Poured 10 mL of the solution into a prechilled 15 mL centrifuge tube. (Everything on ice)
3. Centrifuged at 4700 rpm at 4**°**C for 10 min.
4. Discarded the supernatant.
5. Resuspended the pellet into 2 mL centrifuge tube with 1 mL ice-cold ddH2O
6. Microcentrifuged it at 7000 rpm for 5 minutes.
7. Discarded the supernatant and resuspended it with 1 mL of ice-cold ddH2O.
8. Repeated the steps 6-7 3 times.
9. Final time microcentrifuged at 7000 rpm for 5 minutes.
10. Discarded the supernatant.
11. Resuspended pellet with 50 uL of ice-cold ddH2O.
12. Prepared 200 mL of LB agar by pouring 7.4 g of LB agar powder into 200 mL of distilled water.
13. Autoclaved LB agar.

1. Did nanodrop.

Table 4. Concentration of Duo 2, 3, and 4

| Plasmid | Concentration |
| Duo2 | 13.71 ng/uL |
| Duo3 | 226 ng/uL |
| Duo4 | 138.6 ng/uL |

2. We only need 75 ng -> 1 uL of Duo2 + 99 uL TEv = 100 uL overall. 1uL is 13.71 ng/uL \* 6 uL ≈ 82 ng.

3. Added 6 uL of Duo2 to *E.coli* -> mixed gently.

4. Transferred the solution (*E.coli* + Duo2) to a chilled electroporation cuvette.

5. Capped the cuvette, tapped lightly on the bench -> ice.

6. Turned on electroporator (18 kV, 25 uF, 200 ohms).

7. Wiped the cuvette with Kimtech, then placed the cuvette into an electroporator for 10 sec.

8. Immediately added 1 mL of LB -> quickly pipette.

9. Transferred the mixture to a fresh 1.5 mL tube.

10.Incubated at 150 rpm 37**°**C for 1 hour.

1. Autoclaved 2 tubes with LB agar (slant), 2 tubes with LB broth, 2 tubes with NB
2. Prepared: 100 mL 0.1M CaCl2 (100 mL 0.1M CaCl2 + 20% glycerol)
3. Prepared 2 slant agar (LB agar) + *E.coli*.

11. Inoculated *E.coli* from slant agar tube into 2 plates by streak plate technique.

**!** 1 plate is clean. In the fume hood.

12. 5 mL of LB was inoculated with *E.coli* colony.

13. Cultures were left in incubater at 37**°**C overnight.

### Day 5 - 09.07.2021

1. Nutrient Agar was prepared (28g/1L) - 200 mL.
2. Autoclaved: LB agar 200 mL, NA 200 mL.
3. The UV light in the safety cabinet was turned on for 35-40 minutes.
4. 0.1 mL Kanamycin (07/07/21) was taken and poured into 100 mL LB agar.
5. 100 mL of LB agar was distributed to 4 plates and allowed to solidify. 3 plates were sealed.
6. One plate with kanamycin was inoculated with *E.coli* -> incubation for 72 hours at 30**°**C.

Spectrophotometer (OD).

* DI water - 0.002 abs
* LB broth w/ *E.coli* - 1.341 abs [blanked with water]
* LB broth w/ *E.coli* - 1.312 abs [blanked with LB broth]
* LB broth w/ *E.coli* (diluted 1:2) - 0.705 abs [blanked with water]
* LB broth w/ *E.coli* (diluted 1:2) - 0.693 abs [blanked with LB broth]
* LB broth w/ *E.coli* (diluted 1:3) - 0.524 abs [blanked with LB broth]
* Overnight culture was diluted until OD 0.524

1. 10 mL of the culture solution was transferred into a 15 mL centrifuge tube (preliminary chilled on ice).
2. The tube was centrifuged at 4700 rpm for 10 minutes at 4**°**C.
3. The supernatant was discarded.
4. The pellet was resuspended with 1 mL sterile diH2O
5. Transferred into a cold 1 mL microcentrifuge tube.
6. The tube was centrifuged at 7000 rpm at 4**°**C for 5 min.
7. The supernatant was discarded.
8. The pellet was resuspended with 1 mL of diH2O.
9. The steps 6-8 were repeated 3 times.
10. The final pellet after 4th centrifugation was resuspended with 50 uL of ice-cold ddH2O.
11. Left to be stored at -20**°**C fridge.

## Week 3

### Day 1 - 12.07.2021

1. Turned on the UV light for 30 minutes.
2. Prepared 3 LB agar plates.
3. Prepared 3 LB slants.
4. Prepared 1 LB broth.
5. Proceed inoculation of 2 agar slants with *E.coli* DH5α pRGPDuo2.
6. Inoculated 1 agar slant and 1 LB broth with *E.coli* DH5α.
7. 2 LB agar plates were used for subculturing of transformed *E.coli* DH5α pRGPDuo2 (streak plate).
8. One plate was inoculated with *E.coli* DH5α (streak plate).
9. All agar slants, plates, and broth incubated overnight at 37**°**C.

Plasmid extraction

1. 1 mL of culture tubes (2 tubes) were centrifuged at 7000 rpm for 1 minute at 4**°**C.
2. The supernatant was discarded and the pellet was left to dry.
3. Prepared 10 mL of ALS II by mixing 1 mL of 3(?)N NaOH, 1 mL of 10% SDS and ddH2O.
4. Resuspended the pellet in 100 uL of ice cold ALS I (autoclaved) and mixed by pipetting, making sure the cells dispersed fully.
5. Added 200 uL of ALS II and mixed by inverting the tube 5 times, making sure the content of the tube makes contact with ALS II. Stored the tube on ice for 5 minutes.
6. Added 150 uL of ALS III (autoclaved) and mixed gently by inverting the tube 5 times. Stored the tube on ice for 5 minutes.
7. Centrifuged at 13500 rpm for 5 minutes at 4**°**C.
8. Transferred the supernatant (450 uL) to the new labeled tube.
9. Precipitated DNA from supernatant by adding 2 volumes (900 uL) of 70% ethanol at room temperature. Gently mixed by inverting and incubated 2 minutes at room temperature.
10. Centrifuged at 13500 rpm for 5 minutes at 4**°**C. Discarded the supernatant and dried the pellet.
11. Added 1 mL of 70% ethanol and mixed by inverting.
12. Centrifuged at 13500 rpm for 2 minutes at 4**°**C. Removed the supernatant and left the tube to dry for 10-15 minutes until ethanol will evaporate.
13. Dissolved pellet in 50 uL H2O and checked OD 1 uL of each solution by nanodrop.

Table 5. Concentrations of two samples

| Sample | Concentration | 260/280 |
| 1 | 161.7 ng/uL | 2.12 |
| 2 | 460.3 ng/uL | 1.97 |

* LB broth (250 mL) was prepared
* ddH2O, LB broth new and opened one were autoclaved (30 mL)

### Day 2 - 13.07.2021

1. Turned on the UV light for 30 minutes.
2. OD measurements of colonies - 0.368
3. Microwaved NB agar.
4. Prepared 2 plates (20 mL) and 2 slants (25 mL) with NB agar.
5. Poured 10 mL of cultures into pre-chilled 15 mL centrifuge tubes.
6. Centrifuged 15 mL centrifuge tubes at 4700 rpm at 4**°**C for 10 minutes.
7. Discarded the supernatant and resuspended the pellet with 1 mL of ice-cold ddH2O.
8. Microcentrifuged at 7000 rpm at 4**°**C for 5 minutes.
9. Discarded the supernatant and resuspended the pellet with 1 mL of ice-cold ddH2O.
10. Microcentrifuged at 7000 rpm at 4**°**C for 5 minutes.
11. Discarded the supernatant and resuspended the pellet with 1 mL of ice-cold ddH2O.
12. Microcentrifuged at 7000 rpm at 4**°**C for 5 minutes.
13. Discarded the supernatant and resuspended the peller with 1 mL of ice-cold ddH2O.
14. Microcentrifuged at 7000 rpm at 4**°**C for 5 minutes.
15. Discarded the supernatant and resuspended the pellet with 1 mL of ice-cold ddH2O.
16. Microcentrifuged at 7000 rpm at 4**°**C for 5 minutes.
17. Inoculated NB agar plates and 2 NB agar slants with *E.coli* DHα5.
18. Discarded the supernatant and resuspended the pellet with 50 mL of ice-colled ddH2O.
19. Chilled the tubes on ice.
20. Calculated the volume of required plasmid.
21. Added 0.55 uL of *E.coli* pRGPDuo4 plasmid into all cultures.
22. Transferred these cultures into electroporation cuvette.
23. With Kimwipes wiped electroporation cuvettes.
24. Capped the cuvetted and turned on the electroporator (18 kV, 25uF, 200 ohms).
25. Put electroporation cuvettes into the electroporator for 10 seconds.
26. Added, immediately, 1 mL of LB and mixed by pipetting.
27. Transferred the culture into a new microcentrifuge tube.
28. Incubated at 37**°**C for an hour.
29. Microwaved LB agar with kanamycin.
30. Prepared 4 spread plates with cultures (sample 1, sample 2, diluted sample 1 /1:10, diluted sample 2/1:10).
31. Prepared negative control: spread plate with *E.coli* that was in the initial part of the experiment.
32. Prepared extra 2 plates with kanamycin.
33. Incubated overnight at 37**°**C.

### Day 3 - 14.07.2021

1. 25 mL of LB broth was loaded into centrifuged tubes x3 -> total 3 tubes with LB broth (12.07.21 preparation).
2. 25 **μ**L of kanamycin from the solution (50 ng/mL of kanamycin) was loaded into each tube with LB broth.
3. One single colony was inoculated from plates: Duo4 2nd sample, Duo4 2nd diluted ,Duo4 1st sample (13.07.21).
4. The tubes were incubated for 24 hours at 37**°**C.

![Notebook Figure 14](https://static.igem.org/mediawiki/2021/b/b8/T--NU_Kazakhstan--Notebook_Figure_30.jpg)

Figure 14. Control with Kanamycin [1]

![Notebook Figure 15](https://static.igem.org/mediawiki/2021/4/49/T--NU_Kazakhstan--Notebook_Figure_44.jpg)

Figure 15. Control with Kanamycin [2]

https://static.igem.org/mediawiki/2021/4/4d/T--NU\_Kazakhstan--Notebook\_Figure\_16.jpg
![Notebook Figure 16](https://static.igem.org/mediawiki/2021/2/23/T--NU_Kazakhstan--Notebook_Figure_59.jpg)

Figure 16. E.Coli with Duo 4 on LB+Kanamycin 1st sample [1]

![Notebook Figure 17](https://static.igem.org/mediawiki/2021/a/a6/T--NU_Kazakhstan--Notebook_Figure_73.jpg)

Figure 17. E.Coli with Duo 4 on LB+Kanamycin 1st sample [2]

![Notebook Figure 18](https://static.igem.org/mediawiki/2021/2/2d/T--NU_Kazakhstan--Notebook_Figure_80.jpg)

Figure 18. E.Coli with Duo 4 on LB+Kanamycin 2nd sample [1]

![Notebook Figure 19](https://static.igem.org/mediawiki/2021/0/0e/T--NU_Kazakhstan--Notebook_Figure_35.jpg)

Figure 19. E.Coli with Duo 4 on LB+Kanamycin 2nd sample [2]

![Notebook Figure 20](https://static.igem.org/mediawiki/2021/7/70/T--NU_Kazakhstan--Notebook_Figure_20.jpg)

Figure 20. E.Coli with Duo 4 on LB + Kanamycin (1st sample diluted)

### Day 4 - 15.07.2021

1. Prepared: LB agar 200 mL, LB broth 200 mL, NB 200 mL.
2. The solutions were autoclaved.
3. Prepared ALS II solution mixing SDS 10% + NaOH 2N + di water (10 mL).
4. 1 mL of each overnight incubated *E.coli* samples (1st sample and 2nd sample with pRGPDuo4) were taken and poured into microcentrifuge tubes (in total 2 tubes).
5. The tubes were centrifuged at 14000 rpm for 30 seconds.
6. The supernatant was discarded.
7. 1 mL of sterile distilled water was added to each tube and centrifuged at 12300 rpm for 1 minute.
8. The supernatant was discarded and the tubes were left to air dry inside the safety cabinet.
9. Pellets were resuspended with 100 uL of ice-cold ALS I (pre-autoclaved) and mixed by pipetting.
10. 200 uL of ALS II (doesn't have to be fresh, store at room temperature) was added into each tube and mixed gently by inverting the tube 5 times. Tubes were left on ice for 5 minutes.
11. 150 uL of ALS III (pre-autoclaved) was added into each tube and mixed gently by inverting the tube 5 times. Tubes were left on ice for 5 minutes.
12. Tubes were centrifuged at 12300 rpm for 7 minutes and 450 uL of supernatant from each tube was transferred into new tubes labelled "DNA-supernatant".
13. DNA was precipitated from the supernatant by adding 2 volumes (900 uL) of 70% ethanol chilled on ice. Mixed by inverting and incubated for 2 minutes at room temperature.
14. Tubes were centrifuged at 12300 rpm for 12 minutes and supernatant from each tube was removed, and the tubes were left to air dry.
15. 1 mL of 70% ice-cold ethanol was added to each tube and tubes were centrifuged at 12300 rpm for 3 minutes. Tubes were left to air dry for 10-15 minutes.
16. 50 uL of ultrapure water was added.
17. Nanodrop was applied:

Table 6. Concentration of sample 1 and 2

| Sample | Concentration |
| 1 | 199.6 ng/uL |
| 2 | 527.4 ng/uL |

* LB plate, LB slant agar were prepared.
* *E.coli* DH5α from slant tube (12.07.21 preparation) was inoculated by streak plate technique into 1 LB agar plate.
* *E.coli* from slant tube (12.07.21) were inoculated into 2 slant glass tubes.
* Tubes and plate were allowed for incubation overnight at 150 rpm at 37**°**C.

### Day 5 - 16.07.2021

Preparing electrocompetent E.coli cells

1. Dilute E.coli culture (5mL E.coli culture + 20mL LB broth)
2. Incubate for 30 minutes.
3. Measure OD:

* Calibrate with LB
* 1st trial OD value - 0.233
* Incubate diluted E.coli culture for 20minutes, since OD is not enough
* 2nd trial OD value - 0.342
* Incubate the diluted sample once more for 10 minutes.
* 3rd trial OD value: 0.411

1. Pour 10mL of diluted E.coli solution into a prechilled 15mL centrifuge tube.
2. Centrifuge at 4700 rpm at 4**°**C for 10 minutes.
3. Discard the supernatant.
4. Resuspend pellet in 1.5mL centrifuge tube with 1mL of ice-cold dd H2O.
5. Microcentrifuge at 7000 rpm at 4**°**C for 5 minutes.
6. Repeat 6-8 steps 4 times more.
7. Discard the supernatant.
8. Resuspend the final pellet with 50µL of ice cold dd H2O, put cells on ice.

**Electroporation:**

1. 1µL of Duo3 + 1.5 µL TE buffer.
2. Duo3 concentration:

* nanodrop for Duo3 = 226ng/µL.
* Duo3 conc. = 226ng/µL\*1µL/2.5µL = 90.4 ng/µL.

1. Add 1µL of Duo3 solution into E.coli.
2. Transfer bacteria to chilled electroporation cuvette.
3. Cap cuvette, tap on bench. Put the cuvette on ice.
4. Turn on electroporator (1.8 kV, 25µF, 200 **Ω)**
5. Wipe cuvette with Kimtech, place in electroporator.
6. start and continue for 10 seconds.
7. Remove cuvette, immediately add 1mL of LB broth.
8. Pipette up and down gently.
9. Transfer mixture to a fresh 1.5mL tube.
10. Incubate at 37**°**C for 1 hour.
11. 0.6mL of gentamicin (eye drops) were mixed with 120mL LB agar.
12. 90µL LB agar + gentamicin was poured into 3 plates 30mL each. Allowed to solidify.
13. First plate: 100µL of E.coli with Duo3 spread on LB+gentamicin.
14. Second plate: 100µL of diluted E.coli with Duo3 (10µL+90µL LB) on LB+gentamicin.
15. Third plate: Untransformed E.coli on LB+gentamicin.
16. Incubate plates overnight at 37**°**C.

### Day 6 - 17.07.2021

1. Incubate E.coli Duo3 transformed plates for 2.8 hours more.
2. Ultrapure nuclease free water was autoclaved.
3. PCR tubes were autoclaved.

**PCR for nadE**

1. 12.5µL Master Mix + 1µL F primer nadE + 1µL R primer nadE + 1.5µL gen. DNA from tube 3 LB + 9µL nuclease free H2O.

Table 7. PCR results for nadE

|  | t (°C) | time |  |
| Initial denaturation | 95 | 2 min. | 1 cycle |
| Denaturation | 94 | 30 seconds | 35 cycles |
| Annealing | 63 | 30 seconds |
| Extension | 68 | 1 min. |
| Final Extension | 68 | 5 min. |  |
| Storage | 4 |  |  |

1. 12.5µL Master Mix + 1µL F primer nadE + 1µL R primer nadE + 2µL gen. DNA from tube 1 LB + 8.5µL nuclease free H2O.
2. 12.5µL Master Mix + 1µL F primer nadE + 1µL R primer nadE + 3.3µL gen. DNA from tube 2 LB + 7.2µL nuclease free H2O.
3. 12.5µL Master Mix + 1µL F primer nadE + 1µL R primer nadE + 0.7µL gen. DNA from tube 4 LB + 9.8µL nuclease free H2O.

**Primers mainstock and substock preparation**

**For pRGPDuo-rhlBA-F**

**12.93nmol**

* For main stock: add 129.3µL of sterile ultrapure water
* substock 1: take 10µL of main stock + 90µL sterile ultrapure water
* substock 2: take 10µL of main stock + 90µL sterile ultrapure water

**For pRGPDuo-nadE-F (E5)**

**13.04nmol**

* For main stock: add 130.4µL of sterile ultrapure water
* substock 1: take 10µL of main stock + 90µL sterile ultrapure water
* substock 2: take 10µL of main stock + 90µL sterile ultrapure water

**For pRGPDuo-nadE-R**

**18.96nmol**

* For main stock: add 189.6µL of sterile ultrapure water
* substock 1: take 10µL of main stock + 90µL sterile ultrapure water
* substock 2: take 10µL of main stock + 90µL sterile ultrapure water

![Notebook Figure 21](https://static.igem.org/mediawiki/2021/9/9f/T--NU_Kazakhstan--Notebook_Figure_36.jpg)

Figure 21. E.Coli streak

![Notebook Figure 22](https://static.igem.org/mediawiki/2021/6/66/T--NU_Kazakhstan--Notebook_Figure_22.jpg)

Figure 22. RPCD 04 1st sample

![Notebook Figure 23](https://static.igem.org/mediawiki/2021/c/c5/T--NU_Kazakhstan--Notebook_Figure_62.jpg)

Figure 23. RPCD 04 2nd sample

### Day 7 - 18.07.2021

1. Make a subculture of Diluted Duo3 and Duo3 in 1ml of LB broth + 5µL gentamicin.
2. Incubate them overnight at 37**°**C.

* **Prepare primers stock**

**For pRGPDuo-rhlB-A-R**

**24.28nmol**

* For main stock: add 242.8µL of sterile ultrapure water
* substock 1: take 10µL of main stock + 90µL sterile ultrapure water
* substock 2: take 10µL of main stock + 90µL sterile ultrapure water

**For pRGPDuo-nadE-F (G5)**

**20.25nmol**

* For main stock: add 202.5µL of sterile ultrapure water
* substock 1: take 10µL of main stock + 90µL sterile ultrapure water
* substock 2: take 10µL of main stock + 90µL sterile ultrapure water

![Notebook Figure 24](https://static.igem.org/mediawiki/2021/c/ca/T--NU_Kazakhstan--Notebook_Figure_75.jpg)

Figure 24. E.Coli Duo 3 + gentamicin

![Notebook Figure 25](https://static.igem.org/mediawiki/2021/2/21/T--NU_Kazakhstan--Notebook_Figure_81.jpg)

Figure 25. E.Coli Duo 3 + gentamicin diluted

![Notebook Figure 26](https://static.igem.org/mediawiki/2021/4/4d/T--NU_Kazakhstan--Notebook_Figure_16.jpg)

Figure 26. E.Coli LB broth (5ml E.Coli + 20ml LB)

## Week 4

### Day 1 - 19.07.2021

PCR

1. 12.5µL Master Mix + 1µL F primer nadE + 1µL R primer nadE + 1.5µL gen. DNA from tube 3 LB + 9µL nucleasefree H2O.
2. Set PCR settings as in Table 7 (17.07.2021)

pRGPDuo3 plasmid extraction

1. Centrifuge 1mL of Duo3 colony at 7000 rpm 4**°**C for 1 minute.
2. Discard supernatant and leave the pellet as dry as possible.
3. Resuspend the pellet with 100µL of ice-cold ALS I and mix gently by pipetting.
4. Add 200µL of room temperature ALS II and gently mix by inverting tube 5 times. Put on ice for 5 minutes.
5. Add 150µL of ice-cold ALS III and gently mix by inverting the tube 5 times. Put on ice for 5 minutes.
6. Centrifuge at 13500 rpm 4**°**C for 5 minutes.
7. Transfer 450µL of supernatant to the new labeled tubes.
8. Precipitate DNA from supernatant by adding 900µL of 70% ethanol and incubate tube at room temperature for 2 minutes.
9. Centrifuge tubes at 13500 rpm 4**°**C for 5 minutes and discard supernatant, leave tubes dry for 15 minutes.
10. Add 1mL of 70% ethanol.
11. Centrifuge tubes at 13500 rpm 4**°**C for 5 minutes and discard supernatant, leave tubes dry for 15 minutes.
12. Dissolve plasmids with 50µL of dd water.
13. Nanodrop concentration analysis:

1st sample - 1529ng/µL

2nd sample - 80.30ng/µL

**Preparations for subculturing**

1. Prepared 950mL of LB broth by adding 7g of powder into 950mL of dd water.
2. Prepared 950ml of LB agar by adding 12.95g of powder into 950mL of dd water.
3. Autoclaved LB broth and LB agar.

**PCR conditions calculations**

1. **pRGPDuo-nadE (G5-F5)**

Table 8. pRGPDuo-nadE (G5-F5)

|  | t (°C) | time |  |
| Initial denaturation | 95 | 2 min. | 1 cycle |
| Denaturation | 95 | 30 seconds | 35 cycles |
| Annealing | 66 | 30 seconds |
| Extension | 70 | 1 min. |
| Final Extension | 70 | 5 min. |  |
| Storage | 4 |  |  |

1. **pRGPDuo-rhlB-A (H1-B2)**

Table 9. pRGPDuo-rhlB-A (H1-B2)

|  | t (°C) | time |  |
| Initial denaturation | 95 | 2 min. | 1 cycle |
| Denaturation | 95 | 30 seconds | 35 cycles |
| Annealing | 68 | 30 seconds |
| Extension | 72 | 1 min. |
| Final Extension | 72 | 5 min. |  |
| Storage | 4 |  |  |

1. PCR reaction of 3rd sample genomic DNA with nadE (G5-F5).
2. Mixed 100mL of autoclaved LB agar with 500µL of gentamicin.
3. Prepared 2 LB agar plates and 2 LB agar slants with gentamicin.
4. Inoculation of pRGPDuo3 (subculturing) into 2 LB agar plates and 2 agar slants with gentamicin.
5. Incubate overnight at 37°C
6. Autoclave dd water and dd water nucleasefree

### Day 2 - 21.07.2021

**Performed PCR:**

1. 12.5µL Master Mix + 1µL F primer rhlB-A + 1µL R primer rhlBA + 1.5µL gen. DNA from tube 3 LB + 9µL nuclease free H2O.
2. Conditions for PCR as in the table 9 (19.07.2021)

**Gel electrophoresis**

1. Prepared 1% agarose gel (2g of agarose in 200mL of 1xTAE.
2. Prepared samples for gel electrophoresis:

Table 10. Results of gel electrophoresis

| Sample | TAE volume (µL) | Loading dye volume (µL) | sample volume (µL) |
| DNA ladder | 12.0 | 5.0 | 3.0 |
| Duo 2 | 12.0 | 5.0 | 3.0 |
| Duo 3 | 12.0 | 5.0 | 3.0 |
| Duo 4 | 12.0 | 5.0 | 3.0 |
| nadE (E5-F5) | 12.0 | 5.0 | 3.0 |
| nadE (G5-F5) | 12.0 | 5.0 | 3.0 |
| rhlB-A | 12.0 | 5.0 | 3.0 |

1. 500x FastBlast was diluted by adding 40 ml of it into 160???. This resulted in the final concentratio 100x FastBlast
2. TAEx1 buffer was prepared by adding 16ml of 50x TAE buffer into 784ml of water
3. Hardened agarose gel was placed into the tray
4. TAEx1 buffer was poured enough to make gel submerged in it
5. Samples were poured in order as depicted in Table 11

Table 11. The order of poured samples

| Wells | Samples |
| 2nd | DNA Ladder |
| 4th | Duo2 |
| 6th | Duo3 |
| 8th | Duo4 |
| 10th | nadE (Es-Fs) |
| 12th | nadE (Gs-Fs) |
| 14th | rhla/b |
| 16th | Duo2 |

1. Closed the last and laft it to ??? at 90V
2. Gel was stained with FastBlast 100x for 3min
3. It was washed under tap warm water
4. FastBlast 100x was diluted into 1x
5. Gel with FastBlast 1x was left overnight

Experiment failed

### Day 3 - 22.07.2021

1. 100ml of 1% agarose gel was prepared by adding 1g of agarose powder to newly diluted (yesterday) TAE 1x buffer
2. It was microwaved and poured into the gel cast. Womb was placed in.
3. Loding samples were prepared following way: 5ul of sample, 5ul og loading dye, 10ul of TAE 1x buffer
4. After gel was solidified, samples were loaded into the wells as it is demonstrated in Table 12

Table 12. The order of samples loaded in wells

| Well no. | Sample |
| 1st | DNA ladder |
| 3rd | empty |
| 5th | DNA ladder (concentrated) |
| 7th | rhla/b |
| 9th | nadE (G5-F5) |
| 11th | nadE (E5-F5) |
| 13th | Duo4 |
| 15th | Duo3 |
| 17th | Duo2 |
| 19th | DNA ladder |

1. Gel Electrophoresis machine was set to 90V and run.
2. When band reached 2/3 of the gel, it was stained with FastBlast and shoved
3. Finally, it was washed several times

1. Prepared 1% gel (3g agarose + 300ml TAE)
2. Microwaved gel poured into gel cast. Put comb into it
3. Prepared samples: 10ul TAE + 5ul loading dye + 5 ul sample
4. Prepared TAE buffer (4ml of 50x TAE + 196ml autoclaved H2O)
5. Run gel electrophoresis (40-45min)
6. Prepared Sybr safe solution (7ul Sybr)
7. Poured Sybr safe solution into gel; gel was left for 24 hours of shacking

### Day 4 - 23.07.2021

1. Gel electrophoresis was loaded according to Table 13. it ran at 90V, and passed half of the gel

Table 13. The order of gel electrophoresis samples loaded in wells

| Well no. | Sample |
| 2nd | DNA Ladder |
| 4th | Duo2 |
| 6th | Duo2 ditilled |
| 8th | Duo3 |
| 10th | Duo4 |
| 12th | nadE (G5) |
| 14th | nadE(E5) |
| 16th | rhla/b |
|  |  |

**Samples preparation:**

1. 10ul sample (DNA ladder, extracted plasmids, Duo 2 distilled, PCR products) + 5ul TAE + 5ul loading dye

**Agarose gel preparationC**

1. Prepared 1% agarose gel (1g agarose + 100ml TAE)
2. Addition of 10ul Ethidium Bromide. Calculations:

*C**1**V**1**=C**2**V**2*

*Stock = 10mg/ul*

*1000ug/ml \* V**1**=1ug/ml \*100ml*

*V**1**= 0.01ml=10ul*

Why our electrophoresis worked? (possible explanations)

1. increased concentrations of samples (10ul ladder, gene of interests, PCR products)
2. We used new loading dye (taken from Aigerim)
3. We stained with Ethidium Bromide
4. We decreased voltage to 90V

Previously we used 120V on 22.07.2021 which was too high voltage. It could melt gel smear bands of samples

PCR

1. PCR with nadE (Gs & Es): 12.5 ul Master Mix + 9ul H2O + 1.5ul gen DNA +1ul F primer + 1ul R primer nadE (Annealing temp. = 63°C) (x3 samples, 6 in total)
2. PCR with rhlA: 12.5ul Master Mix + 9ul H2O + 1.5ul gen DNA + 1ul F primer rhlA + 1ul R primer rhlA (Annealing temp.= 57°C) (3 samples)

## Week 5

### Day 1 - 26.07.2021

**Gel Electrophoresis**

1. Prepare 1% agarose gel (1g of agarose in 100mL 1xTAE)
2. Microwave gel (mix each 15-30 seconds to dissolve agarose)
3. Samples preparation:

Table 14. Samples preparation

|  | Sample volume (µL) | 1xTAE (µL) | Loading dye (µL) |
| DNA ladder | 10 | 5 | 5 |
| rhlB-A | 10 | 5 | 5 |
| Duo2,4 | 10 | 5 | 5 |
| Duo3 | 10 | 5 | 5 |
| rhlB-A | 10 | 5 | 5 |
| Duo2,4 | 10 | 5 | 5 |
| Duo3 | 10 | 5 | 5 |

1. Proceed gel electrophoresis at 120V for 30 minutes.

![Notebook Figure 27](https://static.igem.org/mediawiki/2021/c/cb/T--NU_Kazakhstan--Notebook_Figure_28.jpg)

Figure 27. Gel electrophoresis

### Day 2 - 27.07.2021

Table 15. PCR Q5 and PCR TAQx5 composition

| PCR Q5 |  | PCR TAQx5 |
| Forward primers | 1.25µL |  | Forward primers | 0.5µL |
| Reverse primers | 1.25µL |  | Reverse primers | 0.5µL |
| Gen DNA | 1.5µL |  | Gen DNA | 1.5µL |
| Q5 master mix | 12.5µL |  | Q5 master mix | 5µL |
| nuclease free water | 8.5µL |  | nuclease free water | 17.5µL |

1. Prepare 1% agarose gel (1g of agarose in 100mL 1xTAE)
2. Prepare PCR reaction tubes with TAQx5 mastermix 3 tubes (nadEDuo3, nadEDuo2,4,rhlAB) and with NEBQ5 mastermix 3 tubes (nadEDuo3, nadEDuo2,4,rhlAB).
3. Run PCR.
4. Prepare PCR samples for gel electrophoresis.
5. Gel loading:

Table 16. Gel loading

| 2nd well | DNA ladder |
| 4th well | nadE Duo2,4 |
| 6th well | nadEDuo3 |
| 8th well | rhlAB |
| 10th well | nadE Duo2,4 |
| 12th well | nadEDuo3 |
| 14th well | rhlAB |

Started run at 17:16 (90V) till 17:56.

1. Run PCR with rhlAB primers mix.
2. Stained in EtBr.

![Notebook Figure 28](https://static.igem.org/mediawiki/2021/e/e4/T--NU_Kazakhstan--Notebook_Figure_52.jpg)

Figure 28. Gel electrophoresis results

### Day 3 - 28.07.2021

1. Turned on UV light in hood.
2. Observed runned gel with primers under UV exposure.
3. Prepared PCR tubes for Q5 nadEDuo3, Q5 rhlAB, TAQx5 nadEDuo3, TAQx5 rhlAB (12.5µL master mix, 9µL of nucleose free water, 1.5µL of genomic DNA (sample 3), 0.5µL of forward primer, 0.5µL of reverse primer).
4. Runned PCR

Table 17. Running PCR conditions

| Step | t (°C) | time |  |
| Initial denaturation | 95 | 2 min |  |
| Denaturation | 94 | 30 sec | 35 cycles |
| Annealing | 63 | 30 sec |
| Extension | 68 | 1 min |
| Final extension | 68 | 5 min |  |
| Storage | 4 |  |  |

1. Prepared PCR samples for gel electrophoresis (10µL of sample, 5µL of 1xTAE, 5µL of loading dye).

Table 18. Prepared PCR samples for gel electrophoresis

| 2nd well | DNA ladder |
| 4th well | nadEDuo3 TAQx5 |
| 6th well | nadEDuo3 Q5 |
| 8th well | rhlB-A TAQx5 (Ainur's team) |
| 10th well | rhlB-A Q5 (Ainur's team) |
| 12th well | rhlB-A TAQx5 (Anar's team) |
| 14th well | rhlB-A Q5 (Anar's team) |

1. Run electrophoresis (90V for 1hour and 15 minutes)
2. Prepare PCR tubes NEBQ5 nadEDuo2,4 , TAQx5 nadEDuo2,4 (12.5µL master mix, 9µL nuclease free water, 1.5µL of genomic DNA (sample 3), 1µL of F.P. (13.04), 1µL of R.P. (18.96).

Table 19. Conditions of PCR

| Step | t (°C) | time |  |
| Initial denaturation | 95 | 2 min |  |
| Denaturation | 94 | 30 sec | 35 cycles |
| Annealing | 63 | 30 sec |
| Extension | 68 | 1 min |
| Final extension | 68 | 5 min |  |
| Storage | 4 |  |  |

1. Left gel in DNA stain overnight.

![Notebook Figure 29](https://static.igem.org/mediawiki/2021/e/e8/T--NU_Kazakhstan--Notebook_Figure_64.jpg)

Figure 29. Gel electrophoresis results

### Day 4 - 29.07.2021

1. Prepare 1% agarose gel.
2. Preparing DNA samples (10µL of sample, 5µL of 1xTAE, 5µL of loading dye).

Table 20. The order DNA samples loaded in wells

| 2nd well | DNA ladder |
| 4th well | nadEDuo3 TAQx5 |
| 6th well | nadEDuo3 Q5 |
| 8th well | rhlB-A TAQx5 (28.07) |
| 10th well | rhlB-A Q5 (28.07) |
| 12th well | rhlB-A TAQx5 (27.07) |
| 14th well | rhlB-A Q5 (27.07) |
| 16th well | nadEDuo2,4 TAQx5 |
| 18th well | nadEDuo2,4 Q5 |

1. Set electrophoresis (100V for 35 minutes).
2. Left gel in EtBr overnight.

PCR

For master mix TAQx5

* 5µL TAQx5, 1.5µL genetic DNA, 0.5µL forward primer, 0.5 reverse primer, 17.5µL nuclease free water.

Table 21. PCR condition for TAQx5

|  | t (°C) | time |  |
| Initial denaturation | 95 | 2 min |  |
| Denaturation | 95 | 30 sec | 35 cycles |
| Annealing | varies | 45 sec |
| Extension | 68 | 2 min 30 sec |
| Final extension | 68 | 5 min |  |
| Hold | 4 |  |  |

Table 22. Annealing temperatures for PCR samples TAQx5

|  | 1st t (°C) | 2nd t (°C) | 3rd t (°C) |
| NadE TAQx5 Duo3 | 68 | 61 |  |
| NadE TAQx5 Duo2,4 | 61 | 64 | 66 |
| rhlB-A TAQx5 | 67 | 56 | 63 |

For master mix NebQ5

* 12.5µL NebQ5, 1.5µL genetic DNA, 1.25µL forward primer, 1.25 reverse primer, 8.5µL nuclease free water.

Table 23. PCR condition for NebQ5

|  | t (°C) | time |  |
| Initial denaturation | 98 | 2 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Annealing | varies | 20 sec |
| Extension | 72 | 70 sec |
| Final extension | 72 | 2 min |  |
| Hold | 4-10 |  |  |

Table 24. Annealing temperatures for PCR samples NebQ5

|  | 1st t (°C) |
| NadE NebQ5 Duo3 | 72 |
| NadE NebQ5 Duo2,4 | 72 |
| rhlB-A NebQ5 | 72 |

1. Run PCR rhlB-A TAQx5 Ta=56°C.
2. Run PCR rhlB-A TAQx5 Ta=63°C.

### Day 5 - 30.07.2021

Anar's team

1. Run PCR rhlB-A NebQ5 Ta=72°C.
2. Run PCR NadEDuo2,4 NebQ5 Ta=72°C.
3. Run PCR NadEDuo3 NebQ5 Ta=72°C.
4. Run PCR rhlB-A TAQx5 Ta=67°C.
5. Run PCR NadEDuo2,4 TAQx5 Ta=61°C.
6. Run PCR NadEDuo3 TAQx5 Ta=61°C.

### Day 6 - 31.07.2021

1. Run PCR NadEDuo 3 TAQx5 Ta=68°C.
2. Run PCR NadEDuo 2,4 TAQx5 Ta=64°C.
3. Run PCR NadEDuo 2,4 TAQx5 Ta=66°C.

Gel electrophoresis:

1. Prepare agarose gel (1g of agarose in 100mL 1xTAE)
2. Sample preparation (10µL of sample, 5µL of loading dye, 5µL 1xTAE)
3. Prepared 1xTAE (4mL 50xTAE + 196mL water)

Conduct electrophoresis (90V)

Table 25. The order of samples loaded in wells

| 2nd well | DNA ladder |
| 3rd well | rhlB-A TAQx5 56°C |
| 4th well | rhlB-A TAQx5 63°C |
| 5th well | rhlB-A TAQx5 68°C |
| 6th well | rhlB-A NebQ5 72°C |
| 7th well | NadEDuo2,4 NebQ5 72°C |
| 8th well | NadEDuo2,4 TAQx5 66°C |
| 9th well | NadEDuo2,4 TAQx5 64°C |
| 10th well | NadEDuo2,4 TAQx5 61°C |
| 11th well | NadEDuo3 TAQx5 68°C |
| 12th well | NadEDuo3 NebQ5 72°C |
| 13th well | NadEDuo3 TAQx5 61°C |
| 14th well | NadE TAQx5 |
| 15th well | NadEDuo2,4 NebQ5 |

## August

## Week 1

### Day 1 - 02.08.2021

1. Turned on UV light
2. after gel electrophoresis analysis, PCR conditions for rhlAB were recalculated.
3. Prepare 4 PCR tubes: 12.5µL TAQx5, 1.5 gen. DNA(sample 3), 1.25µL forward rhlB-A primer, 1.25µL reverse rhlB-A primer, 8.5 nuclease free water.
4. Prepare 4 PCR tubes: 12.5µL TAQx2, 1.5 gen. DNA(sample 3), 1.25µL forward rhlB-A primer, 1.25µL reverse rhlB-A primer, 8.5 nuclease free water.

Table 26. 1st set of PCR condtitions. For both TAQx5 rhlB-A and TAQx2 rhlB-A

|  | t (°C) | time |  |
| Initial denaturation | 95 | 2 min |  |
| Denaturation | 95 | 45 sec | 35 cycles |
| Annealing | 56 | 1 min |
| Extension | 68 | 3 min |
| Final extension | 68 | 7 min |  |
| Hold | 4 |  |  |

Table 27. 2nd set of PCR condtitions. For both TAQx5 rhlB-A and TAQx2 rhlB-A

|  | t (°C) | time |  |
| Initial denaturation | 95 | 2 min |  |
| Denaturation | 95 | 30 sec | 35 cycles |
| Annealing | 57 | 45 sec |
| Extension | 70 | 2 min 30 sec |
| Final extension | 70 | 5 min |  |
| Hold | 4 |  |  |

Table 28. 3rd set of PCR condtitions. For both TAQx5 rhlB-A and TAQx2 rhlB-A

|  | t (°C) | time |  |
| Initial denaturation | 95 | 2 min |  |
| Denaturation | 95 | 30 sec | 35 cycles |
| Annealing | 55 | 45 sec |
| Extension | 66 | 2 min 30 sec |
| Final extension | 66 | 5 min |  |
| Hold | 4 |  |  |

### Day 2 - 03.08.2021

Table 29. 4th set of PCR condtitions. For both TAQx5 rhlB-A and TAQx2 rhlB-A

|  | t (°C) | time |  |
| Initial denaturation | 95 | 2 min |  |
| Denaturation | 95 | 30 sec | 35 cycles |
| Annealing | 54 | 45 sec |
| Extension | 67 | 2 min 30 sec |
| Final extension | 67 | 5 min |  |
| Hold | 4 |  |  |

Table 30. Run PCR rhlB-A TAQx5

|  | t (°C) | time |  |
| Initial denaturation | 95 | 2 min |  |
| Denaturation | 95 | 30 sec | 35 cycles |
| Annealing | 57 | 1 min |
| Extension | 68 | 2 min 45 sec |
| Final extension | 68 | 5 min |  |
| Hold | 4 |  |  |

Table 31. Run PCR rhlB-A TAQx5

|  | t (°C) | time |  |
| Initial denaturation | 95 | 2 min |  |
| Denaturation | 95 | 30 sec | 30 cycles |
| Annealing | 55 | 1 min |
| Extension | 68 | 2 min 45 sec |
| Final extension | 68 | 5 min |  |
| Hold | 4 |  |  |

### Day 3 - 04.08.2021

1. preparation of 1% agarose gel (2mL 50xTAE + 1g agarose + water up to 100mL)
2. Sample preparation: 10µL sample, 5µL 1xTAE, 5µL loading dye.

**Gel electrophoresis**

Table 32. Prepared PCR samples for gel electrophoresis (90V)

| 2nd well | DNA ladder |
| 3rd well | rhlB-A 54°C TAQx2 |
| 4th well | rhlB-A 54°C TAQx5 |
| 5th well | rhlB-A 55°C TAQx2 (decreased temperature) |
| 6th well | rhlB-A 55°C TAQx5 (decreased temperature) |
| 7th well | rhlB-A 55°C TAQx5 (changed Textension, t. Anar) |
| 8th well | rhlB-A 56°C TAQx2 (changed conditions) |
| 9th well | rhlB-A 56°C TAQx5 (changed conditions) |
| 10th well | rhlB-A 57°C TAQx2 |
| 11th well | rhlB-A 57°C TAQx5 |
| 12th well | rhlB-A 57°C TAQx5 (changed extension time) (Anar) |

1. Gel observed under UV
2. Prepared 8 taqx5 PCR tubes (12.5ul Taq 5x, 1.25 of Fond R prime, 8.5ul of nuclease free water, 1.5 of genomic DNA-4)
3. Runned PCR for one tube (rhlBA taq 5x)

Table 33. PCR conditions for one tube (rhlBA taq 5x)

|  | Temperature (C) | Time |
| Initial denat. | 95 | 2 min |
| Denaturation | 95 | 30 sec |
| Annealing | 57 | 45 sec |
| Extension | 70 | 2min 30sec |
| Final Extension | 70 | 5 min |
| Hold | 4 | infin. |

1. Prepared samples for gel electrophoresis: 10ul sample + 5ul TAE 1X, 5ul loading dye
2. Run gel at

Table 34. Prepared PCR samples for gel electrophoresis

| 2nd well | DNA ladder |  |  |
| 3rd well | rhlab | 54C | taq 2x |
| 4th well |  | 54C | taq 5x |
| 5th well |  | 55C | taq 2x |
| 6th well |  | 55C | taq 5x |
| 7th well |  | 55C | taq 5x |
| 8th well |  | 56C | taq 2x |
| 9th well |  | 56C | taq 5x |
| 10th well |  | 57C | taq 2x |
| 11th well |  | 57C | taq 5x |
| 12th well |  | 57C | taq 5x (changed time (Anar)) |

1. Observed gel under UV

Table 35. Runned PCR per one tube (rhlBA taq 5x)

|  | Temperature (C) | Time |
| Initial denat. | 95 | 3 min |
| Denaturation | 95 | 30 sec |
| Annealing | 58 | 30 sec |
| Extension | 68 | 1 min |
| Final Extension | 68 | 5 min |
| Hold | 4 | infin. |

1. Placed unrunned (en. PCR) tubes in -20 C fridge and runned in 6

### Day 4 - 05.08.2021

Table 36. Runned PCR for one tube (rhl B-A taq5x)

|  | Temperature (C) | Time |
| Initial denat. | 95 | 2 min |
| Denaturation | 95 | 30 sec |
| Annealing | 59 | 1 min |
| Extension | 68 | 2min 45sec |
| Final Extension | 68 | 5 min |
| Hold | 4 | infin. |

Table 37. Runned PCR for one tube (rhl B-A taq 5x)

|  | Temperature (C) | Time |
| Initial denat. | 95 | 2 min |
| Denaturation | 95 | 30 sec |
| Annealing | 60 | 1 min |
| Extension | 68 | 2min 45sec |
| Final Extension | 68 | 5 min |
| Hold | 4 | infin. |

Table 38. Runned PCR for one tube (rhl B-A taq 5x)

|  | Temperature (C) | Time |
| Initial denat. | 95 | 2 min |
| Denaturation | 95 | 30 sec |
| Annealing | 56.5 | 1 min |
| Extension | 68 | 2min 45sec |
| Final Extension | 68 | 5 min (35 cycles) |
| Hold | 4 | infin. |

1. Prepare 5 new PCR tubes: 0.5 ul gen DNA + 5ul Taq 5x + 1ul Forward + 1 ul Reverse + 1 ul dNTP (0.2mM) + 16.5 Nuclease free water

55.5C, 56C, 56.5C, 57C, 57.5C

1. Prepared 50ul of 0.2mM dNTP:

C1V1 = C2V2

1ul \* 10mM = 0.2mM\*x

x = 50ul

Table 39. rhl B-A Taq 5x PCR tubes

|  | Temperature (C) | Time |  |
| Initial denat. | 95 | 2 min |  |
| Denaturation | 95 | 30 sec |  |
| Annealing | ~ | 1 min |  |
| Extension | 68 | 2min 45sec |  |
| Final Extension | 68 | 5 min |  |
| Hold | 4 | infin. |  |
|  |  |  | Cycles: 35x |

1. Annealing temperature: 55.5C, 56C, 56.5C, 57C, 57.5C

### Day 5 - 06.08.2021

1. Run PCR at 57C, 57.5C
2. Prepare gel electrophoresis samples: 10ul DNA sample + 5ul TAE + 5ul loading dye
3. Samples: DNA ladder, PCR at 57C, 58C, 59C, 60C, 56.5C (Ainur's team)
4. PCR at 55.5C, 56C, 56.5C, 57C, 57.5C (Anar's team)

Table 40. Volumes for sample

| Q5 2x | 12.5ul |
| Forward primer | 1.25ul |
| Reverse primer | 1.25ul |
| gen DNA | 1.5ul |
| Nuclease free water | 1.5ul |

Table 41. PCR conditions

|  | Temperature (C) | Time |  |
| Initial denat. | 98 | 3 min |  |
| Denaturation | 98 | 10 sec |  |
| Annealing | 72 | 25 sec |  |
| Extension | 72 | 120 sec |  |
| Final Extension | 72 | 3 min |  |
| Hold | 4 | infin. |  |
|  |  |  | Cycles: 35x |

Previously we runned Q5 sample with T a = 72C with extension time of 70 sec. However, we got our sample of rhl B-A at 1.35Rb:

1.35 - 70sec

2.4 - x

x ~ 120sec = time of extension

Table 42. Prepared PCR tube with rhl B-A Q5 (Total volume = 30ul)

| Q5 2x | 15ul |
| Gen DNA | 1.5 ul |
| Forward primer | 2 ul |
| Reverse primer | 2ul |
| dNTP (0.2mM) | 1ul |
| Nuclease free water | 8.5 ul |

1. We run PCR rhl B-A Q5 with Anneal/ temperature 72, ext. = 120 sec

![Notebook Figure 30](https://static.igem.org/mediawiki/2021/b/bd/T--NU_Kazakhstan--Notebook_Figure_82.jpg)

Figure 30. Results of PCR rhl B-A Q5 with Anneal/ temperature 72, ext. = 120 sec

![Notebook Figure 31](https://static.igem.org/mediawiki/2021/6/65/T--NU_Kazakhstan--Notebook_Figure_2.jpg)

Figure 31. Results of PCR

### Day 6 - 07.08.2021

1. PCR was run with the tube rhl B-A Q5 30 volume

Table 43. PCR conditions for the tube rhl B-A Q5 30 volume

|  | Temperature (C) | Time |  |
| Initial denat. | 98 | 3 min |  |
| Denaturation | 98 | 10 sec |  |
| Annealing | 72 | 25 sec |  |
| Extension | 72 | 120 sec |  |
| Final Extension | 72 | 3 min |  |
| Hold | 4 | infin. |  |
|  |  |  | Cycles: 35x |

## Week 2

### Day 1 - 09.08.2021

1. Runned gel electrophoresis

Table 44. Gel electrophoresis well orders

| 2nd well | DNA ladder |  |
| 3rd well | rhl B-A Taqx5 | 57C |
| 4th well | rhl B-A Taqx5 | 58C |
| 5th well | rhl B-A Taqx5 | 59C |
| 6th well | rhl B-A Taqx5 | 60C |
| 7th well | rhl B-A Taqx5 | 56C |
| 8th well | rhl B-A Taqx5 | 56.5C |
| 9th well | rhl B-A Taqx5 | 58C |
| 10th well | rhl B-A Taqx5 | 63C |
| 11th well | rhl B-A Q5 | 72C |
| 12th well | rhl B-A Q5 | 72C |
| 13th well | rhl B-A Q5 | 72C |

1. Repeated PCR with 58C (9th product)

Table 45. PCR conditions

|  | Temperature (C) | Time |  |
| Initial denat. | 95 | 3 min |  |
| Denaturation | 95 | 30 sec |  |
| Annealing | 59.5 | 1 min |  |
| Extension | 65 | 2 min 45 sec |  |
| Final Extension | 65 | 5 min |  |
| Hold | 4 | infin. |  |
|  |  |  | Cycles: 35x |

1. Staining protocol ???

### Day 2 - 10.08.2021

1. Prepared samples for gel electrophoresis:
2. Gel electrophoresis: redoing gel because it was accidentally super thick
3. Gel was prepared by mixing 1 g of agarose + 100 ml TAE+ 10ul of EthBr. Allowed to solidify
4. Well loading: 90V for about 45 min

### Day 3 - 11.08.2021

1. Gel electrophoresis results:

Table 46. Results of gel electrophoresis

| 1st well | DNA Ladder |  |  |
| 2nd well | rhl B-A Taqx5 | 55.5C |  |
| 3rd well | rhl B-A Taqx5 | 56C |  |
| 4th well | rhl B-A Taqx5 | 56.5C |  |
| 5th well | rhl B-A Taqx5 | 57C |  |
| 6th well | rhl B-A Taqx5 | 57.5C |  |
| 7th well | rhl B-A Taqx5 | 58C |  |
| 8th well | rhl B-A Taqx5 | 56.5C |  |
| 9th well | rhl B-A Taqx5 | 57C |  |
| 10th well | rhl B-A Taqx5 | 58C |  |
| 11th well | rhl B-A Taqx5 | 59C |  |
| 12th well | rhl B-A Taqx5 | 60C |  |
| 13th well | rhl B-A Taqx5 | 59.5C | (extesion 65) |
| 14th well | rhl B-A Taqx5 | 59.5C |  |
| 15th well | rhl B-A Q5 | 72C | Showed some bands |
| 16th well | rhl B-A Q5 | 72C | Showed some bands |

1. Cut bands from 15th and 16th sample
2. Melt them in Thermomixer compact at 65C

The following steps are according to the PureLink Quick Coel Extraction KIt

1. Cut minimal area of gel with a Band
2. Weight gel ~ 0.34g
3. Add Gel Solubilization Buffer (L3) to the gel in a ratio 3:1 (We added 1ml)
4. Place the tube in a thermomixer for 10 min until gel will dissolve. Each 3 min gently mix by inverting
5. Incubate the tube for additional 5 min
6. Purify DNA using a centrifuge
7. Pipet the dissolved gel into the column in wash tube. One column per 400 mg capacity is 850ml, so that we took approximately 700 ml
8. Centrifuge the flow through and place column into the wash tube
9. Add 500ul Wash buffer (W1)
10. Centrifuge at 12000 rpm for 1 min, discard the flow through
11. Centrifuge one more time at 14000 rpm for 2 mins, discard the flow through
12. Place column in a new wash tube (recovery tube). Add 50 ul of Elution Buffer (E5) into the centre of the column. Incubate 1 min at RT
13. Centrifuge the tube at 12 000 rpm for 1 min
14. Store for long term usage at -20C

**Prepare PCR samples**

1. With Q5 one sample (~25ul v) rhl B-A

* 12.5ul Q5, 1.5ul gen DNA (DNA elution product), 1.25 ul forward rhl B-A primer, 1.25ul Reverse rhl B-A primer, 1ul dNTP (0.2mM), 7.5 ul water
* 2 PCR samples for rhl B-A with Taq x5 (25ul), 5ul Taq x5, 1 ul gen DNA (DNA elution product), 1ul Forward rhl B-A primer, 1ul reverse rhl B-A primer, 1ul dNTP (0.2mM), 16 ul water without nuclease

Table 47. PCR conditions for rhl B-A with Q5

|  | Temperature (C) | Time |  |
| Initial denat. | 98 | 3 min |  |
| Denaturation | 98 | 10 sec |  |
| Annealing | 72 | 25 sec |  |
| Extension | 72 | 2 min |  |
| Final Extension | 72 | 3 min |  |
| Hold | 4 | infin. |  |
|  |  |  | Cycles: 35x |

Autoclaved 400 ul of LB agar, and prepared to agar slants and L agar

Table 48. PCR conditions rhl B-A with Taq 5x

|  | Temperature (C) | Time |  |
| Initial denat. | 95 | 2 min |  |
| Denaturation | 95 | 30 sec |  |
| Annealing | 57 | 1 min |  |
| Extension | 68 | 2 min 45 sec |  |
| Final Extension | 68 | 5 min |  |
| Hold | 4 | infin. |  |
|  |  |  | Cycles: 35x |

![Notebook Figure 32](https://static.igem.org/mediawiki/2021/c/c6/T--NU_Kazakhstan--Notebook_Figure_13.jpg)

Figure 32. Results of gel electrophoresis

### Day 4 - 12.08.2021

1. Autoclaved cotton
2. Prepared NA (28g/L) and NB (8gg/L)
3. Prepared media were autoclaved
4. Prepared Gel for electrophoresis (1g agarose +100ul TAE +10 ul of EthBr (stock solution))
5. Gel loading at 120V for 30 min:

Table 49. Results of gel electrophoresis

| 2nd well | DNA ladder |  |
| 4th well | taq 5x |  |
| 6th well | Q5 | gel results, but at ~ 1500Kb |

![Notebook Figure 33](https://static.igem.org/mediawiki/2021/9/93/T--NU_Kazakhstan--Notebook_Figure_24.jpg)

Figure 33. Results of gel electrophoresis

### Day 5 - 13.08.2021

1. Autoclaved distilled water
2. Turn on sec UV on safety cabinet for 30-40 min
3. Prepared 4 Nutrient Agar slants: 3 in glass tubes (15ml) and 1 in plastic tube (25ml)
4. Prepared 2 Nutrient Broth in plastic tubes (30ml)
5. Transferred P. putida to nutrient Broth tube. Put on 30C, 180 rpm, 48 hours shaking

### Day 6 - 14.08.2021

1. PCR sample prepared rheBIA:

* 15 μl Q5 MM
* 2 μl f.primer
* 2 μl r.primer
* 9.25 μl water
* 0.75 μl gen DNA from tube 4
* 1 μl DNTP (0.02 mM)

1. PCR set up for Tsample:

Table 50. PCR set up for Tsample

|  | t (°C) | time |  |
| Initial denaturation | 98 | 5 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Annealing | 72 | 30 sec |
| Extension | 72 | 2 min 5 sec |
| Final extension | 72 | 3 min 30 sec |  |
| Hold | 4 | infinity |  |

1. PCR sample Taq 2x MM rheBIA:

* Taq 2x MM 15 μl
* 1 μl Fpr
* 1 μl Rpr
* 1 μl DNTPs
* 0.75 μl gen DNA
* 11.25 μl water/30

Table 51. Conditions (sample 57)

|  | t (°C) | time |  |
| Initial denaturation | 96 | 3 min |  |
| Denaturation | 96 | 30 sec |  |
| Annealing | 57 | 60 sec |  |
| Extension | 68 | 3 min |  |
| Final extension | ext | 6 min |  |
| Hold | 4 | infinity |  |

1. 2nd sample (56)

* 16.3 MM
* 1 μ F
* 1 μ R
* 1 μ DNTPs
* 0.75 gen DNA
* 13.5 water/32.25 (accidentially)

Conditions: Annealing temperature = 56 °C

Volume = 32.25

1. P.putida grown overnight were inoculated into a new AB tube, 2 slant glass NA tubes and 1 plastic slant agar tube. Left in incubator for ~24 h at temperature = 28°C, 180 rpm.

### Day 7 - 15.08.2021

Because P.putida did not grow well, cultures were left for 48 hours in another incubator at 170 rpm, t = 30°C.

## Week 3

### Day 1 - 16.08.2021

1. Turned on UV light for 30 min.
2. Run PCR with sample Taq 5x prepared 11.08.2021

Table 52. PCR conditions

| Initial den. | 95°C | 2 min |
| Denaturation | 95°C | 30 sec |
| Annealing | 72°C | 1 min |
| Extension | 72°C | 2 min 45 sec |
| Final extension | 72°C | 5 min |
| Hold | 4°C | infinity |

### Day 2 - 17.08.2021

1. Prepared gel for gel electrophoresis (1 g agarose + 100 mL TAE + 10μl ethidium bromide
2. Gel leading:

Table 53. Gel leading

| 2nd well | DNA ladder |  |  |
| 4th well | taq 2x 56°C |  |  |
| 6th well | taq 2x 57°C |  |  |
| 8th well | taq 5 72°C |  |  |
| 10th well | taq 2x 72°C |  |  |

1. Run the gel at 120 V for 35 minutes.
2. Inoculated 1 slant agar in glass with colony of P.putida from another slant agar in plastic and put into the incubator. (from 14.08.2021)
3. Prepared 2 nutrient Agar plates of 30 mL and inoculated them with P.putida from NB.
4. Prepared 2 nutrient Agar plates of 20 mL and inoculated them with P.putida from NB.
5. Prepared new x1 NB of №30mL and inoculated with P.putida from nutrient agar slant in glass that was inoculated on 14.08.2021.
6. All cultures were put into the incubator.
7. Incubator was set to 170 rpm and 30°C.

### Day 3 - 18.08.2021

1. Prepared PCR samples as follows:

Table 54. PCR samples

| 14 μl | Taq enzyme |
| 2 μl | Forward primer |
| 2 μl | Reverse primer |
| 4.25 μl | Nuclease free water |
| 0.75 μl | Genomic DNA (tube 4) |
| 2 μl | dNTPs |

Table 55. Samples were placed into PCR machinese with following conditions

|  | t (°C) | time |  |
| Initial denaturation | 95 | 5 min |  |
| Denaturation | 95 | 30 sec |  |
| Annealing | Sample 1 - 50 | 30 sec |  |
|  | Sample 2 - 52.5 |  |
|  | Sample 3 - 55 |  |
|  | Sample 4 - 60 |  |
|  | Sample 5 - 62.5 |  |
|  | Sample 6 - 65 |  |
| Extension | 72 | 1 min |  |
| Final extension | 72 | 5 min |  |
| Hold | 4 | infinity |  |

### Day 4 - 19.08.2021

1. Turned on UV light in the safety cabinet for 30 min.
2. Prepared 100 mL Nutrient Agar and 50 mL Nutrient Broth.
3. Put Nutrient Agar and Nutrient Broth in Autoclave.
4. Prepared 2 slant agars (20 ml) and 1 nutrient broth (25 ml) with Putida, incubated overnight.

### Day 5 - 20.08.2021

1. Prepared 7 samples for gel electrophoresis. (DNA ladder, 10 μl gen. DNA + 5 μl TAE + 5 μl loading dye.)
2. Prepared 100 ml of gel (1 g agarose + 100 ml TAE)

![Notebook Figure 34](https://static.igem.org/mediawiki/2021/d/db/T--NU_Kazakhstan--Notebook_Figure_46.jpg)

Figure 34. Results of gel electrophoresis

### Day 6 - 22.08.2021

Table 56. Prepared PCR samples for Dream Taq Green DNA polymerase (Total volume: 30 μl)

|  | μl |
| Dream Taq Buffer | 3 |
| Dream Taq polymerase | 0.15 |
| Nuclease free H2O | 22.75 |
| Forward primer | 1.5 (12.93) |
| Reverse primer | 1.5 (24.28) |
| DNTPs | 0.6 μl from 10mM (stock) |
| Template DNA | 0.5 |

How the Dream Taq polymerase volume was calculated:

Stock concentration is 5 U/μl while 1.25 U in 50 μl PCR sample is needed.

5 U - 1 μl

250 U - 50 μl

C1\*V1 = C2\*V2

250 U \* V1 = 1.25 U \* 50 μl

V1 = 0.25 μl

0.25 μl of Taq polymerase in 50 μl sample

0.15 μl in 30 μl sample

Table 57. Conditions: (Dream Taq protocol)

| Initial denaturation | 95°C | 10 min (after add Dream Taq) |  |
| Denaturation | 95°C | 30 sec | 35 cycles |
| Annealing | ~ | 30 sec |
| Extension | 72°C | 2 min |
| Final extension | 72°C | 10 min |
| Hold |  |  |  |

Comment: "We firstly put sample on PCR machine for 10 min without adding Taq polymerase. After, we removed PCR tube, added polymerase & continued PCR."

Table 58. 4 tubes with Dream Taq polymerase

|  | °C |
| Annealing primer (Tm-5) | 67.5 |
| Annealing primer recommended | 72 |
| Annealing part (Tm-5) | 56.1 |
| Annealing part recommended | 60.5 |

Table 59. Prepared PCR samples with 2x Phusion Master Mix. (Total volume: 30 μl)

|  | μl |
| Phusion Master Mix | 15 |
| Nuclease free H2O | 11.5 |
| Forward primer | 1.5 |
| Reverse primer | 1.5 |

Table 60. Conditions (Phusion 2x Master Mix protocol)

| Initial denaturation | 98°C | 3 min |  |
| Denaturation | 98°C | 10 sec | 35 cycles |
| Annealing | ~ | 30 sec |
| Extension | 72°C | 10 sec |
| Final extension | 72°C | 10 min |
| Hold | 4°C | infinity |  |

## Week 4

### Day 1 - 23.08.2021

1. PCR run of 2x Phusion Master Mix samples followed protocol (Annealing 72°C, 60°C)
2. Prepared 50mM MgCl2 solution (autoclaved 1.19 g MgCl2 in 250 ml of DNA-se free water)
3. Prepared 6 agar plates (LB)
4. Inoculated with E.coli, E.coli Duo2, E.coli Duo3, E.coli Duo4, P.Putida
5. Inocubated at 37°C for 24 hours.
6. Following the protocol for Dream Taq polymerase set PCR tubes with annealing temprature 56.1°C, 60.5°C (Put polymerase after initial 10 min)

### Day 2 - 24.08.2021

![Notebook Figure 35](https://static.igem.org/mediawiki/2021/b/b0/T--NU_Kazakhstan--Notebook_Figure_57.jpg)

Figure 35. Prepared 2 samples for gel electrophoresis (Phusion Master Mix)

1. Prepared TAE:

12 ml of 50x TAE + 588 ml of DI

Table 61. Gel electrophoresis

| 2nd well | DNA ladder |
| 3rd well | Dream Taq 67.9°C |
| 4th well | Dream Taq 72°C |
| 5th well | Dream Taq 56.1°C |
| 6th well | Dream Taq 60.5°C |
| 7th well | Phusion MM 66°C |
| 8th well | Phusion MM 72°C |
| 9th well | Dream Taq 67.9°C |
| 10th well | Dream Taq 72°C |
| 11th well | Dream Taq 56.1°C |
| 12th well | Dream Taq 60.5°C |
| 13th well | Phusion MM 66°C |
| 14th well | Phusion MM 72°C |
| 120 V, 30 min |  |

![Notebook Figure 36](https://static.igem.org/mediawiki/2021/8/86/T--NU_Kazakhstan--Notebook_Figure_68.jpg)

Figure 36. Gel electrophoresis

### Day 3 - 25.08.2021

1. 3 PCR samples were prepared as follows:

Table 62. 3 PCR samples

| 14 μl | Taq 2x polymerase |
| 2 μl | Forward primer |
| 2 μl | Reverse primer |
| 4.5 μl | Nuclease free water |
| 3 μl | Genomic DNA (2nd sample) |
| 2 μl | dNTPs |
| 2.5 μl | MgCl2 |
| Total volume = 30 μl |  |

1. Run PCR

Table 63. PCR conditions

| Initial denaturation | 95°C | 7 min |  |
| Denaturation | 95°C | 1 min | 35 cycles |
| Annealing | 52°C, 54°C, 57°C | 45 sec |
| Extension | 72°C | 1 min |
| Final extension | 72°C | 10 min |  |
|  |  |  |  |

Table 64. Gel electrophoresis (10 μl PCR sample + 5 μl loading dye + 5 μl TAE)

| 1st well | DNA ladder |
| 2nd well | 52°C PCR product |
| 5th well | 54.3°C PCR product |
| 7th well | 57°C PCR product |
| 120 V, 30 min |  |

1. Autoclaved LB agar. Prepared 2 plates. Inoculated them with P.putida. Left them in incubator overnight.

![Notebook Figure 37](https://static.igem.org/mediawiki/2021/8/8a/T--NU_Kazakhstan--Notebook_Figure_83.jpg)

Figure 37. Gel electrophoresis

![Notebook Figure 38](https://static.igem.org/mediawiki/2021/0/02/T--NU_Kazakhstan--Notebook_Figure_3.jpg)

Figure 38. Gel electrophoresis

## Week 5

### Day 1 - 31.08.2021

1. Prepare 2 LB agar plater and 2 LB agar slants.
2. Subculture P. putida into 2 LB agar and 2 LB slants.
3. Prepair PCR samples for rhlB-A.

Table 65. Volume composition of phusion Master mix (30μl)

| sample | volume (μl) |
| Forward Primer | 1.5 |
| Reverse Primer | 1.5 |
| Phusion master mix | 15 |
| genetic DNA (2nd sample) | 3 |
| Nuclease free water | 9 |

Table 66. Volume composition of Dream TAQ (30μl)

| sample | volume (μl) |
| Forward Primer | 1.5 |
| Reverse Primer | 1.5 |
| Dream TAQ buffer | 3 |
| Dream TAQ polymerase | 0.15\* |
| Nuclease free water | 20.25 |
| Genomic DNA (2nd sample) | 3 |
| DNTPs (10mM stock) | 0.6 |

Table 67. PCR conditions for Phusion Master Mix

| step | T (°C) | t |  |
| Initial denaturation | 98 | 1 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Annealing | 57, 67, 72 | 10 sec |
| Extension | 72 | 30 sec |
| Final extension | 72 | 10 min |  |
| Hold | 4 | infinity |  |

Table 68. PCR condition for Dream TAQ

| step | T (°C) | t |  |
| Initial denaturation | 95 | 5 min |  |
| Denaturation | 95 | 30 sec | 35 cycles |
| Annealing | 57, 67, 72 | 30 sec |
| Extension | 72 | 3 min |
| Final extension | 72 | 15 min |  |
| Hold | 4 | infinity |  |

1. Prepair PCR samples into 2mL tube.
2. Centrifuge 2mL tubes at 10000 rpm for 2min.
3. Transfer PCR samples into PCR tubes.
4. Run 6 PCR tubes at 6 different condition.

![Notebook Figure 39](https://static.igem.org/mediawiki/2021/0/03/T--NU_Kazakhstan--Notebook_Figure_5.jpg)

Figure 39. P.putida

## September

## Week 1

### Day 1 - 01.09.2021

1. Turn on UV light on 30 min.
2. Prepair 1% agarose gel (mix 1g of agarose with 100mL 1xTAE, gently heat it until agarose will not dissolve).
3. Transfer gel into electrophoresis track.
4. Prepair samples for electrophoresis run.

Table 69. 20μl sample for Phusion M.M. PCR results

| Phusion Master mix PCR sample | 10μl | 20μl sample for Phusion M.M. PCR results |
| 1xTAE | 5μl |
| Loading dye | 5μl |

Table 70. 20μl sample for Dream TAQ PCR results

| Dream TAQ | 10μl | 20μl sample for Dream TAQ PCR results |
| 1xTAE | 10μl |

Table 71. Gel Electrophoresis track order

| 2nd well | DNA ladder (biolab loading dye) |
| 4th well | DNA ladder |
| 5th well | Phusion MM 57°C |
| 6th well | Phusion MM 67°C |
| 7th well | Phusion MM 72°C |
| 8th well | Dream Taq 57°C |
| 9th well | Dream Taq 67°C |
| 10th well | Dream Taq 72°C |
| 11th well | Phusion MM 57°C |
| 12th well | Phusion MM 67°C |
| 13th well | Phusion MM 72°C |
| 14th well | Dream Taq 57°C |
| 15th well | Dream Taq 67°C |
| 16th well | Dream Taq 72°C |

* Run electrophoresis at 100V for 45 minutes.
* Stain the gel for minimum 2 hours.

![Notebook Figure 40](https://static.igem.org/mediawiki/2021/d/dd/T--NU_Kazakhstan--Notebook_Figure_7.jpg)

Figure 40. Results of gel electrophoresis at 100V for 45 minutes

Analysis of the gel under UV:

We gained bends in 9th and 10th wells: which are corresponds to DreamTAQ 67°C and

Dream TAQ 72°C.

7. Gel elution was made according to the protocol of the Quick gel extraction Kit.

1. Cut bands from 9th to 10th well sample (take minimal amount of the gel).
2. Melt them in Thermomixer compact at 65°C
3. Weight gel ~ 0.10g
4. Add Gel Solubilization Buffer (L3) to the gel in a ratio 3:1 (We added 0.3ml)
5. Place the tube in a thermomixer for 10 min until gel will dissolve. Each 3 min gently mix by inverting
6. Incubate the tube for additional 5 min
7. Purify DNA using a centrifuge
8. Pipet the dissolved gel into the column in wash tube. One column per 400 mg capacity is 850ml, so that we took approximately 700 ml
9. Centrifuge the flow through and place column into the wash tube
10. Add 500ul Wash buffer (W1)
11. Centrifuge at 12000 rpm for 1 min, discard the flow through
12. Centrifuge one more time at 14000 rpm for 2 mins, discard the flow through
13. Place column in a new wash tube (recovery tube). Add 50 ul of Elution Buffer (E5) into the centre of the column. Incubate 1 min at RT
14. Centrifuge the tube at 12 000 rpm for 1 min

8. Store extracted DNA samples in the -20°C. fridge.

9. Prepared sample for PCR rhlB-A from extracted DNA.

Table 72. Composition of sample for PCR rhlB-A from extracted DNA

| sample | volume (μl) |
| Forward Primer | 1.5 |
| Reverse Primer | 1.5 |
| Dream TAQ buffer | 3 |
| Dream TAQ polymerase | 0.15\* |
| Nuclease free water | 20.25 |
| Genomic DNA (sample from PCR DreamTAQ 67°C, eluted) | 3 |
| DNTPs (10mM stock) | 0.6 |

10.Run PCR at following conditions:

Table 73. PCR conditions

| step | T (°C) | t |  |
| Initial denaturation | 95 | 5 min |  |
| Denaturation | 95 | 30 sec | 35 cycles |
| Annealing | 67 | 30 sec |
| Extension | 72 | 3 min |
| Final extension | 72 | 15 min |  |
| Hold | 4 | infinity |  |

### Day 2 - 02.09.2021

1. Turn on UV light on 30 minutes.
2. Prepared 1% agarose by adding 1g of agarose into 100mL 1xTAE
3. Gently heat agaroose gel until agarose will dissolve
4. Add 10μl GelRed
5. Run electrophoresis:

Table 74. Gel Electrophoresis track order

| 2nd well | DNA ladder |
| 3rd well | Phusion MM 72°C |
| 4th well | Dream TAQ 57°C |
| 5th well | Dream TAQ 67°C |
| 6th well | Dream TAQ 72°C |
| 7th well | Dream TAQ 67°C (on elution product) |

100V for 45 minutes.

1. Elution product (01.09.2021) was checked by nanodrop on DNA content. Result=15ng/μl

**Not appropriate graph**

![Notebook Figure 41](https://static.igem.org/mediawiki/2021/0/08/T--NU_Kazakhstan--Notebook_Figure_8.jpg)

Figure 41. Gel electrophoresis

### Day 3 - 03.09.2021

1. Turn on UV for 30 minutes.
2. Prepared PCR samples for Phusion Master Mix (4samples)

Table 75. Prepared PCR samples for Phusion Master Mix

| sample | volume (μl) |
| Forward Primer | 1.5 |
| Reverse Primer | 1.5 |
| Phusion master mix | 15 |
| genetic DNA (2nd sample) | 3 |
| Nuclease free water | 9 |

1. Run PCR at following conditions:

Table 76. PCR conditions

| step | T (°C) | t |  |
| Initial denaturation | 98 | 5 min |  |
| Denaturation | 98 | 40 sec | 35 cycles |
| Annealing | 57, 62, 67, 72 | 1 min 30 sec |
| Extension | 72 | 3 min |
| Final extension | 72 | 10 min |  |
| Hold | 4 | infinity |  |

1. Prepared 1% agarose gel (Adding 1g of agarose into 100mL 1xTAE).
2. Prepared PCR samples for gel electrophoresis:

Table 77. 20μl sample for Phusion M.M. PCR results

| Phusion Master mix PCR sample | 10μl | 20μl sample for Phusion M.M. PCR results |
| 1xTAE | 5μl |
| Loading dye | 5μl |

1. Centrifuge samples at 10000 rpm for 2 minutes.
2. Load samples in to the wells.
3. Runned gel at 100V for 45 minutes.
4. Observed Results.

![Notebook Figure 42](https://static.igem.org/mediawiki/2021/6/62/T--NU_Kazakhstan--Notebook_Figure_10.jpg)

Figure 42. Gel electrophoresis

## Week 2

### Day 1 - 06.09.2021

1. Turn on UV light for 30 minutes.
2. Prepare 300mL LB agar by adding 11.1g of LB agar into 300mL water.
3. Heat LB agar and autoclave.
4. Autoclave di water and LB broth.
5. Prepare kanamycin solution by adding 0.5g of kanamycin sulfate into 10mL sterile (autoclaved) water.
6. Microwave used LB agar abd prepare 2LB agar slants and 2 LB agar plates.
7. Subculture P.putida from alsnt and and plate into 2 LB agar slants and 2 LB agar plates (streak plate).
8. Add 0.3mL of kanamycin solution into 300mL autoclaved LB agar and prepare 2 LB agar + kanamycin plates.
9. Subculture E.coli Duo2 and Duo4 into LB agar plates with kanamycin. (streak plate)
10. Prepare 3 PCR samples with Phusion master mix.

Table 78. PCR samples with Phusion master mix

| sample | volume (μl) |
| Forward Primer | 1.5 |
| Reverse Primer | 1.5 |
| Phusion master mix | 15 |
| genetic DNA (2nd sample) | 3 |
| Nuclease free water | 9 |

1. Proceed gradient PCR with conditions:

Table 79. PCR samples with Phusion master mix

| step | T (°C) | t |  |
| Initial denaturation | 98 | 30 sec |  |
| Denaturation | 98 | 15 sec | 35 cycles |
| Annealing | 68.3, 70.1, 72 | 30 sec |
| Extension | 72 | 45 sec |
| Final extension | 72 | 7 min |  |
| Hold | 4 | infinity |  |

1. Store at -20 friedge.

### Day 2 - 07.09.2021

1. Turn on UV on 30 minutes.
2. Placed grown plate with P.putida in -4 friedge.
3. Prepared 300mL LB agar by adding 11.1 g of LB agar into 300mL water.
4. Autoclaved LB agar.
5. Prepared 2 LB broth + kanamycin tubes by adding 25μl of kanamycin solution into 25mL LB broth in centrifuge tube.
6. Transfer E.coli Duo2 and Duo4 into prepared 2 LB broth tubes with kanamycin.
7. Place them in the incubator overnight.
8. Add 0.3mL of kanamycin solution into autoclaved 300mL LB agar.
9. Prepare 2 LB agar + kanamycin plates and subculture E.coli Duo2 and Duo4 from the previous day into new 2 LB agar+kanamycin plates (streak plate).
10. Incubate overnight at 37°C.

### Day 3 - 08.09.2021

1. Turned on UV light on 30 minutes.
2. Prepared PCR samples with TAQ 2x Master Mix.

Table 80. PCR samples with TAQ 2x Master Mix

| sample | volume (μl) |
| Forward Primer | 0.6 |
| Reverse Primer | 0.6 |
| TAQ 2x master mix | 15 |
| genetic DNA (2nd sample) | 1 |
| Nuclease free water | 12.8 |

Table 81. PCR conditions

| step | T (°C) | t |  |
| Initial denaturation | 95 | 2 min |  |
| Denaturation | 95 | 30 sec | 30 cycles |
| Annealing | 72, 67.9, 60.5, 61.2 | 1 min |
| Extension | 68 | 2 min 30 sec |
| Final extension | 68 | 5 min |  |
| Hold | 4 | infinity |  |

1. PCR conditions for annealing temp equal to extension can be proceed in 2 steps.

Table 82. PCR conditions for annealing temp equal to extension

| step | T (°C) | t |  |
| Initial denaturation | 95 | 2 min |  |
| Denaturation | 95 | 30 sec | 30 cycles |
| Annealing +Extension | 68 | 2 min 30 sec |
| Final extension | 68 | 5 min |  |
| Hold | 4 | infinity |  |

1. Prepare 100mL LB agar (3.7g of LB agar in 100mL water) and 100mL LB broth (2g LB broth in 100mL water).
2. Autoclave LB agar and LB broth.
3. Prepare 4 LB agar plates (25mL) and 2 LB broth (25mL).
4. Transfer one colony of E.coli Duo2 and Duo4 from Arlan's plates into new plates (LB agar + kanamycin).
5. 2 LB agar plates put into friedge for future use.
6. Transfer one colony of E.coli Duo 2 and Duo4 into 2 LB broth + kanamycin.

### Day 4 - 09.09.2021

1. Measured OD of the E.coli Duo2 and E.coli Duo4.

E.coli Duo2 = 0.642

E.coli Duo3 = 0.746

1. Prepared electrocompetent E.coli. WE need electrocompitent P.putida, not E.coli. Need to be redone.
2. Prepare Duo3 LB broth by inoculating Duo3 slant into 1mL of LB broth + 5μl of gentamicin.
3. Incubate at 37°C for 24 hours
4. Prepare 1% agarose gel by adding 1g into 100mL 1xTAE.
5. Prepare PCR sample for gel electrophoresis

Table 83. PCR sample for gel electrophoresis

| sample | 10μl |
| 1xTAE | 5μl |
| Loading dye | 5μl |

Proceed gel electrophoresis:

Table 84. Gel Electrophoresis track order

| 2nd well | DNA ladder |
| 3rd well | Phusion MM 68.3°C |
| 4th well | Phusion MM 70.1°C |
| 5th well | Phusion MM 72°C |
| 6th well | TAQ 2X 60.5°C |
| 7th well | TAQ 2X 61.15°C |
| 8th well | TAQ 2X 67.9°C |
| 9th well | TAQ 2X 68°C |
| 10th well | TAQ 2X 72°C |

Run at 100V for 50minutes.

1. Prepare LB agar by adding 8.10g of LB agar into 220mL water. Heat and autoclave.
2. Add 1.1mL of gentamicin into 220mL LB agar.
3. Prepare 1 streak plate of E.coli Duo3 in LB agar + gentamicin.
4. Prepare LB broth with P.putida by inoculating single colony from streak plate into 25mL LB broth.
5. Incubate P.putida at 30°C.

![Notebook Figure 43](https://static.igem.org/mediawiki/2021/5/5c/T--NU_Kazakhstan--Notebook_Figure_11.jpg)

Figure 43. Gel electrophoresis results

### Day 5 - 10.09.2021

**Plasmid extraction of E.coli Duo2, E.coli Duo3 and E.coli Duo4**

1. 1mL of culture tube of E.coli Duo2, Duo3 and Duo4 centrifuge at 10000rpm for 1 minute. Discard the supernatant, leave the pellet as dry as possible.
2. Resuspended the pellet in 100 uL of ice cold ALS I (autoclaved) and mixed by pipetting, makin sure the cells dispersed fully.
3. Added 200 uL of ALS II and mixed by inverting the tube 5 times, making sure the content of the tube makes contact with ALS II. Stored the tube on ice for 5 minutes.
4. Added 150 uL of ALS III (autoclaved) and mixed gently by inverting the tube 5 times. Stored the tube on ice for 5 minutes.
5. Centrifuged at 10000 rpm for 6 minutes at 4**°**C.
6. Transferred the supernatant (450 uL) to the new labeled tube.
7. Precipitated DNA from supernatant by adding 2 volumes (900 uL) of 70% ethanol at room temperature. Gently mixed by inverting and incubated 2 minutes at room temperature.
8. Centrifuged at 10000 rpm for 6 minutes at 4**°**C. Discarded the supernatant and dried the pellet.
9. Added 1 mL of 70% ethanol and mixed by inverting.
10. Centrifuged at 13500 rpm for 2 minutes at 4**°**C. Removed the supernatant and left the tube to dry for 10-15 minutes until ethanol will evaporate.
11. Dissolved pellet in 50 uL H2O and checked OD 1 uL of each solution by nanodrop.

* Duo2 = 260ng/μl
* Duo3 = 603ng/μl
* Duo4 = 458.7ng/μl

1. Prepare LB broth by adding 6g of LB broth into 300mL water. Heat solution.
2. Autoclave LB broth.
3. Prepare 1% agarose gel by adding 1g pf agarose into 100mL 1xTAE.
4. Microwave agarose gel until all agarose will dissolve.
5. Add 10μl of stain into the gel.
6. Prepare samples for gel electrophoresis:

Table 85. PCR sample for gel electrophoresis

| sample | 10μl |
| 1xTAE | 5μl |
| Loading dye | 5μl |

1. Run gel electrophoresis at 120V for 30 minutes.
2. Prepare 2LB broth + kanamycin by adding 25μl of kanamycin into 25mL of LB broth. Inoculate E.coli Duo2 and E.coli Duo 4 into these LB broth + kanamycin tubes.
3. Prepare 1mL LB broth tube + 5μl of gentamicin. Inoculate E.coli Duo3 into LB broth with gentamicin.
4. Prepare 25mL LB agar slant, add immediately 25μl of kanamycin solution. After slant will be prepared, subculture E.coli Duo4 into LB agar + kanamycin slant.

![Notebook Figure 44](https://static.igem.org/mediawiki/2021/a/ac/T--NU_Kazakhstan--Notebook_Figure_12.jpg)

Figure 44. Nanodrop results

![Notebook Figure 45](https://static.igem.org/mediawiki/2021/c/cf/T--NU_Kazakhstan--Notebook_Figure_14.jpg)

Figure 45. Gel electrophoresis

### Day 6 - 11.09.2021

1. OD of P.putida 0.266
2. Leave P.putida in incubator at 30°C for overnight.
3. Prepare PCR samples with TAQ x2:

Table 86. PCR samples with TAQ x2

| sample | volume (μl) |
| Forward Primer | 0.6 |
| Reverse Primer | 0.6 |
| TAQ 2x master mix | 15 |
| genetic DNA (2nd sample) | 1 |
| Nuclease free water | 12.8 |

Table 87. PCR conditions

| step | T (°C) | t |  |
| Initial denaturation | 95 | 3 min |  |
| Denaturation | 95 | 30 sec | 35 cycles |
| Annealing | 72, 60.5, 61, 56, 57 | 1 min |
| Extension | 68 | 2 min 30 sec |
| Final extension | 68 | 7 min |  |
| Hold | 4 | infinity |  |

To improve activity of TAQx2 we decided to add MgCl2

1. Prepare 10mL of 0.1M MgCl2 (0.095g +10mL water).
2. dilute 100 fold to get 1mM.

Prepare electrocompetent P.putida.

1. Measure OD of P.putida = 0.266
2. Pour 10mL of P.putida culture solution into prechilled 15mL centrifuge tube.
3. Centrifuge at 4700 rpm at 4**°**C for 10 minutes.
4. Discard supernatant.
5. Resuspend pellet in 1.5mL centrifuge tube with 1mL of ice-cold dd H2O.
6. Microcentrifuge at 7000 rpm at 4**°**C for 5 minutes.
7. Repeat 6-8 steps 4 times more.
8. Discard supernatant.
9. Resuspend final pellet with 50µL of ice cold dd H2O, put cells on ice.

## Week 3

### Day 1 - 13.09.2021

1. Turn UV light on 30 minutes.
2. Prepare 1% agarose gel by adding 0.3g of agarose into 30mL 1xTAE.
3. Microwave gel until agarose will dissolve.
4. Prepare PCR samples for gel electrophoresis:

Table 88. PCR samples for gel electrophoresis

| sample | 10μl |
| 1xTAE | 5μl |
| Loading dye | 5μl |

Load samples into the gel:

Table 89. Gel Electrophoresis track order

| 1st well | DNA ladder |
| 2nd well | TAQ 2x 56°C |
| 3rd well | TAQ 2x 57°C |
| 4th well | TAQ 2x 60.5°C |
| 5th well | TAQ 2x 61°C |
| 6th well | TAQ 2x 72°C |

1. Prepare 2 more electrocompetent P.putida tubes:

1. Measure OD of P.putida = 1
2. Dilute P.putida culture with LB broth.
3. Measure OD = 0.536
4. Pour 10mL of P.putida culture solution into 2 prechilled 15mL centrifuge tube.
5. Centrifuge at 4700 rpm at 4**°**C for 10 minutes.
6. Discard supernatant.
7. Resuspend pellet in 2 1.5mL centrifuge tube with 1mL of ice-cold dd H2O.
8. Microcentrifuge at 7000 rpm at 4**°**C for 5 minutes.
9. Repeat 6-8 steps 4 times more.
10. Discard supernatant.
11. Resuspend final pellet with 50µL of ice cold dd H2O, put cells on ice.

**Electroporation of P.putida**

Measure Duo plasmids concentration with nanodrop.

* Duo2 = 1492ng/μl
* Duo3 = 844.2ng/μl
* Duo4 = 965.7ng/μl

* We need 75ng of plasmids Duo2:

Dilute Duo 2 plasmids by adding 1µL of Duo2 plasmids with 99µL TAE.

Take 5µL of diluted Duo2 plasmids (74.6ng).

* We need 75ng of plasmids Duo3:

Dilute Duo3 plasmids by adding 1µL of Duo3 + 9µL of TAE

Take 1µL of diluted Duo3 plasmids (84.42ng).

* We need 75ng of plasmids Duo4:

Dilute Duo4 plasmids by adding 1µL of Duo4 + 9µL TAE.

Take 1µL of diluted Duo4 (96.57ng).

1. Mix needed quantity of plasmids with P.putida
2. Turned on electroporator (18 kV, 25 uF, 200 ohms).
3. Wiped the cuvette with Kimtech, then placed the cuvette into electroporator for 4 sec.
4. Immediately added 1 mL of LB -> quickly pipette.
5. Transferred the mixture to fresh 1.5 mL tube.
6. Repeat for all plasmids. Duo2, Duo3 and Duo4.
7. Incubated at 150 rpm 30**°**C for 1 hour.
8. Prepared 6 agar plates

* P.putida + duo2 (LB agar + kanamycine)
* P.putida + duo3 (LB agar + gentamicin)
* P.putida + duo4 (LB agar + kanamycine)
* P.putida + duo2 (LB agar + gentamicin) negative control
* P.putida + duo3 (LB agar + kanamycine) negative control
* P.putida + duo4 (LB agar + gentamicin) negative control
* For kanamycine plates add 25μl of kanamycine solution into 25mL LB agar.
* For gntamicin plates add 125μl of gentamicin solution into 25mL LB agar

Spread 100μl of transformed putida in the plates (spread plate).

Incubate plates overnight at 30**°**C.

**Plasmid extraction of Duo2,3,4 from E.coli** accoding protocol.

1. 1 mL of culture tubes (2 tubes) were centrifuged at 7000 rpm for 1 minute at 4**°**C.
2. The supernatant was discarded and pellet was left to dry.
3. Prepared 10 mL of ALS II by mixing 1 mL of 3N NaOH, 1 mL of 10% SDS and ddH2O.

1. Resuspended the pellet in 100 uL of ice cold ALS I (autoclaved) and mixed by pipetting, makin sure the cells dispersed fully.
2. Added 200 uL of ALS II and mixed by inverting the tube 5 times, making sure the content of the tube makes contact with ALS II. Stored the tube on ice for 5 minutes.
3. Added 150 uL of ALS III (autoclaved) and mixed gently by inverting the tube 5 times. Stored the tube on ice for 5 minutes.
4. Centrifuged at 13500 rpm for 5 minutes at 4**°**C.
5. Transferred the supernatant (450 uL) to the new labeled tube.
6. Precipitated DNA from supernatant by adding 2 volumes (900 uL) of 70% ethanol at room temperature. Gently mixed by inverting and incubated 2 minutes at room temperature.
7. Centrifuged at 13500 rpm for 5 minutes at 4**°**C. Discarded the supernatant and dried the pellet.
8. Added 1 mL of 70% ethanol and mixed by inverting.
9. Centrifuged at 13500 rpm for 2 minutes at 4**°**C. Removed the supernatant and left the tube to dry for 10-15 minutes until ethanol will evaporate.
10. Dissolved pellet in 50 μl H2O and checked OD 1 uL of each solution by nanodrop.

![Notebook Figure 46](https://static.igem.org/mediawiki/2021/1/13/T--NU_Kazakhstan--Notebook_Figure_17.jpg)

Figure 46. Gel electrophoresis

![Notebook Figure 47](https://static.igem.org/mediawiki/2021/d/d6/T--NU_Kazakhstan--Notebook_Figure_18.jpg)

Figure 47. Transformed P.putida

![Notebook Figure 48](https://static.igem.org/mediawiki/2021/4/44/T--NU_Kazakhstan--Notebook_Figure_19.jpg)

Figure 48. Transformed P.putida 2nd

### Day 2 - 14.09.2021

Table 90. Prepared PCR samples

| 1st tube (20μl) | 2nd tube (20μl) | 3rd tube (30μl) | 4th tube (25μl) |
| 1μl Forward primer | 1.5μl Forward primer | 1.5μl Forward primer | 1.875μl Forward primer |
| 1μl Reverse primer | 1.5μl Reverse primer | 1.5μl Reverse primer | 1.875μl Reverse primer |
| 1μl genomic DNA | 1μl genomic DNA | 1μl genomic DNA | 1μl genomic DNA |
| 10μl Phusion Master Mix | 10μl Phusion Master mix | 15μl Phusion Master Mix | 12.5μl Phusion Master Mix |
| 7 μl nuclease free water | 6μl nuclease free water | 8.75μl nuclease free water | 5.875μl nuclease free water |
|  |  | 0.9μl of DMSO | 1.125μl MgCl2 solution |
|  |  | 1.35μl of MgCl2 solution | 0.75μl DMSO |

Proceed 2 step PCR condition:

Table 91. 2 step PCR condition

| step | T (°C) | t |  |
| Initial denaturation | 98 | 3 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Extension | 72 | 1 min 30 sec |
| Final extension | 72 | 10 min |  |
| Hold | 4 | infinity |  |

P.putida Culture:

* Inoculated P.putida into 2 tubes with 25ml of LB broth.
* Incubate for overnight at 30°C.

### Day 3 - 15.09.2021

1. Prepare 30ml 1xTAE by mixing 0.6mL 50xTAE with 29.5mL of di water.
2. Prepare agarose gel by adding 0.3g of agarose into 30ml 1xTAE.
3. Microwave gel until all agarose will dissolve.
4. Add 3μl of stain into the gel.

Prepare samples for gel electrophoresis: 10μl of sample + 5μl loading dye + 5μl 1xTAE.

Table 92. Gel Electrophoresis track order

| 1st well | DNA ladder |
| 3rd well | 1st PCR tube (14.09.21) |
| 4th well | 2nd PCR tube (14.09.21) |
| 5th well | 3rd PCR tube (14.09.21) |
| 6th well | 4th PCR tube (14.09.21) |

Run gel electrophoresis at 120V for 29 minutes

1. 4 plates with LB agar + kanamycine were prepared (0.1mL kanamycine + 100mL LB agar).
2. 2 plates with gentamicin were prepared

![Notebook Figure 49](https://static.igem.org/mediawiki/2021/5/53/T--NU_Kazakhstan--Notebook_Figure_49.jpg)

Figure 49. Gel electrophoresis

### Day 4 - 17.09.2021

1. Measured quantity of extracted plasmids, which were prepared on 13th September, using nanodrop.

Table 93. Measured quantity of extracted plasmids

|  | 260/280 | ng/μ |
| Duo2 | 2.08 | 926.2 |
| Duo3 | 2.14 | 1522 |
| Duo4 | 1.93 | 559.5 |

Prepare PCR samples: 0.6μl DNTPs + 1.5μl Forward primer + 1.5μl reverse primer + 10μl Phusion master mix + 0.6μl DMSO + 1.5μl MgCl2 + 2μl genomic DNA + 8.3μl nuclease free water (Total 25μl)

Table 94. PCR conditions

| step | T (°C) | t |  |
| Initial denaturation | 98 | 1 min |  |
| Denaturation | 98 | 45 sec | 35 cycles |
| Annealing | 57, 62, 66, 70 | 1 min |
| Extension | 72 | 1 min 15 sec |
| Final extension | 72 | 10min |  |
| Hold | 4 | infinity |  |

For transformation we need approximately 75ng of plasmids.

Duo 2 (926ng/μl) - dilute 10 fold by adding 9μl 1xTAE into 1μl Duo2 plasmid = 92,6ng/μl. Take 1μl.

Duo 3 (1522ng/μl) - dilute 100fold by adding 99μl of 1xTAE into 1μl Duo3 plasmid = 15.22ng/μl. Take 5μl.

Duo4 (559.5ng/μl) - dilute 10 fold by adding 9μl 1xTAE into 1μl Duo4 plasmid = 55.95ng/μl. Take 1.5μl.

1. Conduct electroporation of P.putida with Duo2,3,4 at 2500V, 25μF and 200Ω according to protocol.
2. Transfer bacteria to prechilled cuvette, cap cuvette on the bench.
3. Immediately after electroporation add 1ml of LB broth and pipette quickly up and down.
4. Transfer solution into new 1.5ml tube and incubate at 30**°**C for 1 hour.
5. Prepared 6 agar plates

* P.putida + duo2 (LB agar + kanamycine)
* P.putida + duo3 (LB agar + gentamicin)
* P.putida + duo4 (LB agar + kanamycine)
* P.putida + duo2 (LB agar + gentamicin) negative control
* P.putida + duo3 (LB agar + kanamycine) negative control
* P.putida + duo4 (LB agar + gentamicin) negative control
* For kanamycine plates add 25μl of kanamycine solution into 25mL LB agar.
* For gntamicin plates add 125μl of gentamicin solution into 25mL LB agar

Spread 100μl of transformed putida in the plates (spread plate).

Incubate plates overnight at 30**°**C.

3. Prepared LB agar.

4. Inoculated P.aeruginosa on LB agar plate and LB broth.

## Week 4

### Day 1 - 20.09.2021

**DNA extraction of P.aeruginosa**

1. Turn on the UV light for 30 minutes.
2. Measure OD pf LB broth with P.aeruginosa: OD = 0.5
3. Take 2ml of cell suspension and centrifuge at 4700 rpm for 30 minutes. (2 falcon tubes)
4. Discard supernatant and resuspend the pellet with 1ml of TE buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.0) and centrifuge tube at 4700 rpm for 20 minutes.
5. Supernatant was discarded, leaving 300μl of the solution. And was resuspended with 1ml of Lysis solution (Bio-rad). Both tubes were incubated for 1 hour at 37**°**C.
6. Add 1 volume of phenol/chlorophorm/isoamyl (25:24:1 v/v) about 1.3mL into the tube.
7. Tubes allowed to stay or 5 minutes before centrifuge.
8. Centrifuge tubes at 4700 rpm for 33minutes.
9. The upper aqueous solution was carefuly discarded into the new tube. (2 tubes overall)
10. Into each tube 0.1 volume of sodium acetate and 1 volume of absolute ethanol were added:

For 1st tube: 80μl of sodium acetate and 0.8ml of absolute ethanol

For 2nd tube: 90μl of sodium acetate and 0.9ml absolute ethanol

1. Precipitate DNA by putting tubes into -20**°**C friedge overnight.
2. Prepare 300mL of LB agar by adding 11.1g of LB agar into 300mL water then heat and autoclave.
3. Prepare 300mL of LB broth by adding 6g of LB broth into 300mL water then heat and autoclave.
4. Prepare PCR result samples for gel electrophoesis: 10μl of sample +5μl of loading dye + 5μl of 1xTAE

Table 95. Gel Electrophoresis track order

| 1st well | DNA ladder |
| 2nd well | Phusion Master Mix 66°C |
| 3rd well | Phusion Master Mix 57°C |
| 4th well | Phusion Master Mix 62°C |
| 5th well | Phusion Master Mix 70°C |
| 6th well | DNA ladder with another loading dye |

1. Prepare agarose gel by adding 0.3g agarose into 30mL 1xTAE.
2. Microwave gel until agarose will be dissolved.
3. Run gel electrophoresis at 100V for 40 minutes.
4. Wash glassware and clean bench.

![Notebook Figure 50](https://static.igem.org/mediawiki/2021/c/c0/T--NU_Kazakhstan--Notebook_Figure_23.jpg)

Figure 50. Gel electrophoresis

### Day 2 - 21.09.2021

**DNA extraction of P.aeruginosa**

1. Centrifuge precipitated DNA at 10000 rpm for 15 minutes.
2. Add 200μl of 70% ethanol.
3. Centrifuge at 10000 rpm for 15 minutes.
4. Discad supernatant and repeat washing steps with ethanol 3 times.
5. After final centrifugation discard ethanol solution, pellet were left to airdry.
6. Add 100μl of ultrapure water.
7. Observe concentration by nanodrop:

1st tube = 124.1 ng/μl

2nd tube = 33.94ng/μl

**Electroporation of P*****.putida***

1. Measured OD of P.putida: OD = 0.91.
2. Added 4mL of P.putida into 15ml centrifuge tube. (Overall 3tubes)
3. Centrifuge at 4700 rpm for 10 minutes at 4**°**C.
4. Discard the supernatant.
5. Resuspend pellet in 1ml of ice-cold di water.
6. Centrifuge at 4700 rpm for 10 minutes at 4**°**C.
7. Repeat washing 2 times more.
8. Centrifuge at 10000 rpm for 5 minutes at 4**°**C.
9. Remove supernatant, resuspend pellet in 50μl of ice-cold di water.
10. Measure concentration of plasimds.

Duo2 - 738.5ng/μl

Duo3 - 693.2ng/μl

Duo4 - 664.3 ng/μ

We need approximately 75ng of plasmids, so we proceed dilution.

Dilute Duo2 by adding 9μl of 1xTAE into 1μl plasmid, means 73.8ng in 1μl.

Dilute Duo3 by adding 9μl of 1xTAE into 1μl plasmid, means 69.3ng in 1μl

Dilute Duo4 by adding 9μl of 1xTAE into 1μl plasmid, means 66.4ng in 1μl

1. 1μl of each plasmid was added to P.putida and mixed.
2. Transfer bacteria to prechilled cuvette, cap cuvette on the bench.
3. Turn electroporator: 2500V, 25μF and 200Ω 6ms
4. Immediately after electroporation add 1ml of LB broth and pipette quickly up and down.
5. Transfer solution into new 1.5ml tube and incubate at 30**°**C for 1 hour.
6. Spread 100μl of transformed P.putida into LB agar plate (spread plate).
7. 3 LB agar plates with P.putida Duo2, Duo3, Duo4 (only positive control) were prepared. (Duo2,4 with kanamycine, Duo3 with gentamicin)f
8. inoculated P.putida and P.aeruginosa into new LB broth.

![Notebook Figure 51](https://static.igem.org/mediawiki/2021/d/d5/T--NU_Kazakhstan--Notebook_Figure_25.jpg)

Figure 51. LB agar+kanamycin putida+Duo 4

![Notebook Figure 52](https://static.igem.org/mediawiki/2021/b/b7/T--NU_Kazakhstan--Notebook_Figure_26.jpg)

Figure 52. LB agar+gentamicin putida+Duo 3

![Notebook Figure 53](https://static.igem.org/mediawiki/2021/c/c9/T--NU_Kazakhstan--Notebook_Figure_27.jpg)

Figure 53. LB agar+kanamycin putida+Duo 3

### Day 3 - 22.09.2021

1. Buffers from Expand High Fidelity PCR System (Roche) were thowed and incubated at 37**°**C to allow buffers be dissolved.
2. Prepare two mixes MIX1 (sample) and MIX2 (PCR buffer).
3. Mix 1 content (prepared for 5 PCR reactions)

7.5μl Forward Primer + 7.5μl Reverse Primer + 5μl DNTP mix + 7.5μl template DNA + 97.5μl nuclease free water (Total 125μl)

1. Mix 2 content (Prepared for 5 PCR reactions)

25μl Expand high fidelity buffer + 3.75μl Enzyme Mix + 96.25μl (Total 125μl)

Table 96. PCR conditions

| Steps | t (°C) | time |  |
| Initial Denaturation | 94 | 5 min |  |
| Denaturation | 94 | 15 sec | 10 cycles |
| Annealing | 60.5, 66.5, 72 | 30 sec |
| Elongation | 72 | 2 min |
| Denaturation | 94 | 15 sec | 20 cycles |
| Annealing | 60.5, 66.5, 72 | 30 sec |
| Elongation | 72 | 2 min + 5sec/cycle |
| Final elongation | 72 | 7 min |  |
| Hold | 4 | forever |  |

1st tube (60.5**°**C): 25μl of Mix1 + 25μl of Mix2

2nd tube (66.5**°**C): 25μl of Mix1 + 25μl of Mix2

3rd tube (72**°**C): 25μl of Mix1 + 25μl of Mix2

**Gel electrophoresis:**

1. Prepare agarose gel by adding 0.3g into 30mL 1xTAE.
2. Microwave gel until agaroe will dissolve.
3. Prepare samples for gel electrophoresis: 10μl of sample + 5μl loading dye + 5μl 1xTAE

Table 97. Gel Electrophoresis track order

| 1st well | DNA ladder |
| 2nd well | Roche Mix 60.5°C |
| 3rd well | Roche Mix 66.5°C |
| 4th well | Roche Mix 72°C |
| 6th well | DNA ladder |

Run gel electrophoresis at 120V for 29 minutes.

![Notebook Figure 54](https://static.igem.org/mediawiki/2021/7/7b/T--NU_Kazakhstan--Notebook_Figure_29.jpg)

Figure 54. Gel electrophoresis

### Day 4 - 23.09.2021

1. Prepare agarose gel by adding 0.3g of agarose into 30mL 1xTAE.
2. Microwave until agarose will dissolve and add 3μl stain. \*too much stain were used, so gel appeared red
3. Prepare samples for gel electrophoresis: 10μl of sample + 5μl loading dye + 5μl 1xTAE

Table 98. Gel Electrophoresis track order

| 1st well | DNA ladder |
| 2nd well | Roche Mix 60.5°C |
| 3rd well | Roche Mix 66.5°C |
| 4th well | Roche Mix 72°C |

Run gel electrophoresis at 120V for 29 minutes..

### Day 5 - 24.09.2021

1. Turned on UV light for 30 mins.
2. Prepared 2 plates by pouring 20 microliters LB agar + kanamycin and 2 plates with LB agar + gentamycin.
3. Measured the OD of P.putida - 0.61.
4. Prepared 2 tubes with 5 ml of P. putida.
5. Centrifuged tubes 4700 rpm, 4**°**C, 10 min.
6. Discarded the supernatant.
7. Resuspended the pellet in 1 ml of sterile, ice-cold ddH2O and transfer inro a dilluted 1.5 ml microcentrifuge tube.
8. Centrifuged at 4700 rpm, 4**°**C, 10 min.
9. Discarded the supernatant.
10. Resuspended the pellet in 1 ml of ice-cold ddH2O.
11. Repeated steps 8-10 two more times for a total of three washes.
12. Spined final time, at 4700 rpm at 4**°**C, for 10 min, discard the supernatant, resuspend the pellet in 50 microliters of ice-cold ddH2O, and put the cells on ice.
13. Added 0.37 microliters (500 ng) of Gauttam plasmid to one tube, and 0.86 microliters (500 ng) of extracted Duo 2 to another tube.
14. Transfered the bacteria to a chilled electroporation cuvette.
15. Closed with the cap, and put cuvettes back on ice.
16. Turned on the electroporator and set it to 2.5 kV, 25 microF, 200 Ohm.
17. Wiped the cuvette with Kimwipe to remove any residual of ice or water and place cuvette into electroproparation chamber
18. The time for electroporation 5 sec.
19. Immeadiately, added 1 ml of LB broth into the cuvette and piped up and down gently.
20. Transferred the mixture into 1.5 ml microcentrifuge tubes and placed then to incubate at 37**°**C for 1 hour.
21. In the same time, agar gel was prepared by using 3g of agar to 30 ml of TAE.
22. The 20 microliters of samples were loaded.
23. The gel was runned at 100 V, 20 minutes.

### Day 6 - 25.09.2021

1. After dilution, OD of P. putida was 1.137

Creation of competent P. putida.

1. Transfer 5 ml of LB + P.putida into tube
2. Centrifuge at 4700 rpm, 4**°**C, 10 min.
3. Discard the supernatant.
4. Add 5 ml of CaCl2 0.1M, resuspend.
5. Put on ice for 30 min.
6. Centrifuge at 4700 rpm, 4**°**C, 10 min.
7. Discard the supernatant.
8. Add 5 ml of CaCl2 0.1M, 20% glycerl solution, resuspend, adn leave for approximately 10 minutes.
9. Centrifuge at 4700 rpm, 4**°**C, 7-10 min.
10. Remove supernatant.
11. Wash with cold DI water, add 5 ml of DI water, resuspend, centrifuge at 4700 rpm, 4**°**C, 10 min.
12. Remove supernatant.
13. Resuspend pellet in 50 microliters of cold DI water.

Electroporation

1. Add 500 ng of Duo2 (0.86 microliters of Duo2 extracted)
2. Transfer bacteria inro chilled electroporator cuvette. Tap cuvette on bench.
3. Let electroporator: 2500 V, 25 microF, 200 Ohm

1. Put cuvette into electroporator, start and remove after 5 seconds.
2. Immediately, add 1 ml of LB broth, pipette avoiding bubbles.
3. Transfer P.putida + plasmid solution to fresh 1.5 ml tube.
4. Incubate at 30**°**C for 1 hour.
5. Prepare 2 LB agar + kanamycin (25 ml agar + 25 microliters kanamycin)
6. Pour 100 microliters of P.putida + Duo2 onto plates (streak plates)

![Notebook Figure 55](https://static.igem.org/mediawiki/2021/8/86/T--NU_Kazakhstan--Notebook_Figure_31.jpg)

Figure 55. Plates with transformed P.putida. (With extracted Duo2 and Gauttam’s Duo2)

### Day 6 - 25.09.2021

1. After dilution, OD of P. putida was 1.137

Creation of competent P. putida.

1. Transfer 5 ml of LB + P.putida into tube
2. Centrifuge at 4700 rpm, 4°C, 10 min.
3. Discard the supernatant.
4. Add 5 ml of CaCl2 0.1M, resuspend.
5. Put on ice for 30 min.
6. Centrifuge at 4700 rpm, 4°C, 10 min.
7. Discard the supernatant.
8. Add 5 ml of CaCl2 0.1M, 20% glycerl solution, resuspend, adn leave for approximately 10 minutes.
9. Centrifuge at 4700 rpm, 4°C, 7-10 min.
10. Remove supernatant.
11. Wash with cold DI water, add 5 ml of DI water, resuspend, centrifuge at 4700 rpm, 4°C, 10 min.
12. Remove supernatant.
13. Resuspend pellet in 50 microliters of cold DI water.

Electroporation

1. Add 500 ng of Duo2 (0.86 microliters of Duo2 extracted)
2. Transfer bacteria inro chilled electroporator cuvette. Tap cuvette on bench.
3. Let electroporator: 2500 V, 25 microF, 200 Ohm
4. Put cuvette into electroporator, start and remove after 5 seconds.
5. Immediately, add 1 ml of LB broth, pipette avoiding bubbles.
6. Transfer P.putida + plasmid solution to fresh 1.5 ml tube.
7. Incubate at 30°C for 1 hour.
8. Prepare 2 LB agar + kanamycin (25 ml agar + 25 microliters kanamycin)
9. Prepare LB agar + gentamicin plate.
10. Pour 100 microliters of P.putida + Duo2 onto plates (spread plates)
11. Incubate overnight at 30°C.
12. Prepare 2 LB broth tubes. Inoculate P.aeruginosa and P.putida into the broth.

![Notebook Figure 56](https://static.igem.org/mediawiki/2021/6/6d/T--NU_Kazakhstan--Notebook_Figure_32.jpg)

Figure 56. P.putida with Duo2 plasmid in LB agar + kanamycin

![Notebook Figure 57](https://static.igem.org/mediawiki/2021/e/e8/T--NU_Kazakhstan--Notebook_Figure_33.jpg)

Figure 57. P.putida with Duo2 plasmid in LB agar + kanamycin (2nd plate)

![Notebook Figure 58](https://static.igem.org/mediawiki/2021/8/88/T--NU_Kazakhstan--Notebook_Figure_34.jpg)

Figure 58. P.putida with Duo2 plasmid in LB agar + gentamicin (negative control)

## Week 5

### Day 1 - 27.09.2021

1. Turn on UV on 30 minutes.
2. Prepare LB agar plate 20mL (125μl gentamicin)
3. Inoculate P.putida duo2 (25.09.21) 100μl spread plate.

Table 99. PCR conditions mix1 and mix2 (Total volume 50)

| Steps | t (°C) | time |  |
| Initial Denaturation | 95 | 5 min |  |
| Denaturation | 94 | 15 sec | 10 cycles |
| Annealing | 70, 72 | 30 sec |
| Elongation | 72 | 2 min |
| Denaturation | 94 | 15 sec | 20 cycles |
| Annealing | 70, 72 | 30 sec |
| Elongation | 72 | 2 min + 5sec/cycle |
| Final elongation | 72 | 7 min |  |
| Hold | 4 | forever |  |

1. Measure OD P.aeruginosa = 0.224
2. Incubate P.aeruginosa LB broth for overnight.

### Day 2 - 28.09.2021

1. Plates (25.09.21) observed and palced into the +4**°**C friedge.
2. Measure OD P.aeruginosa = 0.802
3. Turned on UV light for 30 minutes.
4. Poured 5mL of LB broth with P.aeruginosa into 15mL tube.
5. Centrifuge at 4700 rpm for 10 minutes at 4**°**C.
6. Discard the supernatant
7. Resuspend with 1mL of ice-cold di water, and transfer into 2mL centrifuge tube.
8. Centrifuge at 7000 rpm for 5 minutes at 4**°**C.
9. Discard supernatant.
10. Resuspend in 1mL ice-cold di water,
11. Repeated the steps 8-10 two times.
12. Centrifuge at 7000 rpm for 5 minutes at 4**°**C, discard supernatant and add 50μl di-water.

## October

## Week 1

### Day 1 - 01.10.2021

1. Run PCR with Q5 for rhlAB

Table 100. PCR with Q5 for rhlAB

| 5xQ5 buffer | 5μl |
| 10mM DNTP | 0.5μl |
| 10μM Forward primer | 1.25μl |
| 10μM reverse primer | 1.25μl |
| genomic DNA | 4μl |
| Q5 polymerase | 0.25μl |
| nuclease free water | 7.75μl |
| Q5 enhancer | 5μl |

Table 101. PCR conditions

| Steps | t (°C) | time |  |
| Initial Denaturation | 98 | 2 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Annealing | 72 | 30 sec |
| Elongation | 72 | 2 min |
| Final elongation | 72 | 2 min |  |
| Hold | 4 |  |  |

### Day 2 - 02.10.2021

1. Run PCR following coditions from 01.10.21
2. Inoculate P.aeruginosa (25.09.21) into two 25mL LB broth tubes and 5mL LB broth tube.
3. Inoculated P.aeruginosa from tube 25.09.21 into 2 plates (spread plate).
4. Plates were incubated for overnight at 37**°**C.

### Day 3 - 03.10.2021

1. LB agar 200mL (7.4g LB agar into 200mL water), LB broth 200mL (4g LB broth into 200mL water) and 1mM MgSO4 solution were prepared and autoclaved.
2. Gel electrophoresis:

a. Agarose gel was prepared by mixing 0.3g of agarose in 30mL 1xTAE.

b. Prepare samples for gel electrophoresis by adding 10μl of sample, 5μl 1xTAE and 5μl loading dye.

Table 102. Gel Electrophoresis track order

| 1st well | DNA ladder |
| 2nd well | DNA ladder (From Dos' lab) |
| 3rd well | Q5 72°C |
| 4th well | Q5 72°C (2.10.21) |
| 5th well | DNA ladder (tube w) |

Run gel electrophoresis at 100V for 20 minutes.

1. P.aeruginosa electroporation.

1. 4mL of grown overnight P.aeruginosa culture were centrifuged at 4700 rpm for 5 minutes at 23**°**C. (2 tubes)
2. discard supernatant and resuspend the pellet with 4mL di water and centrifuge at same condition once more.
3. The cell pellet was resuspended with 1mL of 1mM MgSO4 (room temperature).
4. Centrifuge again at the same speed and discard supernatant.
5. Resuspend the pellet with 1mL of 1mM MgSO4 (room temperature).
6. Centrifuge again at the same speed and discard supernatant.
7. Resuspend the pellet with 50μl of 1mM MgSO4 (room temperature).
8. 5-10μg of plasmid DNA was mixed with 50μl of cell mixture.
9. Measured concentration of extracted Duo2 plasmid by nanodrop:

Duo2 = 693ng/μl, so we take 5μg, means 7.2μl of Duo2 plasmids.

1. Electroporation conditions: 2200V, 25μF, 600Ω, 5ms.
2. Immediately add 1mL of LB broth and resuspend.
3. Incubate at room temperature for 5 minutes.
4. Cells were transfered into falcon tube and shaked at 37**°**C for three hours.
5. Transformed cells are pelleted and 700μl of supernatant discarded.
6. Cells resuspended in remaining mediim and plateed to plates with kanamycin (25mL LB agar + 25μl kanamycin).
7. Plates incubated at 37**°**C for 2 days.

![Notebook Figure 59](https://static.igem.org/mediawiki/2021/1/10/T--NU_Kazakhstan--Notebook_Figure_37.jpg)

Figure 59. Gel electrophoresis results

![Notebook Figure 60](https://static.igem.org/mediawiki/2021/a/af/T--NU_Kazakhstan--Notebook_Figure_39.jpg)

Figure 60. P.aeruginosa with Duo 2 plasmid in LB agar + kanamycin

## Week 2

### Day 1 - 04.10.2021

1. Turn on UV on 30 minutes.
2. Prepare PCR samples:

Table 103. PCR samples

| 5xQ5 buffer | 5μl |
| 10mM DNTP | 0.5μl |
| 10μM Forward primer | 1.5μl |
| 10μM reverse primer | 1.5μl |
| genomic DNA | 7μl |
| Q5 polymerase | 0.25μl |
| nuclease free water | 4.25μl |
| Q5 enhancer | 5μl |

1. Run PCR at following conditions:

Table 104. PCR conditions

| Steps | t (°C) | time |  |
| Initial Denaturation | 98 | 5 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Annealing | 70.56.58 | 30 sec |
| Elongation | 72 | 2 min |
| Final elongation | 72 | 5 min |  |
| Hold | 4 |  |  |

1. Inoculated E.coli+Duo2 into 30mL LB broth + 30μl kanamycin.
2. Inoculate single colony of E.coli+Duo2 into 2mL LB broth + 2μl kanamycin
3. Inoculate P.aeruginosa into LB broth and incubate at 37**°**C.
4. Clean bench, wash all dishes and prepare for autoclave.

### Day 2 - 05.10.2021

* Electroporation of P.aeruginosa

1. OD of overnight P.aeruginosa is 1.127 (540nm)
2. Prepared electrocompetent P.aeruginosa following protocol for E.coli:
3. Poured 5mL of LB broth with P.aeruginosa into 15mL tube.
4. Centrifuge at 4700 rpm for 10 minutes at 4**°**C.
5. Discard the supernatant
6. Resuspend with 1mL of ice-cold di water, and transfer into 2mL centrifuge tube.
7. Centrifuge at 7000 rpm for 5 minutes at 4**°**C.
8. Discard supernatant.
9. Resuspend in 1mL ice-cold di water,
10. Repeated the steps 8-10 two times.
11. Centrifuge at 7000 rpm for 5 minutes at 4**°**C, discard supernatant and add 50μl di-water.
12. Added 2μl of Duo2 (693ng/μl) and 50μl electrocompetent P.aeruginosa into electroporation cuvette.
13. Proceed electroporation at 1600V, 25μF, 100Ω for 5 sec. Add 1mL of LB broth immediatly.
14. Transfer solution into 2mL LB and incubate it for 2 hours at 37**°**C.
15. Inoculate on LB agar + kanamycin
16. Preparation of electrocompetent P.aeruginosa (25.09.21) by washing with CaCl2
17. Transfer 5 ml of LB + P.putida into tube
18. Centrifuge at 4700 rpm, 4**°**C, 10 min.
19. Discard the supernatant.
20. Add 5 ml of CaCl2 0.1M, resuspend.
21. Put on ice for 30 min.
22. Centrifuge at 4700 rpm, 4**°**C, 10 min.
23. Discard the supernatant.
24. Add 5 ml of CaCl2 0.1M, 20% glycerl solution, resuspend, adn leave for approximately 10 minutes.
25. Centrifuge at 4700 rpm, 4**°**C, 7-10 min.
26. Remove supernatant.
27. Wash with cold DI water, add 5 ml of DI water, resuspend, centrifuge at 4700 rpm, 4**°**C, 10 min.
28. Remove supernatant.
29. Resuspend pellet in 50 microliters of cold DI water.

PCR:

1. 10mM solutions each primer was prepared.
2. 3 tubes PCR were prepared according to protocol Q5 with 5μl of genomic DNA, 1μl of DNTP mix and with primers 1+4/5+6/9+10.

Table 105. PCR conditions

| Steps | t (°C) | time rhlAB | time nadE |  |
| Initial Denaturation | 98 | 3 min | 3 min |  |
| Denaturation | 98 | 10 sec | 10 sec | 35 cycles |
| Annealing | 72 | 30 sec | 30 sec |
| Elongation | 72 | 93 sec | 40 sec |
| Final elongation | 72 | 5 min | 2 min |  |
| Hold | 4 |  |  |  |

OD of E.coli with Duo2: OD=0.560

* Extraction of Duo2 plasmid from E.coli:

1. Take 1mL of culture tubes. (2 tubes)
2. Centrifuge at 13500 rpm at 4**°**C for 30 seconds.
3. Discard supernatant, leave pellet to dry.
4. Resuspend pellet in 100μl of ice-cold ALS I, mix by pipetting.
5. Add 200μl of ALS II (room temperature). Mix by inverting 5 times. Store on ice for 5 minutes.
6. Add 150μl of ALS III (ice-cold). Mix gently by inverting tube 5 times. Store tube on ice for 5 minutes.
7. Centrifuge at 13500 rpm at 4**°**C for 5 minutes.
8. Trnsfer 450μl of supernatant into new fresh tubes.
9. Precipitate DNA from supernatant by adding 2 volumes (900μl) of 70% ethanol. Gently mix by inverting . Incubate at room temperature for 2 minutes.
10. Coolect precipitated DNA by centrifuation at 13500 rpm, 4**°**C for 5 minutes.
11. Remove supernatant, dry tubes.
12. Add 1ml of 70% ethanol, mix by inverting.
13. Centrifuge at 13500 rpm at 4**°**C for 2 minutes.
14. We got white pellet (plasmids). Supernatant was removed. Tubes were left to evaporate ethanol (10-15 minutes).
15. Dissolve plasmids in 50μl of DI water.
16. Measure concentration of Duo2 plasmids by nanodrop:
17. tube1 - 637ng/μl
18. tube2 - 606ng/μl

![Notebook Figure 61](https://static.igem.org/mediawiki/2021/7/71/T--NU_Kazakhstan--Notebook_Figure_41.jpg)

Figure 61. Duo 2 plasmid concentrations : tube 1 in G1 and tube 2 on E1

### Day 3 - 06.10.2021

1. Prepare samples for gel electrophoresis: 10μl sample + 5μl loading dye + 5μl 1xTAE.
2. Gel loading:

Table 106. Gel Electrophoresis track order

| 2nd well | DNA ladder |
| 3rd well | rhlAB 56°C |
| 4th well | rhlAB 58°C |
| 5th well | rhlAB 70°C |
| 6th well | rhl 1+4 |
| 7th well | nadE 9+10 |
| 8th well | nadE 5+6 |
| 9th well | rhlAB 56°C |
| 10th well | rhlAB 58°C |
| 11th well | rhlAB 70°C |
| 12th well | rhl 1+4 |
| 13th well | nadE 9+10 |
| 14th well | nadE 5+6 |

Run gel electrophoresis at 90V for 1 hour.

![Notebook Figure 62](https://static.igem.org/mediawiki/2021/d/da/T--NU_Kazakhstan--Notebook_Figure_42.jpg)

Figure 62. Gel electrophoresis results

2. Stained 1 part of the gel (5μl stain+100mL 1xTAE).

Gel elution following the protocol of Purelink Quick Gel Extraction Kit:

1. Cut band (take minimal amount of the gel).
2. Melt them in Thermomixer compact at 65°C
3. Weight gel: Stained gel ~ 0.18g ; Without stain = 0.27g
4. Add Gel Solubilization Buffer (L3) to the gel in a ratio 3:1 (We added 0.54ml and 0.81ml respectively)
5. Place the tube in a thermomixer for 10 min until gel will dissolve. Each 3 min gently mix by inverting (50°C)
6. Add 1 volume of isopropanol into the gel.
7. Incubate the tube for additional 5 min
8. Purify DNA using a centrifuge
9. Pipet the dissolved gel into the column in wash tube. One column per 400 μg capacity is 850μl, so that we took approximately 700 μl
10. Centrifuge the flow through and place column into the wash tube
11. Add 500μl Wash buffer (W1)
12. Centrifuge at 12000 rpm for 1 min, discard the flow through
13. Centrifuge one more time at 14000 rpm for 2 mins, discard the flow through
14. Place column in a new wash tube (recovery tube). Add 50 μl of Elution Buffer (E5) into the centre of the column. Incubate 1 min at RT
15. Centrifuge the tube at 12 000 rpm for 1 min.

Store at +4°C solution in reagent tube.

### Day 4 - 07.10.2021

Table 107. Gel Electrophoresis track order

Table 107. Gel Electrophoresis track order

| 1st well | DNA ladder |
| 2nd well | rhlAB 58°C |
| 3rd well | Duo2 |
| 4th well | nadE eluted |
| 5th well | nadE + EtBr |
| 6th well | rhlAB 57°C |
| 7th well | double digested Duo2 |
| 8th well | rhlAB 58°C (1-4) |
| 9th well | rhlAB 59°C |
| 10th well | rhlAB 60°C |
| 11th well | - |
| 12th well | Double digested Duo2 |
| 13th well | rhlAB 58°C (1-4) |
| 14th well | rhlAB 58°C |
| 15th well | rhlAB 57°C |
| 16th well | rhlAB 59°C |
| 17th well | rhlAB 60°C |
| 18 well | - |

One half of the gel was left in a stain for overnight.

1. Measure concentration of nadE:

nadE with EtBr (eluted) = 21.41 ng/μl

nadE without (eluted) = 17.37ng/μl

**Restriction Digestion (Sac I, Sal I)**

keep reagents on ice; add restriction enzymes last

**For plasmid Duo2**

DNA 1μg = 1.57μl / C(Duo2)=637ng/μl

10x Cutsmart Buffer - 5μl (1x)

Sac I - 1μl

Sal I - 1μl

nuclease free water - 41.43μl (total 50μl)

37°C incubation for 15 minutes

65°C heat inactivation for 20 minutes

**for nadE**

DNA 200ng = 11.8μl / C(nadE)=17.37ng/μl

10x Cutsmart Buffer - 1.5μl (1x)

Sac I - 0.2μl

Sal I - 0.2μl

nuclease free water - 1.3μl (total 15μl)

37°C incubation for 15 minutes

65°C heat inactivation for 20 minutes

Inoculation of only colony with P.aeruginosa+pRGPDuo2 (streak plate)

negative control - P.aeruginosa in LB agar + kanamycin

![Notebook Figure 63](https://static.igem.org/mediawiki/2021/3/31/T--NU_Kazakhstan--Notebook_Figure_43.jpg)

Figure 63. Gel electrophoresis results

![Notebook Figure 64](https://static.igem.org/mediawiki/2021/c/c5/T--NU_Kazakhstan--Notebook_Figure_45.jpg)

Figure 64. P. aeruginosa with pRGPDuo2

### Day 5 - 08.10.2021

1. Prepared 2 tubes for digestion Duo2:

1st tube: Duo2 plasmid - 2μl (637ng/μl)

CutSmart - 5μl

Sal I - 1μl

Sac I - 1 μl

nuclease free water - 41μl

2nd tube: Duo2 plasmid diluted - 2μl (70ng/μl)

CutSmart - 5μl

Sal I - 1μl

Sac I - 1 μl

nuclease free water - 41μl

1. Prepared 1 tube for digestion of nadE gene:

nadE - 19.8μl

CutSmart - 1.5μl

Sal I - 0.2μl

Sac I - 0.2μl

nuclease free water - 1.3μl

1. Tubes incubated fro 1 hour at 37°C.
2. heated at 65°C for 20 minutes in order to inactivate.
3. Prepared agarose gel solution: 0.3 of agarose were added into 30mL 1xTAE. Microwaved in order to dissolve and 3μl of GelRed stain have been added.
4. DNA (sample 2) was observed by nanodrop: resulted in 25-33ng/μl. Average concentration = 29ng/μl.

| 1st well | DNA ladder | (5μl ladder, 5μl loading dye, 10μl 1xTAE) |
| 2nd well | Duo 2 (5.10.21) | (5μl sample, 5μl loading dye, 10μl 1xTAE) |
| 3rd well | Double digested Duo2 | (5μl sample, 5μl loading dye, 10μl 1xTAE) |
| 4th well | Diluted Duo2 | (7μl sample, 5μl loading dye, 8μl 1xTAE) |
| 5th well | DOuble digested diluted Duo2 | (7μl sample, 5μl loading dye, 8μl 1xTAE) |

Gel electrophoresis at 90V for 30 minutes.

Prepared 6 PCR samples:

1. old rhlBA (25μl)
2. new rhlBA (1-4) (25μl)
3. nadE (5-6) (25μl)
4. rhlA (3-4) (25μl)
5. 5 rhlB (1-2) (25μl)
6. nadE (9-10) (50μl)

Table 109. The volumes for 25μl samples

| 5xQ5 buffer | 5μl |
| 10mM DNTP | 1μl |
| 10μM Forward primer | 1.5μl |
| 10μM reverse primer | 1.5μl |
| genomic DNA | 3.5μl |
| Q5 polymerase | 0.25μl |
| nuclease free water | 7.25μl |
| Q5 enhancer | 5μl |

Table 110. Volumes for 50μl sample

| 5xQ5 buffer | 10μl |
| 10mM DNTP | 2μl |
| 10μM Forward primer | 3μl |
| 10μM reverse primer | 3μl |
| genomic DNA | 7μl |
| Q5 polymerase | 0.5μl |
| nuclease free water | 19.5μl |
| Q5 enhancer | 5μl |

Table 111. PCR conditions for nadE (5-6)

| Steps | t (°C) | time |  |
| Initial Denaturation | 98 | 3 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Annealing | 72 | 30 sec |
| Elongation | 72 | 40 sec |
| Final elongation | 72 | 2 min |  |
| Hold | 4 |  |  |

Table 112. PCR conditions for old rhlBA and rhlB (1-2)

| Steps | t (°C) | time |  |
| Initial Denaturation | 98 | 3 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Annealing | 72 | 30 sec |
| Elongation | 72 | 93 sec |
| Final elongation | 72 | 5 min |  |
| Hold | 4 |  |  |

rhlBA (1-4) should be placed at 56°C annealing temperature.

rhlA (3-4) should be placed at 58°C annealing temperature.

nadE (9-10) (50μl)should be placed at 72°C annealing temperature.

Table 113. rhlBA (1-4) PCR conditions

| Steps | t (°C) | time |  |
| Initial Denaturation | 98 | 3 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Annealing | 72 | 30 sec |
| Elongation | 72 | 2 min |
| Final elongation | 72 | 5 min |  |
| Hold | 4 |  |  |

Performed elution of double digested Duo2:

1. Cut band (take minimal amount of the gel).
2. Melt them in Thermomixer compact at 50°C
3. Weight gel~ 0.17g
4. Add Gel Solubilization Buffer (L3) to the gel in a ratio 3:1
5. Place the tube in a thermomixer for 10 min until gel will dissolve. Each 3 min gently mix by inverting (50°C)
6. Add 1 volume of isopropanol into the gel.
7. Incubate the tube for additional 5 min
8. Purify DNA using a centrifuge
9. Pipet the dissolved gel into the column in wash tube. One column per 400 μg capacity is 850μl, so that we took approximately 700 μl
10. Centrifuge the flow through and place column into the wash tube
11. Add 500μl Wash buffer (W1)
12. Centrifuge at 12000 rpm for 1 min, discard the flow through
13. Centrifuge one more time at 14000 rpm for 2 mins, discard the flow through
14. Place column in a new wash tube (recovery tube). Add 50 μl of Elution Buffer (E5) into the centre of the column. Incubate 1 min at RT
15. Centrifuge the tube at 12 000 rpm for 1 min.

Store at -20°C for long term usage

**Ligation:**

plasmid Duo2 - 2μl - 36.12ng

nadE - 26.85ng - 2.3μl of diluted nadE

T4 DNA ligase buffer - 2μl

T4 DNA ligase - 1μl

Nuclease free water - 13.8μl (Total 20μl)

1. Resuspend gently up and down, Incubated at room temperature for 10 minutes.
2. Incubate at 4°C overnight.

![Notebook Figure 65](https://static.igem.org/mediawiki/2021/3/32/T--NU_Kazakhstan--Notebook_Figure_47.jpg)

Figure 65. DNA extracted from P.aeruginosa

![Notebook Figure 66](https://static.igem.org/mediawiki/2021/e/ed/T--NU_Kazakhstan--Notebook_Figure_48.jpg)

Figure 66. Gel electrophoresis

![Notebook Figure 67](https://static.igem.org/mediawiki/2021/4/4f/T--NU_Kazakhstan--Notebook_Figure_50.jpg)

Figure 67. Nanodrop

### Day 6 - 09.10.2021

1. Tube with ligation l mix was incubated at 65°C for inactivation.
2. 5μl of the mixture was added to 50μl competent P.aeruginosa and electroporated at 1600μl for 5 seconds.
3. immideately 1mL of LB broth solution was added after electroporation.
4. 3ml of transformed P.aeruginosa solution left in incubator for 2 hours at 37°C.
5. 100μl of transformed P.aeruginosa were plated on 3 plates of LB agar + kanamycin.
6. negative control: LB agar without kanamycin was used to grow P.aeruginosa from tube (2.10.21)
7. The plates were left for overnight incubation at 37°C.

Ligation reaction quantity calculation:

Quanatity of insert = (size of insert/size of plasmid)\*quantity of DNA plasmid\*molar ratios

Quantity of insert = (842/7928)\*2(18.06)\*7=26.85

nadE concentration 2353, dilution 100x, so that concentration become 23.53

.We take 1.2μl of nadE for ligation mixture.

Gel electrophoresis:

1. Prepare samples for gel electrophoresis:

We took 15μl of sample or DNA ladder + 5μl loading dye for all samples, but for extracted Duo2 and ligated nadE+Duo2 we take 5μl sample + 5μl loading dye + 10μl 1xTAE.

Table 114 Gel Electrophoresis track order

| 2nd well | DNA ladder |  |
| 3rd well | rhlBA old | no results |
| 4th well | rhlBA (1-4) | not preper band |
| 5th well | rhlA (3-4) | got at 800-900bp |
| 6th well | rhlB (1-2) | got at 1400 bp |
| 7th well | nadE (5-6) | got at 800-300bp |
| 8th well | nadE (9-10) | no results |
| 9th well | ligated nadE Duo2 | no results |
| 10th well | Duo2 extracted | got at 7-8 kbp |

Run Gel electrophoresis at 80V for 1 hour.

![Notebook Figure 68](https://static.igem.org/mediawiki/2021/5/5e/T--NU_Kazakhstan--Notebook_Figure_51.jpg)

Figure 68. P.aeruginosa with pRGP Duo2 + nadE gene in LB agar+kanamycin 1st

![Notebook Figure 69](https://static.igem.org/mediawiki/2021/e/ed/T--NU_Kazakhstan--Notebook_Figure_53.jpg)

Figure 69. P.aeruginosa with pRGP Duo2 + nadE gene in LB agar+kanamycin 2nd

![Notebook Figure 70](https://static.igem.org/mediawiki/2021/6/61/T--NU_Kazakhstan--Notebook_Figure_55.jpg)

Figure 70. P.aeruginosa with pRGP Duo2 + nadE gene in LB agar+kanamycin 3d

### Day 7 - 10.10.2021

Fresh colony culturing for plasmid extraction:

1. One colony of P.aeruginosa was taken from plates LB agar + kanamycin (9.10.21) Duo2 + nadE (from 3 plates) and from negative control plate LB agar + kanamycin (9.10.21) and were inoculated in 1ml LB broth + 1μl kanamycin solution (overall 4 tubes).
2. 4 tubes were left for overnight incubation at 37°C.
3. Gel elution following protocol:

Cut bands (take minimal amount of the gel).

1. Melt them in Thermomixer compact at 50°C
2. Weight gel:

rhlA = 0.14g

rhlB = 0.12g

Duo2 = 0.12g

nadE = 0.16g

1. Add Gel Solubilization Buffer (L3) to the gel in a ratio 3:1
2. Place the tube in a thermomixer for 10 min until gel will dissolve. Each 3 min gently mix by inverting (50°C)
3. Add 1 volume of isopropanol into the gel.
4. Incubate the tube for additional 5 min
5. Purify DNA using a centrifuge
6. Pipet the dissolved gel into the column in wash tube. One column per 400 μg capacity is 850μl, so that we took approximately 700 μl
7. Centrifuge the flow through and place column into the wash tube
8. Add 500μl Wash buffer (W1)
9. Centrifuge at 12000 rpm for 1 min, discard the flow through
10. Centrifuge one more time at 14000 rpm for 2 mins, discard the flow through
11. Place column in a new wash tube (recovery tube). Add 50 μl of Elution Buffer (E5) into the centre of the column. Incubate 1 min at RT
12. Centrifuge the tube at 12 000 rpm for 1 min.

Use nanodrop to calculate concentration of eluted DNA samples:

rhlA = 20.70ng/μl

rhlB 41.68ng/μl

nadE 36.93ng/μl

Duo2 = 16.26ng/μl

* Inoculation of P.putida into LB broth and LB agar
* Restriction Digestion: (Total 15μl)

for nadE

DNA 150ng = 4.06μl

10x CutSmart Buffer = 1.5μl

Nhe I = 0.6μl

nuclease free water = 9.14μl

for rhlA

DNA 150ng = 7.25μl

10x CutSmart Buffer = 1.5μl

Sal I = 0.3μl

Sac I = 0.3μl

nuclease free water = 5.65μl

for rhlB

DNA 150ng =3.60μl

10x CutSmart Buffer = 1.5μl

Sal I = 0.3μl

Sac I = 0.3μl

nuclease free water =9.3μl

for Duo2 (Total 50μl)

DNA 140ng =2μl (70ng/μl)

10x CutSmart Buffer = 5μl

Sal I = 1μl

Sac I = 1μl

nuclease free water = 41μl

Incubation for 2.5 hours at 37°C.

Heat inactivation at 65°C for 20 minutes.

**Gel electrophoresis:**

Gel preparation by adding 0.3g og agarose into 30mL 1xTAE. Dissolve solution by microwaving and then add 3μl of Gel Red.

Samples preparation:

Table 115 Gel Electrophoresis track order

| 1st well | DNA ladder | 15μl DNA ladder + 5μl loading dye |
| 2nd well | Duo2 (70ng/μl) | 15μl sample + 5μl loading dye |
| 3rd well | double digested Duo2 | 15μl sample + 5μl loading dye |
| 4th well | rhlA digested | 10μl sample + 5μl loading dye + 5μl 1xTAE |
| 5th well | rhlB digested | 10μl sample + 5μl loading dye + 5μl 1xTAE |
| 6th well | nadE digested | 10μl sample + 5μl loading dye + 5μl 1xTAE |

Run gel electrophoresis at 90V for 50 minutes.

**Gel elution:**

Cut bands (take minimal amount of the gel).

1. Melt them in Thermomixer compact at 50°C
2. Weight gel:

rhlA = 0.08g

rhlB = 0.09g

nadE = 0.11g

1. Add Gel Solubilization Buffer (L3) to the gel in a ratio 3:1
2. Place the tube in a thermomixer for 10 min until gel will dissolve. Each 3 min gently mix by inverting (50°C)
3. Add 1 volume of isopropanol into the gel.
4. Incubate the tube for additional 5 min
5. Purify DNA using a centrifuge
6. Pipet the dissolved gel into the column in wash tube. One column per 400 μg capacity is 850μl, so that we took approximately 700 μl
7. Centrifuge the flow through and place column into the wash tube
8. Add 500μl Wash buffer (W1)
9. Centrifuge at 12000 rpm for 1 min, discard the flow through
10. Centrifuge one more time at 14000 rpm for 2 mins, discard the flow through
11. Place column in a new wash tube (recovery tube). Add 50 μl of Elution Buffer (E5) into the centre of the column. Incubate 1 min at RT
12. Centrifuge the tube at 12 000 rpm for 1 min.

**Ligation:**

T4 DNA ligase buffer (10x) - 2μl

Vector dna - 2.8μl

Insert DNA:

rhlA - 0.5μl

rhlB - 1.8μl

T4 DNA ligase - 1μl

nuclease free water:

for rhlA - 13.7μl

for rhlB - 12.4μl

incubate overnight at 16°C.

**50xTAE preparation:**

1. Prepare 100ml EDTA pH 8.0 solution: In 80ml dd water add 14.61g of EDTA, place in magnetic stirrer, add NaOH pellets one by one until all EDTA will dissolve.
2. In total we got 125ml of 0.4M EDTA, just put 12.5ml 0.4M EDTA instead of 10ml 0.5M EDTA.
3. Make 50x TAE by adding 24.2g Tris base into 60ml dd water, mix Tris with stir bar until it will dissolve.
4. Add to the solution 5.71ml Acetic acid 99.7% and 12.5ml 0.4M EDTA. Bring final volume to 100ml with dd water.

measured concentration of genes (DNA) by nanodrop:

rhlA - 91.50ng/μl

rhlB - 31.98 ng/μl

nadE - 16.32ng/μl

![Notebook Figure 71](https://static.igem.org/mediawiki/2021/7/77/T--NU_Kazakhstan--Notebook_Figure_56.jpg)

Figure 71. Nanodrop 1

![Notebook Figure 72](https://static.igem.org/mediawiki/2021/3/39/T--NU_Kazakhstan--Notebook_Figure_58.jpg)

Figure 72. Nanodrop 2

## Week 3

### Day 1 - 11.10.2021

1. Heat inactivation of rhlA+Duo2 and rhlB+Duo2 for 20 minutes at 65°C.
2. OD of P.putida (10.10.21) = 0.268 at 578nm
3. Extraction of plasmids from P.aeruginosa (9.10.21) by Aigerim's protocol.
4. 1 mL of culture tubes (4 tubes) were centrifuged at 7000 rpm for 1 minute at 4**°**C.
5. The supernatant was discarded and pellet was left to dry.
6. Prepared 10 mL of ALS II by mixing 1 mL of 3N NaOH, 1 mL of 10% SDS and ddH2O.
7. Resuspended the pellet in 100 uL of ice cold ALS I (autoclaved) and mixed by pipetting, makin sure the cells dispersed fully.
8. Added 200 uL of ALS II and mixed by inverting the tube 5 times, making sure the content of the tube makes contact with ALS II. Stored the tube on ice for 5 minutes.
9. Added 150 uL of ALS III (autoclaved) and mixed gently by inverting the tube 5 times. Stored the tube on ice for 5 minutes.
10. Centrifuged at 13500 rpm for 5 minutes at 4**°**C.
11. Transferred the supernatant (450 uL) to the new labeled tube.
12. Precipitated DNA from supernatant by adding 2 volumes (900 uL) of 70% ethanol at room temperature. Gently mixed by inverting and incubated 2 minutes at room temperature.
13. Centrifuged at 13500 rpm for 5 minutes at 4**°**C. Discarded the supernatant and dried the pellet.
14. Added 1 mL of 70% ethanol and mixed by inverting.
15. Centrifuged at 13500 rpm for 2 minutes at 4**°**C. Removed the supernatant and left the tube to dry for 10-15 minutes until ethanol will evaporate.
16. Dissolved pellet in 50 μl H2O and checked OD 1 uL of each solution by nanodrop.

negative control - 71.82ng/μl

1st sample - 464.2ng/μl

2nd sample - 371.6ng/μl

3rd sample - 333.7ng/μl

Gel electrophoresis:

Prepare gel by adding 1g of agarose into 100ml 1xTAE, dissolve agarose by heating and then add 10μl of GelRed.

Prepare samples for gel electrophoresis: (15μl sample + 5μl loading dye)

Table 116. Gel Electrophoresis track order

| 2nd well | DNA ladder |
| 4th well | negative control of P.aeruginosa |
| 6th well | 1st sample |
| 8th well | 2nd sample |
| 10th well | 3rd sample |
| 12th well | ligation product Duo2 + rhlA |
| 14th well | ligation product Duo2 + rhlB |
| 16th well | nadE digestion product (5,6) |
| 18th well | nadR digestion product sample 2 (5,6) |

Created competent P.putida with CaCl2 (2tubes):

1. Transfer 5 ml of LB broth + P.putida into tube
2. Centrifuge at 4700 rpm, 4**°**C, 10 min.
3. Discard the supernatant.
4. Add 5 ml of CaCl2 0.1M, resuspend.
5. Put on ice for 30 min.
6. Centrifuge at 4700 rpm, 4**°**C, 10 min.
7. Discard the supernatant.
8. Add 5 ml of CaCl2 0.1M, 20% glycerl solution, resuspend, adn leave for approximately 10 minutes.
9. Centrifuge at 4700 rpm, 4**°**C, 7-10 min.
10. Remove supernatant.
11. Wash with cold DI water, add 5 ml of DI water, resuspend, centrifuge at 4700 rpm, 4**°**C, 10 min.
12. Remove supernatant.
13. Resuspend pellet in 50 microliters of cold DI water.

Inoculated P.aeruginosa into new LB broth.

Gel elution of P.aeruginosa plasmid samples:

Cut bands (Take minimal amount of gel)

1. Melt them in Thermomixer compact at 50°C
2. Weight gel:

1st sample = 0.17g

2nd sample = 0.16g

3rd sample = 0.13g

Unknown band at 800-900bp = 0.1g

1. Add Gel Solubilization Buffer (L3) to the gel in a ratio 3:1
2. Place the tube in a thermomixer for 10 min until gel will dissolve. Each 3 min gently mix by inverting (50°C)
3. Add 1 volume of isopropanol into the gel.
4. Incubate the tube for additional 5 min
5. Purify DNA using a centrifuge
6. Pipet the dissolved gel into the column in wash tube. One column per 400 μg capacity is 850μl, so that we took approximately 700 μl
7. Centrifuge the flow through and place column into the wash tube
8. Add 500μl Wash buffer (W1)
9. Centrifuge at 12000 rpm for 1 min, discard the flow through
10. Centrifuge one more time at 14000 rpm for 2 mins, discard the flow through
11. Place column in a new wash tube (recovery tube). Add 50 μl of Elution Buffer (E5) into the centre of the column. Incubate 1 min at RT
12. Centrifuge the tube at 12 000 rpm for 1 min.

Prepare 3 PCR samples. (nadE 5-6, rhlB 1-2, rhlA 3-4)

Q5 buffer - 5μl

10mM DNTP mix - 1μl

10mM forward primer - 1.5μl

10mM reverse primer - 1.5μl

Template DNA - 3.5μl

Q5 polymerase - 0.25μl

Q5 enhancer - 5μl

nuclease free water - 7.25μl (Total 25μl)

Table 117. PCR conditions for nadE (5,6) and rhlA (3,4)

| Steps | t (°C) | time |  |
| Initial Denaturation | 98 | 3 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Annealing | 72 | 30 sec |
| Elongation | 72 | 40 sec |
| Final elongation | 72 | 2 min |  |
| Hold | 4 |  |  |

Table 118. PCR conditions for rhlB (1,2)

| Steps | t (°C) | time |  |
| Initial Denaturation | 98 | 3 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Annealing | 72 | 30 sec |
| Elongation | 72 | 93 sec |
| Final elongation | 72 | 5 min |  |
| Hold | 4 |  |  |

![Notebook Figure 73](https://static.igem.org/mediawiki/2021/1/16/T--NU_Kazakhstan--Notebook_Figure_60.jpg)

Figure 73. Nanodrop

![Notebook Figure 74](https://static.igem.org/mediawiki/2021/2/27/T--NU_Kazakhstan--Notebook_Figure_61.jpg)

Figure 74. Gel electrophoresis

### Day 2 - 12.10.2021

1. Measured OD of P.putida; OD=1.184
2. Prepared electrocompetent P.putida (2tubes).
3. Poured 5mL of LB broth with P.putida into 15mL tube.
4. Centrifuge at 4700 rpm for 10 minutes at 4**°**C.
5. Discard the supernatant
6. Resuspend with 1mL of ice-cold di water, and transfer into 2mL centrifuge tube.
7. Centrifuge at 7000 rpm for 5 minutes at 4**°**C.
8. Discard supernatant.
9. Resuspend in 1mL ice-cold di water,
10. Repeated the steps 7-9 two times.
11. Centrifuge at 7000 rpm for 5 minutes at 4**°**C last time, discard supernatant and add 50μl di-water.

Digestion and ligation of pRGPDuo2 plasmid. Digestion with Nhe I, ligation of rhlA;rhlB.

**Gel electrophoresis**

1. Prepared agarose gel (1g agarose + 100ml 1xTAE + 10μl GelRed).
2. Prepared samples for gel (15μl sample + 5μl loadig dye).

Table 119 Gel Electrophoresis track order

| 2nd well | DNA ladder |
| 4th well | rhlA |
| 6th well | rhlB |
| 8th well | nadE |
| 10th well | Duo2 diluted |
| 12th well | Duo2 digested |

Run gel electrophoresis at 100V for 50 minutes.

Prepared 6 plates with kanamycin inoculate E.coli to LB agar and broth + 5 plates with LB agar.

![Notebook Figure 75](https://static.igem.org/mediawiki/2021/5/54/T--NU_Kazakhstan--Notebook_Figure_63.jpg)

Figure 75. Gel electrophoresis

### Day 3 - 13.10.2021

1. Heat inactivated ligation mix (rhlB + Duo2, rhlA + Duo2)
2. Double digestion of Duo2 and nadE with Bmt I
3. gel elution for 4th, 6th and 8th well.
4. Melt them in Thermomixer compact at 50°C

1. Weight gel: For rhlA ~ 0.32g

For rhlB = 0.34g

For nadE = 0.22g

1. Add Gel Solubilization Buffer (L3) to the gel in a ratio 3:1
2. Place the tube in a thermomixer for 10 min until gel will dissolve. Each 3 min gently mix by inverting
3. Incubate the tube for additional 5 min
4. Purify DNA using a centrifuge
5. Pipet the dissolved gel into the column in wash tube. One column per 400 μg capacity is 850μl, so that we took approximately 700 μl
6. Centrifuge the flow through and place column into the wash tube
7. Add 500μl Wash buffer (W1)
8. Centrifuge at 12000 rpm for 1 min, discard the flow through
9. Centrifuge one more time at 14000 rpm for 2 mins, discard the flow through
10. Place column in a new wash tube (recovery tube). Add 50 μl of Elution Buffer (E5) into the centre of the column. Incubate 1 min at RT
11. Centrifuge the tube at 12 000 rpm for 1 min

5. Transformed P.putida with ligation mix rhlB+Duo2; rhlA + Duo2, left for incubation on plates (LB agar + kanamycin).

6. Prepared gel electrophoresis samples for tomorrow:

15μl DNA ladder + 5μl loading dye

15μl Duo2 Bmt I + 5μl loading dye

15μl Duo2 diluted Bmt I + 5μl loading dye

15μl nadE Bmt I + 5μl loading dye

10μl ligated rhlA + Duo2 + 5μl 1xTAE + 5μl loading dye

10μl ligated rhlB + Dup2 + 5μl 1xTAE + 5μl loading dye

10μl Duo2 stock (607ng) + 5μl 1xTAE + 5μl loading dye

![Notebook Figure 76](https://static.igem.org/mediawiki/2021/f/f0/T--NU_Kazakhstan--Notebook_Figure_65.jpg)

Figure 76. P.putida with pRGPDuo2 + rhlA

![Notebook Figure 77](https://static.igem.org/mediawiki/2021/f/f6/T--NU_Kazakhstan--Notebook_Figure_66.jpg)

Figure 77. P.putida with pRGPDuo2 + rhlB

![Notebook Figure 78](https://static.igem.org/mediawiki/2021/1/11/T--NU_Kazakhstan--Notebook_Figure_67.jpg)

Figure 78. Nanodrop

### Day 4 - 14.10.2021

1. [Measure concentration of eluted rhlA, rhlB (13.10.21) by nanodrop.

Table 120 Gel Electrophoresis track order

| 5th well | DNA ladder |
| 6th well | Duo2 stock |
| 7th well | Duo2 Bmt I digested |
| 8th well | Duo2 diluted Bmt I digested |
| 9th well | nadE Bmt I |
| 10th well | rhlA + Duo2 ligated |
| 11th well | rhlB + Duo2 ligated |

Run gel electrophoresis at 90V for 1 hour

2. Measure concentration of eluted Duo2 + nadE extracted from P.aeruginosa

1st sample - 38.62ng/μl

2nd sample - 14.34ng/μl

3rd sample - 14.41ng/μl

**Duo2 + nadE (P.putida) digestion**

Bmt I - 2μl

DNA - 2μl (606ng/μl)

10x CutSmart buffer - 5μl

nadE - 26μl (2.49ng/μl)

nuclease free water - 16μl

**Duo2 + nadE (P.aeruginosa) digestion**

Sac I - 1μl

Sal I - 1μl

DNA - 35μl

10x CutSmart buffer - 5μl

Nuclease free water - 8μl

PCR for nadE (5-6) amplification:

Q5 buffer - 5μl

10mM DNTP mix - 2μl

forward primer 10mM - 1.5μl

reverse primer 10mM - 1.5μl

Template DNA - 4μ

Q5 polymerase - 0.25μl

Q5 enhancer - 5μl

nuclease free water - 5.75μl (2 tubes of 25μl)

Table 121. PCR conditions

| Steps | t (°C) | time |  |
| Initial Denaturation | 98 | 3 min |  |
| Denaturation | 98 | 10 sec | 35 cycles |
| Annealing | 72 | 30 sec |
| Elongation | 72 | 40 sec |
| Final elongation | 72 | 2 min |  |
| Hold | 4 |  |  |

![Notebook Figure 79](https://static.igem.org/mediawiki/2021/9/9a/T--NU_Kazakhstan--Notebook_Figure_69.jpg)

Figure 79. Nanodrop

* Gel have too little concentration of samples, that is why band is not clear on photo, but we got results.
![Notebook Figure 80](https://static.igem.org/mediawiki/2021/2/25/T--NU_Kazakhstan--Notebook_Figure_71.jpg)

Figure 80. Gel electrophoresis

### Day 5 - 15.10.2021

Gel electrophoresis: (15μlsample + 5μl loading dye)

Prepare agarose gel (1g agarose + 100ml 1xTAE + 10μl GelRed)

Table 122. Gel Electrophoresis track order

| 2nd well | DNA ladder |
| 3rd well | PCR nadE 1 |
| 4th well | PCR nadE 2 |
| 5th well | Duo2 + nadE digested (P.aeruginosa) |
| 6th well | Duo2 + nadE digested (P.putida) |

GRun gel electrophoresis at 90μl for 1 hour.

2. Prepare LB agar (11.1g + 300mL water) and LB broth (6g + 300mL water) and autoclave for an hour.

3. Inoculation of P.aeruginosa, P.aeruginosa with Duo2 and P.aeruginosa with Duo2 + nadE. (They are ready for electrophermentation)

4. Inoculate 2 plates with P.putida with Duo2 + rhlA and P.putida with Duo2 + rhlB.

5. 5μl of the mixture was added to 50μl competent P.aeruginosa and electroporated at 1600V for 5 seconds.

6. Immideately 1mL of LB broth solution was added after electroporation.

6. Prepared fresh LB broth with P.aeruginosa.

![Notebook Figure 81](https://static.igem.org/mediawiki/2021/2/26/T--NU_Kazakhstan--Notebook_Figure_72.jpg)

Figure 81. Gel electrophoresis

### Day 6 - 16.10.2021

1. Prepared 2xLB agar 25ml + 25μl kanamycin.
2. Prepare electrocompetent P.aeruginosa, OD=1.0
3. Pour 5mL of diluted E.coli solution into prechilled 15mL centrifuge tube.

1. Centrifuge at 7000 rpm at 4**°**C for 5 minutes.
2. Discard supernatant.
3. Resuspend pellet in 1.5mL centrifuge tube with 1mL of ice-cold dd H2O.
4. Microcentrifuge at 7000 rpm at 4**°**C for 5 minutes.
5. Repeat c-d steps 4 times more.
6. Discard supernatant.
7. Resuspend final pellet with 50µL of ice cold dd H2O, put cells on ice.

4. Inoculated P.aeruginosa + pRGPDuo2 + rhlB into a plate with kanamycin.

5. Transformed P.aeruginosa with Duo2 + rhlA (1600V)

6. After electroporation immediately added 1mL LB broth and Incubated for 3 hours.

7. Inoculated transformed P.aeruginosa with Duo2 + rhlA into LB agar plates with kanamycin.

8. Incubate plates at 37**°**C overnight.

![Notebook Figure 82](https://static.igem.org/mediawiki/2021/b/b9/T--NU_Kazakhstan--Notebook_Figure_74.jpg)

Figure 82. *P.aeruginosa* with pRGPDuo2+rhlA

![Notebook Figure 83](https://static.igem.org/mediawiki/2021/b/b2/T--NU_Kazakhstan--Notebook_Figure_77.jpg)

Figure 83. *P.aeruginosa* with pRGPDuo2+rhlB

## Week 4

### Day 1 - 18.10.2021

Table 123. Measured concentrations

| Duo2 (1) | 5.326ng/μl |
| Duo2 (2) | 144.4ng/μl |
| Duo2 + rhlA (1) | 889.3ng/μl |
| Duo2 + rhlA (2) | 106.8ng/μl |
| Duo2 + rhlB (1) | 62.98ng/μl |
| Duo2 + rhlB (2) | 433.6ng/μl |
| Duo2 + nadE (1) | 29.51ng/μl |
| Duo2 + nadE (2) | 164.2ng/μl |

### Day 2 - 19.10.2021

Planning gel electrophoresis:

Table 124. Gel Electrophoresis track order

| 2nd well | DNA ladder |
| 3rd well | Duo2 |
| 4th well | Duo2 + nadE |
| 5th well | Duo2 + rhlA |
| 6th well | Duo2 + rhlB |
| 8th well | DNA ladder |
| 9th well | Duo2 double digested |
| 10th well | Duo2 + nadE double digested |
| 11th well | Duo2 + rhlA double digested |
| 12th well | Duo2 + rhlB double digested |

2.

Table 125. Double digestions of plasmids

| plasmid | Duo2 | Duo2 + rhlA | Duo2 + rhlB | Duo2 + nadE |
| CutSmart buffer | 5μl | 5μl | 5μl | 5μl |
| DNA | 1.4μl | 1.9μl | 0.5μl | 1.2μl |
| nuclease free water | 41.6μl | 41.1μl | 42.5μl | 41.8μl |
| Sac I | 1μl | 1μl | 1μl | 1μl |
| Sal I | 1μl | 1μl | 1μl | 1μl |

heated 1 hour at 37**°**C, heat inactivation at 65**°**C for 20 minutes.

Table 126. Volumes for digestion with Nhe I

| Plasmids | Duo2 + rhlA | Duo2 + rhlB |
| CutSmart buffer | 5μl | 5μl |
| DNA | 5μl | 8μl |
| nuclease free water | 38μl | 35μl |
| Nhe I | 2μl | 2μl |

heated 1 hour at 37**°**C, heat inactivation at 80**°**C for 20 minutes.

### Day 3 - 20.10.2021

1. Performed Double digestion of Duo2+nadE, Duo2+rhlA and Duo2+rhlB.

Table 127

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Plasmids | Duo2 | Duo+nadE | Duo2+rhlA | Duo2+rhlB |
| CutSmart buffer 10X | 5μl | 5μl | 5μl | 5μl |
| DNA | 1.1μl | 2.4μl | 3.83μl | 5.01μl |
| SacI | 1μl | 1μl | 1μl | 1μl |
| SalI | 1μl | 1μl | 1μl | 1μl |
| nuclease free water | 41.9μl | 40.6μl | 39.17μl | 37.09μl |

1. Heated 1 hour at 37**°**C, heat inactivation at 65**°**C for 20 minutes in a thermomixer.
2. Performed PCR of extracted plasmids:

Table 128

|  |  |  |  |
| --- | --- | --- | --- |
| DNA | Duo2 + rhlA (2) (0.71 microliters) | Duo2 + rhlB(1) (1.2 microliters) | Duo2 + nadE (1) (2.6 microliters) |
| Nuclease free water | 10.04 microliters | 9.55 microliters | 8.15 microliters |
| Q5 Buffer | 5 microliters | 5 microliters | 5 microliters |
| Q5 enhancer | 5 microliters | 5 microliters | 5 microliters |
| 10 mM DNTP mix | 1 microliters | 1 microliters | 1 microliters |
| 10 mM primer forward | 1.5 microliters | 1.5 microliters | 1.5 microliters |
| 10 mM primer reverse | 1.5 microliters | 1.5 microliters | 1.5 microliters |
| Q5 Polymerasr | 0.25 microliters | 0.25 microliters | 0.25 microliters |

1. Treatment with acetone tubes with rhamnolipid for 10h to precipitate them. To scale the mass of precipitated rhamnolipids.

Table 129

|  |  |
| --- | --- |
| Foil | 0.43g |
| Duo2+NB | 0.64g |
| Duo2+rhlB+NB | 0.63g |
| Duo2+rhlA+NB1 | 0.62g |
| Duo2+rhlA+NB2 | 0.63g |
| Duo2+NB | 0.63g |
| Duo2+rhlB+NB | 0.62g |
| Plain | 0.62g |
| Duo2+nadE+LB1 | 0.77g |
| Duo2+nadE+LB2 | 0.87g |
| Duo2+rhlA+LB1 | 0.83g |

Running gel (15ul DNA sample + 5ul loading dye)

Table 130

|  |  |
| --- | --- |
| 2nd well | DNA ladder |
| 3rd well | duo2 |
| 4th well | duo2+nadE |
| 5th well | duo2+rhlA |
| 6th well | duo2+rhlB |
| 8th well | DNA ladder |
| 9th well | dd duo2+nadE |
| 10th well | dd duo2+rhlB |
| 11th well | dd duo2+rhlB |
| 13th well | pcr nadE |
| 14th well | pcr rhlA |
| 15th well | pcr rhlB |

Table 131

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Steps | t (°C) | time rhlB | time nadE and rhlA |  |
| Initial Denaturation | 98 | 3 min | 3 min |  |
| Denaturation | 98 | 10 sec | 10 sec | 35 cycles |
| Annealing | 72 | 30 sec | 30 sec |
| Elongation | 72 | 93 sec | 40 sec |
| Final elongation | 72 | 5 min | 2 min |  |
| Hold | 4 |  |  |  |

### Day 4 - 21.10.2021

1. Extraction of plasmid by Aigerim's protocol
2. Double digestion of obtained plasmids

Table 132

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Plasmids | Duo2 | Duo+nadE | Duo2+rhlA | Duo2+rhlB |
| r1.1 NEB buffer | 5μl | 5μl | 5μl | 5μl |
| DNA | 2.1μl | 2.69μl | 1.84μl | 1.22μl |
| SacI | 1μl | 1μl | 1μl | 1μl |
| SalI | 1μl | 1μl | 1μl | 1μl |
| nuclease free water | 40.9μl | 40.31μl | 41.16μl | 41.78μl |

Incubate at 37C for 1h. Heat inactivated at 65C for 20 min.

1. PCR

Table 133

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Steps | t (°C) | time rhlB | time nadE and rhlA |  |
| Initial Denaturation | 98 | 3 min | 3 min |  |
| Denaturation | 98 | 10 sec | 10 sec | 35 cycles |
| Annealing | 72 | 30 sec | 30 sec |
| Elongation | 72 | 93 sec | 40 sec |
| Final elongation | 72 | 5 min | 2 min |  |
| Hold | 4 |  |  |  |

1. Running 0.8% agarose gel. Loaded 16ul DNA samples + 4ul loading dye. Set for 1h at 90V

Table 134

|  |  |
| --- | --- |
| 2nd well | DNA ladder |
| 3rd well | Duo2 |
| 4th well | Duo2 digested with SacI (1h digestion) |
| 5th well | Duo2 + nadE (1h digestion) |
| 6th well | Duo2 + rhlA (1h digestion) |
| 7th well | Duo2 + rhlB (1h digestion) |
| 8th well | Duo2 digested with SacI (15 min digestion) |
| 9th well | Duo2 + nadE (15 min digestion) |
| 10th well | Duo2 + rhlA (15 min digestion) |
| 11th well | Duo2 + rhlB (15 min digestion) |
| 13th well | DNA ladder |
| 14th well | PCR nadE |
| 15th well | PCR rhlA |
| 16th well | PCR rhlB |
| 17th well | extracted Duo2+nadE |
| 18th well | extracted Duo2+rhlA |
| 19th well | extracted Duo2+rhlB |

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# Team:NU Kazakhstan/Engineering

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## Summary

The central aim of our project is to enhance the rhamnolipid production on the basis of the engineering modification of the *Pseudomonas putida* bacteria by adding **pRGPDuo2 a  plasmid that has a dual expression system for the co-expression of two different inducible genes**[1]. In our case we targeted the genes; ***nadE*** and ***rhlA/B*** genes as inserts for dual inducible co-expression. We hypothesized that the insertion of these genes should enhance the rhamnolipid synthesis under an electrofermentative system. The logic was based on the fact that a simultaneous expression of the respiratory gene ***nadE*** with rhamnolipid genes ***rhlA/B*** in an oil-challenged system could lead to improved remediation of the oil. This would merely be functional under the employment of electrofermentation to target elevated respiration of the bacterial system.

Throughout the project, we went through several iterations of the engineering design cycle, concurrently solving the issues that appeared during the engineering process. This engineering cycle is comprised of the following steps: Research, Imagine, Design, Build, Test, Learn, and Improve. Overall, we have 3 cycles.

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Figure 1. The Engineering Design Cycle

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CONTACT

igem@nu.edu.kz

Kabanbay batyr av., 53, Nur-Sultan, Kazakhstan

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# Team:NU Kazakhstan/Experiments

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# Background

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## Project Steps

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## Used Protocols

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CONTACT

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Kabanbay batyr av., 53, Nur-Sultan, Kazakhstan

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## **Strain modification**

**1. Gel electrophoresis**

![](https://static.igem.org/mediawiki/2021/9/95/T--NU_Kazakhstan--iGEM_Figure_1_1_Gel_electrophoresis_of_PCR_products.jpg)

Figure 1.1 Gel electrophoresis of PCR products

A - *rhlA/B*

B - *rhlA/B*

C - *rhlA*

D - *rhlB*

E - *nadE* for restriction digestion with Nhe1 (insert for MCS2)

The Figure 1.1 shows the picture of gel run after PCR amplification of genes aimed to be inserted into plasmid. Although there are smears at the bottom of the gel that might be represented by the DNA fragment contaminations or formed primer dimers, the gel provided confirmation of successful amplification of *rhlA* (C) and *rhlB* (D) and *nadE* (E) genes. These genes were eluted and gel purified for further use in genetic cloning. *nadE* in this case was amplified using primers that have restriction sites of Nhe1 restriction enzyme, which is not suitable for insertion into MCS1.

![](https://static.igem.org/mediawiki/2021/6/6d/T--NU_Kazakhstan--iGEM_Figure_1.2_Gel_electrophoresis_of_PCR_products.jpg)

Figure 1.2 Gel electrophoresis of PCR products

A - *rhlA/B* (Ta=56°C)

B - *rhlA/B* (Ta=58°C)

C - *rhlA/B* (Ta=70°C)

D - *rhlA/B* (Ta=72°C)

E - *nadE* for restriction digestion with SalI and SacI (insert for MCS1)

A, B, C and D represent PCR products aimed to isolate the cluster of *rhlA* and *rhlB* genes at different annealing temperatures; however, the amplification was not successful. The wells E represents successfully isolated *nadE* gene using primers with restriction sites suitable for insertion into MCS1.

![](https://static.igem.org/mediawiki/2021/8/86/T--NU_Kazakhstan--iGEM_Figure_3.jpg)

Figure 1.3. Gel electrophoresis of double digested plasmids

A - pRGPDuo2 plasmid undigested

B - pRGPDuo2 digested with SacI and SalI (digestion at 37° for 1 hour)

C - pRGPDuo2 + *nadE* digested with SacI and SalI (digestion at 37° for 1 hour)

D - pRGPDuo2 + *rhlA* digested with SacI and SalI (digestion at 37° for 1 hour)

E - pRGPDuo2 + *rhlB* digested with SacI and SalI (with digestion at 37° for 1 hour)

F - pRGPDuo2 digested with SacI and SalI (digested at 37° for 15 min)

G - pRGPDuo2+ *nadE* digested with SacI and SalI (digested at 37° for 15 min)

H - pRGPDuo2+ *rhlA* digested with SacI and SalI (digested at 37° for 15 min)

I - pRGPDuo2+ *rhlB* digested with SacI and SalI (digested at 37° for 15 min)

To confirm that the restriction and ligation was performed successfully, the plasmids pRGPDuo2 + *nadE,* pRGPDuo2 + *rhlA* and pRGPDuo2 + *rhlB* were extracted from *P. aeruginosa* cells transformed with corresponding plasmids and grown overnight in selective medium solution of Luria Bertani broth and kanamycin. The extracted DNA plasmids were incubated with SalI and SacI restriction enzymes for double digestion. The digestion reaction products were run on gel represented on Figure 1.3. It can be seen that the uncut plasmid (A) is localized higher than the cut linearized plasmid. The uncut plasmid (A) is circular which, due to the clustering of genes, travels distance less than allowed by its size (7928 bp). Meanwhile, the linearized plasmids resulting from the double digestion are located in the line with the ladder band representing 8 kbp. The digestion reaction mixture of plasmids extracted from strains pRGPDuo2 + *nadE,* pRGPDuo2 + *rhlA* and pRGPDuo2 + *rhlB* also showed bands indicating inserted genes. The size of the *rhlA* gene is 916 bp (including region outside of the gene complementary to the primer sequence) and can be observed in the wells D and H lower than the ladder band representing 1000 bp, which confirms the digest of the *rhlA* gene from the plasmid extracted from the engineered strain of *P. aeruginosa*. *nadE* gene of size 846 bp cut from the plasmid (C) was identified lower than the band of the ladder (1000 bp) and of *rhlA* gene (916 bp). *rhlB* gene of size 1320 bp (wells E and I) also was detected between ladder bands of 1 kbp and 1.5 kbp. Overall, the gel confirmed that the insertion of the genes into plasmids was successful as they were double digested again after transformation of *P. aeruginosa*.

**2. Colonies on selective plate**

![](https://static.igem.org/mediawiki/2021/f/f1/T--NU_Kazakhstan--iGEM_Figure_2_1_E_coli_transformed_with_pRGPDuo2_on_LB_agar_with_kanamycin.jpg)

Figure 2.1 *E. coli* transformed with pRGPDuo2 on LB agar with kanamycin

As it was expected, *E .coli* carrying pRGPDuo2 with kanamycin resistance gene was able to grow on selective media containing kanamycin. It indicates that the transformation of RGPDuo2 plasmid into *E. coli* was completed and *E. coli* carries pRGPDuo2.

![](https://static.igem.org/mediawiki/2021/3/39/T--NU_Kazakhstan--iGEM_Figure_2_2_Wild-type_E_coli_on_LB_agar_with_kanamycin_%28negative_control%29.jpg)

Figure 2.2 Wild-type *E. coli* on LB agar with kanamycin (negative control)

Wild-type *E. coli* does not have resistance to kanamycin, therefore it was not able to grow on selective media plates.

![](https://static.igem.org/mediawiki/2021/9/97/T--NU_Kazakhstan--iGEM_Figure_2_3_Wild-type_P_aeruginosa_on_LB_agar_with_kanamycin_%28negative_control%29.jpg)

Figure 2.3 Wild-type *P. aeruginosa* on LB agar with kanamycin (negative control)

Similarly, wild-type *P. aeruginosa* does not carry resistance genes to kanamycin. Therefore, it did not grow on LB agar containing kanamycin. It serves as a negative control for transformation of *P. aeruginosa* with different plasmids.

![](https://static.igem.org/mediawiki/2021/b/bb/T--NU_Kazakhstan--iGEM_Figure_2_4_P_aeruginosa_transformed_with_pRGPDuo2_on_LB_agar_with_kanamycin.jpg)

Figure 2.4 *P. aeruginosa* transformed with pRGPDuo2 on LB agar with kanamycin

![](https://static.igem.org/mediawiki/2021/4/4c/T--NU_Kazakhstan--iGEM_Figure_2_5_P_aeruginosa_transformed_with_pRGPDuo2-nadE_on_LB_agar_with_kanamycin.jpg)

Figure 2.5 *P. aeruginosa* transformed with pRGPDuo2-*nadE* on LB agar with kanamycin

![](https://static.igem.org/mediawiki/2021/d/d2/T--NU_Kazakhstan--iGEM_Figure_2_6_P_aeruginosa_transformed_with_pRGPDuo2-rhlA_on_LB_agar_with_kanamycin.jpg)

Figure 2.6 *P. aeruginosa* transformed with pRGPDuo2-*rhlA* on LB agar with kanamycin

![](https://static.igem.org/mediawiki/2021/a/a4/T--NU_Kazakhstan--iGEM_Figure_2_7_P_aeruginosa_transformed_with_pRGPDuo2-rhlB_on_LB_agar_with_kanamycin.jpg)

Figure 2.7 *P. aeruginosa* transformed with pRGPDuo2-*rhlB* on LB agar with kanamycin

Figures 2.4-2.7 depict that pRGPDuo2 carrying *nadE*, *rhlA* and *rhlB* genes respectively were incorporated into *P. aeruginosa*. Single colonies from the given plates were used in the following experiments.

## **Electrofermentation experiment**

**3. Bioelectrochemical methods**

![](https://static.igem.org/mediawiki/2021/2/25/T--NU_Kazakhstan--iGEM_Figure_3.1_Cyclic_voltammograms.png)

Figure 3.1 Cyclic voltammograms (CV) at 10mV/s scan rate between -400mV and 400mV for the different electro fermentative setups for production of rhamnolipids by the different test *P. aeruginosa* strains (engineered and wild type). Media used: MSM (minimum salt media) + oil + casein

CV plots show distinct oxidation (upwards) and reduction (downward) curves which imply that the setups are redox active (electrochemically active). The gaps between the curves show the extent of electrochemical activity measured in degree of current outputs quantified in microamperes. The wider the gaps between the curves, the higher the current differences, and the higher the electroactivity. At time 0h (beginning of experiment) there was a very minimal electroactivity in all the test setups with the different strains as there was almost 0 uA current output. However after 24 h of incubation, there were clearly distinct oxidation and reduction curves showing increased current outputs as the cells grew. Further examining the 24 h curves for each of the strains, it can be observed that there were protrusions on the oxidation curve (approximately 100 mV) and reduction curve (0 mV). This peak is attributed to a specific mechanism of electroactivity the bacteria are using most likely attributed to increased biomass or electroactive biofilm formation. It is quite obvious that *P. aeruginosa nadE* (red line) had the highest peak among the test strains after 24h incubation. This could be attributed to the increased respiratory activity of the strain caused by the influence of increased NAD synthetase production. Increased NAD synthetase directly influences increased cellular metabolic activities and could directly affect rhamnolipids yields. In this case, all other strains *P. aeruginosa rhlA*, *P. aeruginosa rhlB* and the wild type strains though they showed slight peaks at similar oxidation reduction points, they did not have overexpressed NAD synthetase, therefore showed much less respiratory activity in comparison with the *P. aeruginosa nadE* strain. We hypothesised that this increased respiratory activity could be influential on enhanced biosurfactant yields as well.

![](https://static.igem.org/mediawiki/2021/1/1f/T--NU_Kazakhstan--iGEM_Figure_3.2_Chronoamperometry_data.png)

Figure 3.2 Chronoamperometry data at (400mV poised potential) for the different electro fermentative setups for production of rhamnolipids by the different test *P. aeruginosa* strains (engineered and wild type). Media used: MSM + oil + casein

From the data it was observed that all the engineered strains yielded higher current outputs than the wild type strain. In specific terms, the highest current was generated by the *P. aeruginosa nadE* set up. This data also shows that this strain had the highest respiratory activity measured here as extracellular electron transportation quantified as electrical currents. Over the course of 24 hours, the current production was highest for the *P. aeruginosa nadE* strain at about 22 hours yielding approximately 0.2 µA. The strain eliciting the second highest current production was *P. aeruginosa rhlA* (0.16 µA), closely followed by the wild type strain (0.15 µA) and then *P. aeruginosa rhlB* (0.10 µA). For all the strains, there was an initial lag period between 0h and 3h of incubation. This could be attributed to the initial adaptation time the strains would need to be able to acclimatize with the medium environment before cellular growth and metabolism begins. After 3 h on incubation however, there was a gradual rise in current produced by all strains which directly correlates with improved metabolic activities.

![](https://static.igem.org/mediawiki/2021/f/f0/T--NU_Kazakhstan--iGEM_Figure_3_3_Total_electrical_charge_production.png)

Figure 3.3 Total electrical charge production at 24h incubation time for the different electro fermentative setups for production of rhamnolipids by the different test *P. aeruginosa* strains (engineered and wild type). Media used: MSM + oil + casein

Total charge produced after 24 h incubation was shown above. This relates to the amount of electrical charges in Coulombs produced by the different test setups carrying the different strains. The graph shows that *P. aeruginosa nadE* setup produced the highest charge of 10.8 mC at 24 h. The trend in total charge production was thus: *P. aeruginosa nadE* > *P. aeruginosa* *rhlA* > *P. aeruginosa* wild type > *P. aeruginosa rhlB.* This result is synonymous with the data of current produced over a 24 h period for all the test strains. *P. aeruginosa nadE* elicited overall over 40% increase in charge as compared to its wild type strain, while *P. aeruginosa rhlB* showed a reduced amount of charge produced in comparison with the wild type.

## Extraction and analysis of biosurfactant

**4. Extraction of biosurfactant**

Table 4.1 Masses of the precipitate from the cell-free broth containing bacterial cell products

|  |  |
| --- | --- |
| *P.aeruginosa* strains supernatant of which was used for precipitation | Average mass of extracted precipitate (mg/ml of cell-free supernatant) |
| *P. aeruginosa* wild-type | 19.0 |
| *P. aeruginosa* pRGPDuo2 | 20.5 |
| *P. aeruginosa* pRGPDuo2 + *rhlB* | 24.0 |
| *P. aeruginosa* pRGPDuo2 + *rhlA* | 29.6 |
| *P. aeruginosa* pRGPDuo2 + *nadE* | 39.0 |

After the cold acetone precipitation, the precipitates from each sample were hot air dried. The resulting dried substances were weighted. The average of the obtained values for each type of strain was calculated and recorded in Table 4.1. The largest mass value was observed in the sample from the *P. aeruginosa* pRGPDuo2 + *nadE*, while the lowest mass of the precipitate was yielded from supernatant of wild-type *P. aeruginosa.* From the data it can be observed that the greater production of cell product involving rhamnolipids is correlated with the overexpression of *nadE* genes. Wildtype *P. aeruginosa* and *P. aeruginosa* pRGPDuo2 provided values close to each other. *P. aeruginosa* pRGPDuo2 does not carry inserted genes; therefore, the production is lower. However, the increased value relative to wildtype can be caused by the additional gene load provided by the plasmid. Strains carrying **rhlA** and **rhlB** also show increased production of substances; however, the fact that the yield is lower than from *P. aeruginosa* pRGPDuo2 + *nadE* might imply that the overexpression of rhlA and rhlB genes is not enough for the enhancing rhamnolipid production under electrefermentative conditions.

**5. Fourier Transform Infrared (FTIR) Spectroscopy**

![](https://static.igem.org/mediawiki/2021/4/46/T--NU_Kazakhstan--iGEM_Figure_5.1_FTIR_spectra_of_extracted_rhamnolipids.png)

Figure 5.1 FTIR spectra of extracted rhamnolipids from *P. aeruginosa* strains tested

The FTIR spectra of the rhamnolipid products extracted from production setups utilizing all the test strains showed that there was almost 100% uniformity in spectra pattern from all strains, confirming the chemical similarity of the rhamnolipids produced by all the strains.

The spectra bands between 1600 and 1740 cm-1 indicating a C=O stretching was caused by ester and acidic groups. Spectra bands around 2930 cm-1, 2857 cm-1 and 1460 cm-1 were as a result of the symmetrical and asymmetrical stretching vibrations of C–H in aliphatic bonds and aliphatic bending corresponding to lipids. The band on 3430 cm-1 represented the hydroxyl group free O-H stretch as a result of hydrogen bonding. A peak observed around 1380 cm-1 was due to the COO– antisymmetric stretching,

A signature stretching of C-O-C bond was observed at 1042 cm-1 and it was indicative of the rhamnose sugar. This is explained by the fact that C-O-C vibrations in the cyclic structures of carbohydrates were as a result of the presence of bonds formed between Carbon atom and hydroxyl groups present in chemical structures of rhamnose rings. A band was observed around 703 cm-1 which is a signature region identical with rhamnolipids.

These absorption bands were all characteristic of glycolipids, especially rhamnolipids and FTIR spectra are similar to the spectra of previously reported rhamnolipids (Eraqi et al., 2016 & Deepika et al.*,* 2015).

## Bioremediation

**6. Laboratory-scale bioremediation of soil saturated with crude oil**

![](https://static.igem.org/mediawiki/2021/1/15/T--NU_Kazakhstan--iGEM_Figure_6_1_Bioremediation.jpg)

Figure 6.1 Bioremediation treatment of crude oil contaminated soils with crude biosurfactants from the various production set-ups (electrofermentation) using the different *P. aeruginosa* strains

A: Treatment *with P. aeruginosa* Wild type crude biosurfactant (from conventional fermentation)

B: Untreated control

C: Treatment with *P. aeruginosa nadE* crude biosurfactant (electrofermentation)

D: Treatment with *P. aeruginosa rhlA* crude biosurfactant (electrofermentation)

E: Treatment with *P. aeruginosa rhlB* crude biosurfactant (electrofermentation)

Table 6.1 Analysis of emulsification assay results after 24 hours of incubation

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | *P. aeruginosa* Wild Type | Untreated control | *P. aeruginosa* with pRGPDuo2 + *nadE* | *P. aeruginosa* with pRGPDuo2 + *rhlA* | *P. aeruginosa* with pRGPDuo2 + *rhlB* |
| Emulsification index, % | 39.53 | - | 74.23 | 33.52 | 36.71 |

Emulsification index was identified using the ImageJ software. The remaining crude oil on top of the media of untreated control was taken as a control . Compared to the given value, the emulsification index was identified for other strains of *P. aeruginosa*. Emulsification index for wild-type *P. aeruginosa* (39.53%), *P. aeruginosa rhlA* (33.52%) and *P. aeruginosa rhlB* (36.71%) was approximately similar, indicating to the fact that *P. aeruginosa* already contains both *nadE* and *rhl* genes and electrochemically active. Furthermore, it indicates that overexpression of any genes (either *rhlA* or *rhlB*) would not lead to the consequent increase of rhamnolipid production.

However, as the results have shown, overexpression of *nadE* gene increased the emulsification index 1.88 times, from 39.53% to 74.23%. It is possible to conclude that the *nadE* gene overexpression in *P. aeruginosa* was able to increase the electron pull of the bacteria and elevate rhamnolipid production.

## Successes & Failures

Under Covid-19 pandemic, reagents shortage, slow logistics, expired kits and unexpected obstacles our team showed maximal effort. We believe that our hard work was worthwhile.

Successes

* Designed and expressed [*rhlA*](http://parts.igem.org/Part%3ABBa_K4083007), [*rhlB*](http://parts.igem.org/Part%3ABBa_K4083006), [*nadE*](http://parts.igem.org/Part%3ABBa_K4083004).
* Inserted genes in a newly designed [pRGPDuo2](http://parts.igem.org/Part%3ABBa_K4083000)
* Confirmed that *nadE* overexpression increases rhamnolipid production rate under electrofermentative conditions.
* Designed 3D models of our synthesized proteins.

Failures

* We first planned to use *E.coli* DH5α as intermediate of our vector plasmids. But because of time limits, we decided to transform plasmids directly to the *P.putida* and *P.aeruginosa*. However we used *E.coli* DH5α to amplify the number of our pRGPDuo2 plasmids.
* Our *rhlAB* gene sequence wasn’t successfully extracted due to its large size (2244bp) and absence of required polymerases, so we didn’t get results after spending more than two months on PCR of *rhlAB*. Alternatively, we designed new primers for *rhlA* and *rhlB* separately. So that we can design two pRGPDuo2 plasmids with only *rhlA* and only *rhlB* genes. The *rhlB* protein works properly in the presence of *rhlA* protein, but does not require coexpression of *rhlA* (Wittgens et al., 2017). It means that *rhlA* and *rhlB* proteins participate in rhamnolipid passway separately and theoretically *rhlA* and **rhlB** protein synthesis gene sequence can be inserted on different multiple cloning sites.
* We also found that our *P.putida* KT2440 doesn't want to uptake our plasmids with *rhlA* and rhlB. Thus we took *P.aeruginosa* PAO1 as a model in order to conduct experiments with *nadE* gene overexpression, since *P.aeruginosa* contains wild *rhlAB* gene. We confirmed that *nadE* overexpression increases rhamnolipid synthesis, thus our mutant *P.putida* with *rhlAB* gene should have the same trend.
* Unable to confirm efficiency of dual expression plasmid pRGPDuo2, because *rhlAB* wasn’t extracted. We expect that plasmid with dual expressed *rhlAB* and *nadE* will significantly increase rhamnolipid synthesis rate.

## Future tests

* Insert of *rhlA/rhlB* and *nadE* genes into pRGPDuo2 plasmid, therefore testing efficiency of dual expression system. Transform plasmid in nontoxic *P. putida* KT2440 strain and observe emulsification index in electrofermentative bioremediation of crude oil.
* Designing pRGPDuo3 and pRGPDuo4 mutant plasmids, which are more efficient in protein synthesis gene expression. Design new primers and insert *rhlAB*, *nadE* genes in order to conduct electrofermentative bioremediation with crude oil (Gauttam et al., 2021).
* Investigate further models with Michaelis Menten equation. Proceed experiments on enzymes quantification, identify concentration of NAD synthetase and plot Michaelis Menten graph. Calculate the efficiency of overexpressed *nadE* gene in rhamnolipid synthesis compared to wild type *P.aeruginosa* based on Vmax of NAD synthetase production*.*

## Conclusion

Our model of rhamnolipid synthesis based on *P. aeruginosa* PAO1 confirmed that *nadE* overexpression substantially increases rhamnolipid synthesis under electrofermentative conditions. However, plasmids with only *rhlA* and only *rhlB* genes showed relatively the same rhamnolipid synthesis rate as wild type *P. aeruginosa*. *P. aeruginosa* already has *rhlA/B* gene sequence and overexpression of one of these two proteins will not affect the overall rate. This is because RhlA and RhlB proteins work cooperatively and cannot synthesize rhamnolipid in the absence of each other. In order to increase rhamnolipids concentration we need to insert *rhlA* and *rhlB* genes in one *P. putida* strain either via dual expression plasmid or two plasmids at once.

**Reference List:**

Eraqi, W.A., Yassin, A. S., Ali, A. E. and Amin, M. A., (2016). Utilization of Crude Glycerol as a Substrate for the Production of Rhamnolipid by *Pseudomonas aeruginosa*. Biotechnology Research International Volume 2016, Article ID 3464509, <http://dx.doi.org/10.1155/2016/3464509>

Deepika, K.V., Sridhar, P. R. and Bramhacjari, P.V. (2015). Characterization and antifungal properties of rhamnolipids produced by mangrove sediment bacterium *Pseudomonas* *aeruginosa* strain KVD-HM52. Biocatalysis and Agricultural Biotechnology 4: 608 – 615. <http://dx.doi.org/10.1016/j.bcab.2015.09.009>

Wittgens, A., Kovacic, F., Müller, M.M. et al. (2017) Novel insights into biosynthesis and uptake of rhamnolipids and their precursors. *Appl Microbiol Biotechnol* 101, 2865–2878. <https://doi.org/10.1007/s00253-016-8041-3>

Gauttam, R., Mukhopadhyay, A., Simmons, B. A., & Singer, S. W. (2021, May 19). *Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in pseudomonas putida KT2440*. Society for Applied Microbiology. https://doi.org/10.1111/1751-7915.13832

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