iGEM 2024 — Team iiser-tvm

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# NoteBook

**## Notebook**

*### “our Magic book”*

-Shrushti, Gouri, Irin, Paarth

* <
  >

  May 2024
  + Sun
  + Mon
  + Tue
  + Wed
  + Thu
  + Fri
  + Sat

  Week 1- 28
  - 29
  - 30
  - 1
  - 2
  - 3
  - 4
  Week 2- 5
  - 6
  - 7
  - 8
  - 9
  - 10
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  Week3- 12
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  Week 4- 19
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  - 21
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  - 24
  - 25
  Week 5- 26
  - 27
  - 28
  - 29
  - 30
  - 31
  - 1

  June 2024
  + Sun
  + Mon
  + Tue
  + Wed
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  + Fri
  + Sat

  Week 6- 26
  - 27
  - 28
  - 29
  - 30
  - 31
  - 1
  Week 7- 2
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  - 4
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  Week 8- 9
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  Week 9- 16
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  Week 10- 23
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  Week 11- 30
  - 1
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  July 2024
  + Sun
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  Week 12- 30
  - 1
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  Week 13- 7
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  Week 14- 14
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  Week 15- 21
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  Week 16- 28
  - 29
  - 30
  - 31
  - 1
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  August 2024
  + Sun
  + Mon
  + Tue
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  Week 17- 28
  - 29
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  Week 18- 4
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  Week 19- 11
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  Week 20- 18
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  Week 21- 25
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  September 2024
  + Sun
  + Mon
  + Tue
  + Wed
  + Thu
  + Fri
  + Sat

  Week 22- 1
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  - 3
  - 4
  - 5
  - 6
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  Week 23- 8
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  Week 24- 15
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  Week 25- 22
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  Week 26- 29
  - 30
  - 1
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  - 3
  - 4
  - 5

  Collapse all

  Expand all
* ### May

  Week 1

  # Day 1-30/04/2024

  ---

  + Standard LB broth (for one litre= 25 ml of LB)
    250ml = 6.25 gm of LB
    250ml = 6.25 gm of LB
  + For the LB agar (for 1 litre = 15 gm agar + 25 gm LB)
    200ml = 3 gm of agar + 5 gm of LB -
  + Since there was a premix of LB+agar of 40 gm in 1 litre, we took 8 gm.
  + Each plate is 25 ml approx of LB agar (8 plates).

  # Day 2 - 01/05/2024

  ---

  + 100% glycerol stock autoclaved for reviving the bacteria.
  + Revived the cells by plating (Quadrant streaking ) and culture in a falcon
    tube(15ml) overnight culture for the TA7-EG and TA7.
  + Shaker incubator at 37°C.
  + Primary strain in 4°C.

  # Day 3 - 02/05/2024

  ---

  + The plates had good bacterial growth, and the falcon was contaminated.
  + Made the glycerol stock (for 1ml of stock)
    - 900ul of culture
    - 100ul of glycerol
    For the strains TA7 & TA7-EG
  + Kept in -80°C cryovials
  + The falcon culture we made was contaminated since there was a growth in the
    control tube.
  + We prepared one more culture media by picking one colony from the plate with
    the same calculations and shaker incubation for 12 hrs

  # Day 4 - 03/05/2024

  ---

  + The control had no growth, and the other tubes looked turbid, signifying the
    bacterial growth.
  + We made a glycerol stock of the confirmed bacterial growth and kept it at -80°C
    in the same place as the last time we kept it.

  Week 2

  # Day 5 -06/05/2024

  ---

  + Modified M9 minimal media( for 1 litre)
    5x M9 salt - 200ml
    1M MgSO4 - 2 ml
    1M CaCl2 - 100ul
    Carbon source -
    Trace metals-
  + M9 minimal salt stock (for 1 litre)
    47.7 mM Na2HPO4·7H2O - 64 gm
    22 mM KH2PO4- 15 gm
    18.7 mM NH4Cl- 5 gm
    8.5 mM NaCl- 2.5 gm
  + Made 50ml of 1M MgSO4
  + Made 50 ml of 1M CaCl2

  # Day 6 - 08/05/2024

  ---

  + TPA-200mM = 6.644 gm of TPA in 200 ml water.
  + Add the NaOH to mix TPA well in the water and to maintain the pH.
  + The pH was adjusted to 7.56-7.57.
  + A new plate with the culture (TA7, TA7-EG) and a new glycerol stock solution
    were streaked.
  + Ethylene glycol was filtered since it had dust particles
  + Got the trace metals, but the composition was incomplete
  + Since the composition was incomplete, we’ll try to grow without it.

  # Day 7 - 91/05/2024

  ---

  + Make a liquid culture for the streaking and refill the glycerol stock.
  + 250 ml of LB broth
  + Made the minimal M9 media
    The concentrations were as follows:

  | TPA | EG | TPA & EG |
  | --- | --- | --- |
  | 5mM | 5mM | 20mM+20mM |
  | 10mM | 10mM | 10mM+10mM |
  | 15mM | 15mM | 15mM+15mM |
  | 20mM | 20mM | 10mM+15mM |
  |  |  | 15mM+10mM |
  |  |  | 5mM+10mM |
  |  |  | 10mM+5mM |

  **The calculations:**

  For 25ml

  5x M9 salt = 5ml

  1M Mgso4 = 0.05ml

  1M Cacl2 = 0.0025ml

  For the carbon source (TPA and EG)

  | Concentrations | TPA | EG |
  | --- | --- | --- |
  | 5mM | 0.635ml | 7ul |
  | 10mM | 1.25ml | 14ul |
  | 15mM | 1.875ml | 21ul |
  | 20mM | 2.5ml | 28ul |

  # Day 8 - 10/05/2024

  ---

  + Glycerol stock was prepared and kept at -80C
  + Streaking of the TA-7 and TA-7&EG in various concentration
  + Kept the plates at 37°C for the overnight incubation

  # Day 9 - 11/05/2024 (GOURI’S BIRTHDAY- she went home smh smh)

  ---

  + There was no growth in any plates.
  + The reasons could be the pH for the entire media or the temperature should be
    around 30C.

  Week 3

  # Day 10 - 12/05/2024

  ---

  + This time, we made a liquid culture (broth) with the following concentrations:

  | TPA | EG | TPA & EG |
  | --- | --- | --- |
  | 20mM | 20mM | 20mM+20mM |
  | 10mM | 10mM | 15mM+15mM |
  |  | 15mM(TA-7) | 10mM+10mM |

  - We made three sets of the cultures:(20mM TPA, 20mM EG, 20mM+20mM TPA & EG)
  * With TAP water
  * With trace metals (from VV lab), there are three components missing
  * With just distilled water- For 15ml media:
  M9 media = 3ml = 51ul

  Mgso4 = 30ul = 510 ul

  Cacl2 = 1.5ul = 25.5ul

  TAP water/Trace metals = 25ul = 3.0315ml- The calculations:
  The M9 media salts- 3ml+30ul+1.5ul = 3.0315 ml

  (1)- TAP/Trace metals

  (2) - Just distilled water

  | Concentrations | TPA/EG or TPA&EG | TAP/Trace metals | The total amount of water that needs to be added |
  | --- | --- | --- | --- |
  | 20mM TPA(1) | 1.5ml | 25ul | 10.4435ml |
  | 20mM TPA(2) | 1.5ml | - | 10.4685ml |
  | 20mM EG (1) | 16.8ul | 25ul | 11.9267ml |
  | 20mM EG(2) | 16.8ul | - | 11.9517ml |
  | 20mM+20mM (1) | 1.5ml+0.0168ml | 25ul | 10.4267ml |
  | 20mM+20mM(2) | 1.5ml+0.0168ml | - | 10.4517ml |
  | 10mM+10mM(1) | 0.75ml+0.0084ml | 25ul | 11.1857ml |
  | 10mM+10mM (2) | 0.75ml+0.0084ml | - | 11.2101ml |
  | 15mM EG | 12.6ul | - | 11.9559ml |
  | 10mM TPA(1) | 0.75ml | 25ul | 11.1935ml |
  | 10mM TPA(2) | 0.75ml | - | 11.2185ml |
  | 10mM EG(1) | 8.4ul | 25ul | 11.9351ml |
  | 10mM EG(2) | 8.4ul | - | 11.9601ml |
  | Control |  |  | 11.9685ml |

  * Inoculation of liquid culture in 50ml falcon tubes

# Day 11 - 13/05/2024

---

* The liquid culture has no growth
* We’ll keep it for 24-48 hrs
* Dr Phale KT2440 streaking (reviving)
* We had slight turbidity in some of the falcons, so we kept another 12hrs.
* The falcons that have the growth were:
  1) 20mM+20mM = Trace metals
  2) 20mM ethylene glycol = Trace metals

# Day 12 - 14/05/2024

---

* Revived strains were inoculated in the liquid culture to make glycerol stock.
* A minimal media culture was used for the shaking incubation. We checked those,
  and the colonies were seen in the 15mM EG (TA-7).
* Glycerol stock was made of KT2440 and kept at -80°C.

# Day 13 - 15/05/2024

---

* There was growth in 5 tubes, but the problem is that the growth is seen only in
  TPA-EG and EG cultures.
* There was no growth in the only TPA cultures.
* This could be because TPA is not fully protonated, making it hard for the bacteria
  to consume.
* To confirm whether the turbidity from the culture is bacteria or chemical
  contamination, we streaked the plates on the agar plates -250ml
* We made a new set of trace metals
  1) Boric acid - 2gm (VV lab)
  2) Nickel chloride - anhydrous (Sukhendu Mandal)
  3) Zinc sulphate - anhydrous (Sukhendu Mandal)
* Preparation of the trace metals:
  1) Dissolve 1.352gm of Fecl3.6H2O in 50 ml of 0.1 M HCl.
  2) Add 36 ml of H2O
  3) Dissolve 294 mg of CaCl2
  4) 198 mg of Mncl2.4H2O
  5) 288 mg of ZnSO4.7H2O
  6) 47.8 mg of CoCl2.6H20
  7) 34.1 mg of CuCl2.6H2O
  8) 45.5 mg of NiCl2.6H2O
  9) 12.4 mg of Boric Acid
  10)48.9 mg of Na Molybdate
  11)34.6 mg of Na selenite
  12) Add H2O to the remaining volume to make up 100 ml.

# Day 14 - 16/05/2024

---

* Both the agar media plates and the liquid culture were made.
* The calculations:
For the 25ml trace metals
1) Salts = 5.0550 ml
2) Agar = 0.462 ml

| Concentration | Agar+M9 salts+ TPA/EG/TPA and EG | Total water needed to add to make up to 25 ml |
| --- | --- | --- |
| 10mM EG | 5.5310ul | 19.469ml |
| 10mM TPA | 6.7670ul | 19.455ml |
| 20mM EG | 5.5450ul | 19.455ml |
| 20mM TPA | 8.0170ul | 16.983ml |
| 10mM+10mM | 6.7810ul | 18.219ml |
| 20mM+20mM | 8.0450ul | 16.955ml |
 Control | 5.5170ul(only agar+salts) | 19.483ml |

# Day 15 - 18/05/2024

---

* There was fungal contamination in the agar plates.
* liquid culture was shaken in the incubator till 20/05/24

Week 4

# Day 16 - 19/05/2024

---

* Outreach in Vithura.

# Day 17 - 20/05/2024

---

* Made a fresh TPA with the pH balanced at 7.47-7.48.
* Made the liquid M9 culture media of:
1) 20mM TPA -(30°C/37°C)

2) 20mM EG - (30°C/37°C)

3) 20mM+20mM - (30°C/37°C)

4) 10mM+10mM - (30°C)

5) 10mM TPA - (30°C)

6) 10mM EG - (30°C)* Two sets were made to compare the temperature difference, and for that, we used the same
  bacteria we streaked on 15/05/2024.

# Day 18 - 21/05/2024

---

* The bacterial growth was not seen, and the culture was shaken more for a day.

# Day 19 - 22/05/2024

---

* The bacterial density OD was taken for 2 hrs.

1) 4:45 evening:

20mM TPA- 0.049

20mM EG- 0.112

10mM+10mM- 0.086

10mM TPA- 0.089

10mM EG- 0.076

20mM+20mM- 0.064

2) 5:45 evening:

20mM TPA- 0.100

20mM EG- 0.074

10mM+10mM- 0.078

10mM TPA- 0.087

10mM EG- 0.124

20mM+20mM- 0.045

# Day 20 - 23/05/2024

---

* According to Dr Yashraj Chavan, we should serial dilute the culture for the bacteria to
  force growth within 12 hrs (maintain the bacteria in the exponential growth phase).
* He also suggested that instead of using the falcons, we should use the conical flask to
  provide more volume for the growth.
* Our PhD mentor suggested that the growth assay and dilution should start when there is
  turbidity and that readings should be taken every 12 hours.
* We decided to make a fresh new culture but keep the old culture to shake in the
  incubator to check how long the bacteria (TA-7) will take to grow in the TPA.
* We made a fresh media of 15ml:
Calculations:
M9 Media- 3ml
MgSo4- 30ul
CaCl2 - 1.5 ul
Trace metal - 15 ul

| Concentration | TPA / EG/ TPA & EG | Water |
| --- | --- | --- |
| 20mM TPA | 1.5 ml | 10.4535ml |
| 20mM EG | 16.8 ul | 11.9367ml |
| 20mM TPA and EG | 1.5 ml+16.8ul | 10.4367ml |
| 10mM TPA | 0.75ml | 11.2035ml |
| 10mM EG | 8.4 ul | 11.9421ml |
| 10mM TPA & EG | 0.75 ml+8.4ul | 11.1951ml |

* The plasmid resuspension was done; the Eppendorf was spun for 2:30 min at 5000 rpm.
* Then 100ul of new microbial DNA water in the tubes was centrifuged for 2:30 min by
  5000rpm.
* The OD for bacterial density:

+ 10mM TPA+EG- 0.085
+ 10mM TPA- 0.009
+ 10mM EG- 0.366
+ 20mM TPA+ EG- 0.047
+ 20mM TPA- 0.064
+ 20mM EG- 0.112

* We streaked the plates made on 20/05/24 cause there was contamination to check for
  bacterial, fungal, and chemical contamination.

# Day 21 - 24/05/2024

---

* In the afternoon the readings were:

+ 10mM TPA+EG- 0.131
+ 10mM TPA- 0.141
+ 10mM EG- 0.038
+ 20mM TPA+ EG- 0.110
+ 20mM TPA- 0.076
+ 20mM EG- 0.020

* The plasmid purity was checked in nanodrop.
1) SaG2- SaSSy Fpps-Leu2d:

ng/ul = 6.1

A260/A280 = 1.85

A260/A230 = 0.87

2) SGFpps: SaSSy PCF DUET:

ng/ul = 8.4

A260/A280 = 1.76

A260/A230 = 0.62

3) SaG1 - SaSSy-Leu2d

ng/ul = 6.4

A260/280 = 1.81

A260/A230 = 0.94

4) SaG4 - SaCPR2-HIS

ng/ul = 2.0

A260/A280 = 1.98

A260/A280 = 0.60* There is contamination in the plasmid sample
* The bacteria streaked on the previous day showed bacterial contamination.

# Day 22 - 25/05/2024 ( SHRUSHTI’S BIRTHDAY - she might have cried)

---

* The OD was checked at 2:00 PM
Concentration TPA & EG TPA EG

10mM 0.060 0.134

20mM 0.041 0.117

10mM+10mM 0.033

20mM+20mM 0.021* The OD checked at 2:00 AM.
Concentration TPA & EG TPA EG

10mM -0.012 0.212

20mM 0.102 -0.102

10mM+10mM 0.246

20mM+20mM 0.117* There was contamination in the control TPA+EG bottle. We need to check what the
  contamination is

Week 5

# Day 23- 26/05/2024

---

* Since there was contamination, we troubleshooted to figure out where it went wrong. In that, we understood that our M9 media, MgSo4, and CaCl2 could be contaminated.

# Day 24 - 27/05/2024

---

* We prepared 1L stock of M9 media and CaCl2 and autoclaved it because we thought the M9 media could be contaminated.

# Day 25 - 28/05/2024

---

* The autoclaved M9 media also had a dead fungus and there was growth in CaCl2.
* Prepared 100ml of M9 media on the suggestion of Nithya Ma’am.
* Everything was done in the LAF and autoclaved.
* After autoclaving, there was no contamination.

### June

Week 6

# Day 26 - 29/05/2024

---

* For the transformation, the LB plates were made (250ml).
* Out of curiosity, we did a gram staining experiment for the bacteria we found in the
  control.
* It says it’s gram-negative.

# Day 27 - 30/05/2024

---

* 50ng plasmids in 50ul of competent cells

Plasmid standard concentration = 8.4 ng/ul

8.4 ng- 1 ul

50 ng - x ul

= 50/8.4

= **5.95 ul of plasmid.**

* LB broth was prepared (100ml)=2.5 gm
* The OD was checked in afternoon was:

+ 20mM TPA+ EG- -0.041
+ 20mM TPA- 0.053
+ 20mM EG- -0.013

* The samples were turbid, but we decided to keep them shaking.
* We talked to Anjali (PhD) from Dr Sabari's lab, who told us that the turbidity could be a problem due to the CaCl2. She suggested we use the dihydrate one and try it once.
* We prepared one more fresh media this time. Everything was autoclaved: flasks, cotton, M9 media, Milli Q, MgSo4, Cacl2, and cotton. The newly prepared media was plugged into cotton.
* The 2:30 OD reading :

+ TPA + EG 20mM- -0.016
+ TPA 20mM- -0.018
+ EG 20mM- -0.013

# Day 28 - 31/05/2024

---

* The new media got soaked up by the cotton, and some beakers were open
* We decided to make the whole media again.
* The OD of the previous media that was running was:

+ TPA + EG 20mM- -0.041
+ TPA 20mM- 0.053
+ EG 20mM- -0.013

# Day 29 - 1/06/2024

---

* At night the OD was checked at 2:30 PM

+ TPA + EG 20mM- -0.016
+ TPA 20mM- -0.018
+ EG 20mM- -0.013

* In the afternoon the OD was checked at 2:30 AM.

+ -TPA + EG 20mM- -0.167
+ -TPA 20mM- 0.020
+ -EG 20mM- -0.008

* The media was prepared in the conical flask at night since the previous one was
  contaminated.

Week 7

# Day 30 - 2/06/2024

---

* The new conical flask culture fell off the beaker because the cotton was not intact.

+ -TPA + EG 20mM- 0.455
+ -TPA 20mM- 2.427
+ -EG 20mM- -0.04

* After the growth, we decided to do a serial dilution of the 20mM TPA as it was so turbid.
* The dilution will be by 1/10

**Calculations:**

13.5 ml - New media

1.5 ml - of Alicot (bacterial culture)

For 13.5 culture media (For the TPA):

M9 - (3/15)\*13.5 = 2.7 ml

MgSo4 - (30\*10^-3/15)\*13.5 = 27 ul

CaCl2 - (1.5/15)\*13.5 = 1.35 ul

Trace = 13.5 ul

TPA- 1.35ml

Water- 9.399ml

* We will streak the bacteria on the agar plates to confirm that the turbidity is due to
  bacteria.

# Day 31 - 3/06/2024

---

* We transformed the plasmids on the spectinomycin plates once again.

# Day 32 - 4/06/2024

---

* E.coli DH5 alpha was needed to make the cells competent.
* The plates with the spectinomycin had no growth and there were so many bubbles in it,
  the possible reason for this is that LB could be less warmer while pouring.
* Allen (PhD) from Dr. Sadanand lab suggested we run the gel and check if the plasmid is
  intact. She also gave a proper protocol for the preparation of competent cells.
* For the gel preparation
0.35 g of agarose

35 ml of autoclaved water

10 ul EtBr

2 ul Ladder (Second well)

80 volts

35 ml of buffer TAE/TBS

50 ng of samples for loading* The OD of culture at 2:30 was

+ -TPA + EG 20mM- 0.080
+ -TPA 20mM- 1.035
+ -EG 20mM- 0.044

# Day 33 - 5/06/2024

---

* The gel was run with all the plasmids by 50 ng of plasmid
  The calculation goes like:
  1) pCDFDuet - 5.95 ul (Total 15 ul loaded)
  2) SaSSy-Leu 2d - 8.1 ul ( 12 ul loaded)
  3) SaSSy-Leu 2d - 7.4 ul (10 ul loaded)
  4) CPR- (-HIS) - 25ul (20 ul loaded)
* The gel was running at 80V for 1:30 hrs.
  The bands were intact for all except the CPR-HIS; the possible reason that the band was
  not visible was mostly because the concentration was low.
* We got the E.coli comp cells from Rohit and stored them at -20°C ( we learnt it the hard
  way) LMAO
* We got E.coli Dh5 Alpha from the SMS lab.
* The comp cells were streaked on the normal LB control and spectinomycin and its
  control.
* Also, the E.coli bacterial cells were streaked in the morning.
* At night, there was no growth in control of both LB and spectinomycin plates
* This result proved that the comp cells were not having any problems.
* We decided to run the experiment again. We got the comp cells again from Rohit.
* From the E.coli bacterial comp cells, there was uniform growth in the plate. We
  inoculated it with kanamycin and ampicillin to check if it was contaminated.
* The plate is parafilmed and kept in 4.
* The OD for the culture was checked.
  Concentration TPA & EG TPA EG
  20mM 0.002 -0.023
  20mM+20mM 0.362
* A stock solution of spectinomycin was prepared:
  0.2 g of spectinomycin
  2 ml of water
* The new spectinomycin plates were prepared.
  - Prepared 50% glycerol
  - Total vol 90ml
  - 45 ml of glycerol in 45 ml of H2O

# Day 34 - 6/06/2024

---

* We transformed the plasmid again, but we tried to use only 1 ng of plasmid this time.
* For the plates- 200 ul of comp cells
  1 ng/ul - 4 plates - 2 Amp, 2 Spec
* Working concentration:
  Spectinomycin - 50 ul
  Ampicillin - 100 ul
* For SaSSy Leu 2d
  Plasmid master mix (10 ul)
  6.4 ul - 1 ul
  1 ng - x
  x\* 6.4 ng/ul = 1 \* 1 ng/ul
  x= 1/ 6.4 ul
  X = 0.162 ul
  For 1 ng/ul:
  10 ng = 1.6 ul of plasmid + 8.4 ul NFW
  Take 1 ul for 50 ul comp cells
* The OD was checked
  Concentration TPA & EG TPA EG
  20mM 0.0031 0.044
  20mM+20mM 0.993
* The serial dilution of the TPA+EG was done at 1/100
  13.5 - New media
  1.5 ml of the bacterial aliquot
* For the spectinomycin (50 ul), ampicillin (100 ul) and control
* The transformation was done, and the primary culture was inoculated from comp cells.

# Day 35 - 7/06/2024

---

* Transformed plates - The growth was there except in normal LB control.
* The primary culture of E.coli was contaminated as there was growth in control
* The fresh inoculation was done in the morning.
* Transformation of SaSSy- Leu2d is done again.
* Comp cells from Dr. Nishant's lab
* Amp plates also from the same lab
* We plated two sets
  ● Ampicillin:
  1) 1 ng/ml
  2) 50 ng/ml
* Controls:
  One with nothing, no antibiotic plates (37°C)
* Comp cell preparation was done and kept.
* Preparation for the media:
  1) Stock solution, stock solution for primary culture.
  2) 0.1 M Cacl2 - 200ml
  3) Primary and secondary culture (LB culture)
  4) Ice buckets

# Day 36 - 8/06/2024

---

* The primary culture for preparing the comp cells went wrong because the OD exceeded
  0.6, which was 1. We have to start again.
* The transformation was done again with the FPPS-SaSSy Leu 2d.

Week 8

# Day 37 - 9/06/2024

---

* We got colonies in our ampicillin plates and no growth in the plates from GIrija
* We streaked the colonies we got in our plates to test our hypothesis.
* And plated it on the plates from Girija
* After 12 hours of incubation, we saw growth in it.
* We did an isolation process of these colonies.
* The values we got are:
  Paarth- 1) ng/ul- 0.045
  2) A260/A280 - 1.85
  3) A260/A230 - 1.41
  Gouri - 1) ng/ul - 0.013
  2) A260/280 - 1.70
  3) A260/A230 - 0.61
  Irin - 1) ng/ul - 0.013
  2) A260/A280 - 1.70
  3) A260/A230 - 0.51
* The values for isolation are not optimum, so we have to do it again.

# Day 38 - 10/06/2024

---

* Team meeting

# Day 39 - 11/06/2024

---

* For transformation by electroporation in E.coli
* Calculations are like:
  SaSSy-FPPS Leu 2d
  6.1 ng/ul\*x = 5 ng/ul \* 2 ul
  X = 5⁄6
  = 1.6 ul
* Add 0.4 NFW
* Electroporation of SaSSy-FPPS Leu 2d
  1) E.coli - Mach1 (100ul)
* Plating in the ampicillin and spectinomycin plates

# Day 40 - 12/06/2024

---

* There was growth in the ampicillin plates in 20ul and 160ul
* There was no growth in the spectinomycin plates in 20ul and 160ul
* The LB plates were also having no growth, showing that there was no contamination
* The E.coli cells were Mach1
* They grow within 3 hrs after primary inoculation
* The restriction digestion experiment
  DNA plasmid - 12.5 ul
  NOT 1 - 0.2 ul
  BamH1-HF - 0.2 ul
  Xhol - 0.2 ul
  Bgl2- 0.2 ul
  Buffer - 2 ml
  Milli Q - 4.7 ul
  Total reaction = 20 ul
* Plasmid isolation values:
  1) ng/ul - 41 ng/ul
  2) A260/A280 - 1.83
  3) A260/A230 - 1.13

# Day 41 - 13/06/2024

---

* Plasmid isolation was done
* We ran gel 1% agarose gel
  15 ul of digested plasmid+5 ul of loading dye
  10 ul of original plasmid + 3 ul of loading dye
  Order :
  1 2 3 4
  5 ul of ladder Digested plasmid Original plasmid 12 ul of ladder
* Intact plasmids were there, but digestion didn't happen
* 2 bands - 1) 9kb
  2) 6.5 kb
* Other bands were so faint we are not sure

# Day 42 - 14/06/2024

---

* Restriction digestion again:
  1) Ng/ul - 146 ng/ul
  2) A260/A280 - 1.88
  3) A260/A230 - 2.18
  1) ng/ul - 148 ng/ul
  2) A260/A280 - 1.90
  3) A260/A230 - 2.19
* For the restriction digestion:
  146 ng/ul
  0.146 ug
  0.146 ug \* x = 1 ug
  X = 6.85 ul
  (NEB ladder 1 kb = 10 ul)
* Restriction enzymes:
  Xho 1 - 20 units/ul
  BamH1 - 20 units/ul
  Not 1 - 10 units/ul
  Bgl2 - 10 units/ul
* Streaking of the Mach1 cells from Dr. Vijay's lab

# Day 43 - 15/06/2024

---

* Preparation of the competent cells of Mach1 cells.

Week 9

# Day 44 - 16/06/2024

---

* Break for a day

# Day 45 - 17/06/2024

---

* Transformation of pCDFduet 4 ul
  1.62- water
  2.38 - plasmid

# Day 46 - 18/06/2024

---

* Transformation was not done with our cells since there was no growth
* But for the Vijay lab cells, good colonies were present which indicated that
  transformation occurred.
* We isolated the new LB and one with the old LB
* The isolation process
  (x ul)
  ng/ul - 42.2
  A260/A280 - 1.88
  A260/A230 - 1.47
  (20 ul )
  ng/ul - 41.9
  A260/A280 - 1.95
  A260/A230 - 2.01
  (280 ul)
  ng/ul - 46.4
  A260/A280 - 1.95
  A260/A230 - 1.60

# Day 47 - 19/06/2024

---

* We discussed the protocol for the Pseudomonas putida electrocomponent cells with Dr.Vijay.
* We streaked the TA7-EG and TA7.

# Day 48 - 20/06/2024

---

* The growth in the plates of P.putida TA7-EG was very stressed
* P.putida TA-7 was also stressed
* The TA7-EG and KT2440 were streaked again.
* 10% glycerol was made
* Kept the autoclaved tips and falcons in the 4°C
* We went to the School of chemistry to learn the procedure for GC-MS

# Day 49 - 21/06/2024

---

* Made glycerol stocks of Mach1

# Day 50 - 22/06/2024

---

* The competent cells were made of TA7-EG and KT2440 and stored at -80°C

Week 10

# Day 51 - 23/06/2024

---

* We discussed the further plans for the wet lab, and some mailing work was done.
* We spoke about the NMR and GCMS in the CSB.

# Day 52 - 24/06/2024

---

* We did a transformation of pCDFduet into P.putida TA7-EG and KT2440.
* We made spectinomycin and streptomycin plates.

# Day 53 - 24/06/2024

---

* We talked to PhDs about identifying the protein in our bacteria, and they suggested we do western blotting and coomassie staining for the detection.
* We checked the plates, and there was no growth, so we thought the pCDF was incompatible with P.putida.
* After some time, there was a slight growth in the plates, so we let them grow a little more to check. At night, we found that there was more growth in the plates.

# Day 54 - 26/06/2024

---

* We isolated the plasmid from the transformed P.putida.
* After getting the results, we decided to do it again.

# Day 55 - 27/06/2024

---

* Outreach

# Day 56 - 28/06/2024

---

* Outreach

# Day 57 - 29/06/2024

---

* Outreach

Week 11

# Day 58 - 30/06/2024

---

* Streaking of the TA7-EG to prepare the comp cells at night

# Day 59 - 1/07/2024

---

* The colonies from streaked plates of TA7-EG were inoculated into LB and after 12 hours a secondary culture was prepared and its OD was measured till it reached 0.5
* The protocol for TA7-EG competent cells was followed and the competent cells were prepared and kept at -80°C

Week 12

# Day 60 - 2/07/2024

---

* Made 250ml of LB agar
* 5 plates of spectinomycin and 5 plates of streptomycin.

# Day 61 - 3/07/2024

---

* The DXS and DXS\_DXR plasmids were transformed into Mach1 cells for amplification.
* The pCDFduet was transformed into the P.putida TA7-EG and KT2440.
* Aliquots for the DXS - Plasmid - 0.133ul
  - Water- 3.86 ul
  DXS\_DXR - plasmid - 0.15
  - water - 3.84 ul

# Day 62 - 4/07/2024

---

* There was a growth in all the plates except in the control for the pCDFDuet.
* DXS\_DXR and DXS showed growth. Their plasmid isolation was done and stored at -20°C
* The primary inoculation was done of TA7-EG and KT2440 to induce SaSSy\_FPPS.
* The LB broth and terrific broth were prepared for induction purposes.

# Day 63 - 5/07/2024

---

* The secondary culture of the KT2440 and TA7-EG was prepared in two different samples - LB and Terrific broth.
* The induction of the pCDFDuet by the IPTG was done at around 4 OD.
* After the induction, the samples were grown for 18 hr.
* Transformation of the DXS\_DXR and DXS in the P.putida TA7-EG and KT2440.
* Due to some confusion, for the P.putida TA7-EG, both DXS\_DXR and DXS were done, but for the KT2440, only DXS\_DXR was used.

# Day 64 - 6/07/2024

---

* The growing P.putida TA7-EG and KT2440 with the pCDFDuet were pelleted down and kept at -80C, and the supernatant was kept at 4°C.
* For the DXS, there was no growth, and DXS\_DXR had good growth.
* We kept the DXS plates in the incubator to see if they grew.
* At night, the set of the DXS\_DXR was kept for primary inoculation to induce the plasmid.

### July

Week 13

# Day 65 - 7/07/2024

---

* We checked the primary culture, but there was no growth since the incubator shut down
* We again prepared the sample for the primary culture.

# Day 66 - 8/07/2024

---

* Secondary inoculation was done for the DXS-DXR in terrific broth and LB.
* The glycerol stock was made and stored at -80C.
* There was no significant growth in the DXS-DXR and DXR culture of P.putida TA7-EG and KT2440.
* We decided to do a fresh primary inoculation again.
* We got the glass beads from Dr. Ravi's lab for GCMS.

# Day 67 - 9/07/2024

---

* 5.8 M HCL was prepared for acid-washed beads.
* The beads were acid-washed and kept for drying.

# Day 68 - 9/07/2024

---

* GCMS was done for the pCDFDuet in the TA7-EG strain and we got the following result

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-13-gcms-pcdf-duet-day-67.jpeg)

# Day 69 - 10/07/2024

---

* To confirm that the peak in the analysis report of GCMS is santalene only, we will give non-transformant bacteria sample of the TA7-EG.
* For the same, we inoculated the TA7-EG from an old plate.

# Day 70 - 11/07/2024

---

* There was no growth in the plates of the TA7-EG.
* Yeast transformed by SaSSy-FPPS, and pure hexane.
* The TA7-EG was streaked from the glycerol stock once again.

# Day 71 - 12/07/2024

---

* The primary inoculation was done for the TA7-EG.
* The glycerol stocks were made for the TA7-EG.

Week 14

# Day 72 - 14/07/2024

---

* The empty vector TA7-EG (not transformed with the pCDFduet) was given for GCMS.
* The GCMS results analysis: There was a peak of 204 m/z in the pCDFDuet TA7-EG report, and there was no peak in the non-transformed for the same retention time.
* The gel was made and preserved.

# Day 73 - 15/07/2024

---

* We need to run the western blot to confirm the result of the GCMS.
* The lysis buffer was made
* Plasmids arrived from the pSEVA collection in the form of agar stabs
* The pSEVA424 was streaked onto LB plates

# Day 74 - 16/07/2024

---

* Sonication was done for both the pellet KT2440 and TA7-EG transformed by pCDFDuet.
* The previous gel got dry, so we must prepare a new one.
* Got the loading dye from Dr Tapas's lab.
* Streaked the pCDFduet TA7-EG and KT2440
* Made plates with Gentamicin, Streptomycin and Kanamycin for the new plasmids for isolation since they were given in agar stabs.
* The primary inoculation for the pSEVA424 was done.

# Day 75 - 17/07/2024

---

* Made M9 media for TPA EG and inoculated the pCDFDuet transformed in it.
* The KT2440 was not grown, so we streaked again.
* The western blot was run with the primary antibody overnight.
* The plasmid isolation was done for pSEVA424.
* The other set was streaked for pSEVA241 and pSEVA631.

# Day 76 - 18/07/2024

---

* A secondary antibody was used for the western blot.
* The blot image showed nothing, as there was a mistake in the preparation of the secondary antibody. For that, our mentor suggested first going with the coomassie experiment and then going for western blotting.
* Since the plasmid isolation values were not good, we inoculated the pSEVA424 with another set of plasmids, pSEVA241 and pSEVA631.
* For the coomassie, we prepared a gel and kept it at 4C.

# Day 77 - 19/07/2024

---

* We made samples of P.putida TA7-EG, KT2440, and two yeast samples to load in the gel. The samples were made in different concentrations.
* The plasmid isolation was done for all the pSEVA424, pSEVA424, pSEVA631.
* There was no growth in the pCDFDuet-transformed P.putida, so we decided to make a control and check the issue.
* After the coomassie gel was done, the bands were very smeared, and the possible reason could be improper denaturation

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-14-day-76-coomassie-yeast-ta7-eg-pcdf-ta7-eg.jpeg)

1.ladder

2. yeast (1)

3. yeast (2)

4. KT2440 1:3 (loading dye:sample)

5. TA7-EG 1:3

6. TA7-EG 1:2

7. KT2440 1:2

8. KT2440 1:1

9. TA7-EG 1:1

# Day 78 - 19/07/2024

---

* To understand the issue, we made a media with glucose as a carbon source.
* Since the gel was very clear, we decided to make another gel and run it for more time, and the gel was made. The samples were denatured correctly and loaded.

Week 15

# Day 79 - 21/07/2024

---

* We understood there was a fungus in the ethylene glycol, so we had to prepare everything again.
* The KT2440 and TA7-EG were inoculated in the glucose.
* The gel was running in the destaining solution as there were a lot of stains on the gel.

# Day 80 - 22/07/2024

---

* We made new plates for the streptomycin.
* There was still no growth in any of the media.
* To keep control, we decided to make a media of just *P.putida* TA7-EG.
* The media was prepared, and the pH was maintained at 7- 7.5.

# Day 81 - 23/07/2024

---

* The inoculation of *P.putuda* TA7-EG was done using an untransformed one in the TPA-EG media.

# Day 82 - 24/07/2024

---

* Checked if there is growth in *P.putida* TA7-EG untransformed and transformed (no growth)

# Day 83 - 25/07/2024

---

* Checked if there is growth in *P.putida* TA7-EG untransformed and transformed (no growth)

# Day 84 - 26/07/2024

---

* Talked to Vijay and Anandhu regarding the plate reader set up

# Day 85 - 27/07/2024

---

* The plate reader was set up but due to some contamination issue the reading was stopped mid-way

Week 16

# Day 86 - 28/07/2024

---

* Set up the plate reader using a new plate
* The growth was measured for 12 hours

# Day 87 - 29/07/2024

---

* No work since everyone went for visa appointment

# Day 88 - 30/07/2024

---

* The growth curve obtained was analysed. As the results were not satisfactory we planned on repeating the experiment again
* After troubleshooting the problem, we realised that the problem maybe due to the M9 media

# Day 89 - 31/07/2024

---

* Further plans regarding the Gibson Assembly and plasmid transformation were made

Week 17

# Day 90 - 01/08/2024

---

* Fresh new M9 media was prepared and autoclaved

# Day 91 - 02/08/2024

---

* New TPA was made and adjusted to pH 7.5
* *P.putida* TA7-EG was inoculated in the newly prepared M9 media and maintained at 7-7.5 pH
* The inoculum was kept at 30 degree celsius

# Day 92 - 03/08/2024

---

* YAY THERE IS GROWTH IN THE MEDIA!!!
* There was little growth in the media and no growth in the controls. We let it grow throughout the day

### August

Week 18

# Day 93 - 04/08/2024

---

* Kept *P.putida* TA7-EG growing in the incubator and the turbidity was observed every few hours

# Day 94 - 05/08/2024

---

* Kept *P.putida* TA7-EG in plate reader at around 1pm to obtain the growth curve
* We kept the plate reader running for around 8 hours and the following growth curve was observed

![](https://static.igem.wiki/teams/5181/notebook/week18-day93-growth-curve-ta7-eg-in-tpa-eg-tpa-eg.jpg)

* We talked with Aswathy regarding Gibson Assembly and set a time to do the experiment

# Day 95 - 06/08/2024

---

* Completed Gibson Assembly for pRGPDuo3 with the assistance of Aswathy and transformed it into *P.putida* TA7-EG
* As our previous growth curve was not satisfactory we contacted Dr.Oliver Branderberg to help us figure out why the P.putida TA7-EG is not growing in M9 media. He mentioned that the strain struggles to switch from LB to minimal media and that if you grow the bacteria in LB till it reaches the stationary phase and suddenly switch it to minimal media it may not grow. He suggested us the following method to grow the strain

  - Grow P.putida TA7-EG LB overnight

  - Dilute it again in fresh LB in the morning till it reached 0.1 OD and let it grow for another 2 hours so that the cells are active

  -Then inoculate in M9 media
* Along with this, we came up with another plan to grow the bacteria in M9 media continuing TPA+EG+5% glucose and subsequently decrease the concentration of glucose so that the bacteria gets adapted to growing in TPA and EG

# Day 96 - 07/08/2024

---

* We started growing P.putida TA7-EG in minimal media containing TPA, EG and 5% glucose. By night, the media was turbid so we diluted it to a media containing TPA, EG and 4% glucose
* Side-by-side we inoculated P.putida TA7-EG in LB and let it grow overnight
* We transformed E.coli Mach1 with pRGPDuo3 and let it grow overnight in gentamicin plates

# Day 97 - 08/08/2024

---

* We serial diluted the *P.putida* TA7-EG growing in minimal media containing TPA, EG and 4% glucose to another M9 media with TPA,EG and 3% glucose
* We noticed turbidity at night and hence further diluted it to a media with 2% glucose
* The *P.putida* TA7-EG which was grown in LB was diluted to a fresh media containing LB. It was allowed to grow for 2 more hours after its OD reached 0.1, The bacteria was then inoculated in M9 media.
* No growth was observed in the plates. After troubleshooting with the dry lab team we came to know that the ori region has a problem and hence the plasmid will not replicate in the host

# Day 98 - 09/08/2024

---

* We serial diluted the *P.putida* TA7-EG growing in minimal media containing TPA, EG and 2% glucose to another M9 media with TPA,EG and 1% glucose
* We began growing *P.putida* TA7-EG transformed with pCDFDuet in M9 media containing TPA, EG and 5% glucose. At night we noticed some turbidity hence we diluted it to another media with 4% glucose

# Day 99 - 10/08/2024

---

* We serial diluted the *P.putida* TA7-EG growing in minimal media containing TPA, EG and 1% glucose to another M9 media with only TPA and EG
* We serial diluted *P.putida* TA7-EG transformed with pCDFDuet in M9 media containing TPA, EG and 4% glucose. At night we noticed some turbidity hence we diluted it to another media with 3% glucose

Week 19

# Day 100 - 11/08/2024

---

* We serial diluted the *P.putida* TA7-EG growing in minimal media containing TPA and EG to another M9 media
* We serial diluted *P.putida* TA7-EG transformed with pCDFDuet in M9 media containing TPA, EG and 3% glucose. At night we noticed some turbidity hence we diluted it to another media with 2% glucose

# Day 101 - 12/08/2024

---

* In the morning we realised that there was some contamination in the control. We decided to repeat the experiment with more care

# Day 102 - 13/08/2024

---

* Our cultures spilled inside the shaker incubator. The experiments will have to be conducted again

# Day 103 - 14/08/2024

---

* A new M9 media containing 5% glucose, TPA and EG was prepared and inoculated with *P.putida* TA7-EG
* We decided to do the restriction digestion of pSEVA241 using EcorI and HindIII. 50ul reaction was prepared using the following values
Plasmid (pSEVA241)- 11.44ul

EcoRI- 0.67ul

HindIII- 0.67ul

NFW- 32.22ul

2.1 buffer- 5ul* The reaction was kept at 37 degrees celsius for 1 hours before performing gel electrophoresis using 1% agarose gel

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-19-day-102-13-08-2024-gel.jpeg)

1. Ladder

2. digested pSEVA241* As per our plasmids only one band should be visible since the 56bp fragment should have ran out of the gel due to its very small size. But our results suggest otherwise indicating that the digestion did not occur properly
* A standard gel- clean up protocol was followed. Nanodrop was done to check the concentration of plasmid. It showed really poor concentration
* We decided to repeat the experiment the next day

# Day 104 - 15/08/2024

---

* The 5% glucose M9 media was diluted to another M9 media containing 4% glucose, TPA and EG
* In the morning another restriction digestion of pSEVA241 was conducted

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-19-day-103-14-08-2024-gel.jpeg)

1. ladder

2. digested pSEVA241

3. undigested pSEVA241* The gel shows a very light band indicating a very low plasmid concentration
* We did the gel clean-up process and the nanodrop results showed poor values again
* Upon discussing with our lab technician we came to know that one of the buffers used in the gel clean up was not mixed with ethanol.
* We decided to do the restriction digestion followed by gel clean up again.
* Upon getting poor results again we talked with Dr. B M N Ramesh from Dr.Ravi’s lab. He suggested the following points

+ Use a bigger well so that we can load a larger quantity of the reaction mixture
+ Use 2ng of plasmid instead of 1ng

# Day 105 - 16/08/2024

---

* The 4% glucose M9 media was diluted to another M9 media containing 3% glucose, TPA and EG. This time 50ul of streptomycin was added to the media to ensure only our bacteria will be grown in the media
* We decided to do the digestion of both pSEVA241 and pSEVA424 in 50ul reaction but with 2ng of plasmid. The calculations were as follows:

+ Plasmid (pSEVA241)- 22.88ul
+ EcoRI- 1.33ul
+ HindIII- 1.33u
+ NFW- 19.46u
+ 2.1 buffer- 5u

+ Plasmid (pSEVA424)- 28ul
+ EcoRI- 1.33ul
+ HindIII- 1.33ul
+ NFW- 14.34ul
+ 2.1 buffer- 5ul

|  | pSEVA241 | pSEVA424 |
| --- | --- | --- |
| Plasmid | 22.88ul | 28ul |
| Ecor1 | 1.33ul | 1.33ul |
| HindIII | 1.33ul | 1.33ul |
| 2.1 Buffer | 5ul | 5ul |
| NFW | 19.46ul | 14.34ul |

* After talking with Vaishakh from Dr.Ravi’s lab we came to know that we were supposed to load the entire 50ul reaction into the well and incubate it at 37 degrees celsius for 3-6 hours. Until now we were only loading 7.5ul of the reaction and incuabting it for only 1-2 hours
* We ran the gel and got good results

# Day 106 - 17/08/2024

---

* We decided to stop the Oliver plan since the glucose dilution method worked
* We diluted the M9 media containing 3% glucose to another media with 1% glucose

Week 20

# Day 107 - 18/08/2024

---

* Induced the P.putida TA7-EG transformed with pCDFDuet growing in 50ml M9 media

# Day 108 - 19/08/2024

---

* We pelleted the induced bacteria and stored it at -80 degrees.
* We made a new 250ml culture of P.putida TA7-EG- pCDFDuet and induced it at 11pm
* Steamed  *E.coli* Mach1 in LB plates to make competent cells

# Day 109 - 20/08/2024

---

* The 250ml culture broke:((
* We decided to use the previously pelleted bacteria for western blot and GCMS
* We Gibson assembled the SaSSy-FPPS fragments with pSEVA241 and CPR-P450 with pSEVA424
* We transformed the assembled plasmids into E.coli Mach1 cells and kept them for growing overnight (pSEVA241 in kanamycin plates and pSEVA424 in streptomycin plates)
* We inoculated the previously streaked E.coli Mach1 in LB and left them growing overnight

# Day 110 - 21/08/2024

---

* The assembled plasmids did not grow in the kanamycin and streptomycin plates. Instead there was some contamination in the plates. We decided to make new plates.
* We treated the *P.putida* TA7-EG (transformed with pCDFDuet) pellets with cell lysis followed by sonication. Then the santalene was purified using Ni2+-NTA.
* SDS-PAGE was conducted and was kept for staining overnight
* PCR was conducted for amplifying the Gibson assembled SaSSY-FFPS fragments using the following calculations:
* Gel electrophoresis was conducted for the PCR fragments. We did not get any fragments indicating that something went wrong with either the Gibson assembly or PCR. After troubleshooting we realised that the issue could be due to the fact that we had made four 25ul vials (each vial for different temperatures, 68 °C, 70 °C, 72 °C and 74 °C of the reaction mixture). In a 25ul reaction, the amount of DNA polymerase and primers is very low and we thought that maybe the required amount is not added correctly due to some pipetting error.
* We checked the OD of the PCR fragments. The concentration of the DNA is very low indicating that PCR did not occur properly
* *E.coli* Mach1 was inoculated in LB and kept growing overnight. This is for making competent cells

# Day 111 - 22/08/2024

---

* After destaining the SDS-PAGE gel we got the following results

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/pcdf-coomasie-23-08-24.jpg)

1. ladder

2. Flow through

3. Eluted protein* The marked band corresponds to SaSSy. From this we concluded that pCDFDuet is working in P.putida TA7-EG YAYYYYYYYYYY!!!!
* After yesterday’s troubleshooting, we made a 100ul PCR reaction and divided it into four 25ul vials and kept them for PCR at different temperatures in order to find the optimal annealing temperature

# Day 112 - 23/08/2024

---

* The *E.coli* was inoculated into secondary culture and the OD was checked at regular intervals. Once it reached 0.5 it was kept in ice and a standard protocol for making competent cells was followed
* To check the competency of our E.coli Mach1 we transformed the cells with pCDFDuet and it was kept for growing overnight Before conducting another PCR we decided to run a gel for the previously Gibson assembled and we got the following results

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-20-day-111-22-08-2024-gel.jpeg)

1. Ladder

2. Gibson assembled fragments

3. Diluted Gibson assembled fragments (2 fragments+ 8ul NFW)* We got a band at 4kb which means GIBSON ASSEMBLY WORKED YAYYYYYY!!! From this we came to the conclusion that the PCR is not occurring properly. Also there are 3 other bands which means only a small fraction of the fragments are getting assembled. We decided to perform Gibson assembly again
* During the preparation of the PCR reaction vials we had diluted the 2ul of the Gibson assembled fragments in 8ul of NFW and used that to make a 100ul reaction. Further we divided the reaction mixture into 4 vials containing 25ul each. This may have led to a very small amount of template in the reaction mixture and hence the failed PCR

# Day 113 - 24/08/2024

---

* After talking about PCR to Vaishakh he suggested we should make an entire 20ul reaction instead of dividing the 100ul reaction mixture into four vials. He also suggested we always let a reagent fully thaw before using it in order to get even concentration.
* We also assembled the SaSSY-FPPS fragments again using Gibson Assembly
* After taking a note of his suggestions we conducted another PCR experiment and GOT BANDS!!!! (staying up till 6am was definitely worth it)

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-20-day-112-23-08-2024.jpeg)

1. Ladder

2. PCR at 65 °C

3. PCR at 64 °C

4. PCR at 68 °C

5. Old Gibson assembled fragment at 68 °C* From this result we optimised the PCR protocol and also came to know that our GIbson Assembly is working properly

Week 21

# Day 114 - 25/08/2024

---

* We assembled CPR-P450 fragments using Gibson Assembly and kept them for PCR and got results AGAINNNN!!!

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-20-day-113-24-08-2024.jpeg)

1. Ladder

2. 64°C with DMSO (no band)

3. 67°C with DMSO

4. 67°C without DMSO

5. 68°C without DMSO

6. Diluted CPR-P450 Gibson assembled* From this result we came to the conclusion that 64°C is not an optimum temperature for annealing

# Day 115 - 26/08/2024

---

* We got quite low values for nanodrop of the PCR product so we decided to PCR the fragments again

# Day 116 - 27/08/2024

---

* The nanodrop value of the second PCR value became even lower. We thought something was going wrong during the PCR clean-up
* We decided to do the PCR all over again

# Day 117 - 28/08/2024

---

* We kept for PCR of CPR-P450 Gibson assembled fragments again
* The nanodrop values came slightly better (but still low for a PCR product) but we still decided to continue with it since it was sufficient enough for restriction digestion
* We kept Mach1 transformed with pSEVA631 and pSEVA241 to grow overnight for plasmid isolation

# Day 118 - 29/08/2024

---

* We isolated the plasmids pSEVA631 and pSEVA241. The nanodrop values for pSEVA241 was good but the values for pSEVA631 was low. When we had done the plasmid isolation of pSEVA631 before we got similar low values suggesting that the plasmid is not replicating properly within the bacteria
* The restriction digestion of the CPR-P450 fragment and pSEVA241 was performed using EcorI-HF and HindIII in 2.1 buffer. The digestion was done for almost 6 hours
* Clean up of the restriction digestion products was done and they were kept for ligation overnight at 16°C using T4 DNA ligase

# Day 119 - 30/08/2024

---

* A gel was run for the ligated product to see if the fragments ligated correctly

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-21-day-118-29-08-2024.jpeg)

1. Ladder
2. Ligated pSEVA241-CPR-P450* A very faint smear was observed. We were not sure if this meant the plasmid did not get assembled. After talking to Vaishakh from Dr.Ravi’s lab, he suggested that we should still try to transform and see if we get colonies
* We transformed Mach1 cells with the ligated plasmid, streaked them on Kanamycin plates and let then grow overnight

# Day 120 - 31/08/2024

---

* In the morning we observed a large number of colonies. This did not confirm whether our plasmid has been assembled or not since there is a chance that the pSEVA241 backbone got reassembled on its own
* In order to confirm whether our plasmid has been ligated or not, we isolated the plasmid and ran a gel

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-21-day-119-30-08-2024.jpeg)

1 and 2) Ladder

3) Ligated pSEVA241-CPR-P450

4) Isolated pSEVA241-CPR-P450 from Mach1* In the fourth lane there is a band above 10kb and there is a bright band at 4kb. We were not sure as to why we got these results. The ladder also looks like it has not run properly. We assumed there was some issue with the TBE buffer. Hence we decided to make a new one and run the gel again

### September

Week 22

# Day 121 - 01/09/2024

---

* We kept the isolated pSEVA241-CPR-P450 for single restriction digestion using EcoR1 so that we can confirm whether the plasmid has been ligated or not (we should get a single band around 7kb)
* We ran the gel and got the following results

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-21-day-120-31-08-2024.jpeg)

1) Ladder

2)Isolated pSEVA241-CPR-P450

3) to 5) Single digested plasmid

* The lanes 3 to 5 suggest that the single digested vector corresponds to 4kb. Since our plasmid should be around 7kb, there should be a band at 7kb after single restriction digestion. This indicated that the plasmid did not get ligated properly
* We also decided to ligate pSEVA631 with SaSSy-FPPS insert. The vector and the insert was digested using EcoRI-HF and HindIII
* At night it was kept for ligation at 16°C

# Day 122 - 02/09/2024

---

* We ran a gel for pSEVA241-CPR-P450 again just to get a final confirmation that it has not been ligated. Instead of doing only single digestion, we decided to do a double digestion using EcoRI and XhoI

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-22-day-121-01-09-2024.jpeg)

1. Ladder

2. Isolated plasmid

3. Double digested plasmid* After running the gel, we understood that the plasmid has not been ligated. After digestion of the vector pSEVA241, we did clean up instead of running a gel and eluting it from there. In the case of just clean-up the 56 bp MCS region would not have been removed and hence during ligation it must have got re-ligated with the vector backbone.
* We decided that the whole ligation process had to be repeated from the beginning. Hence we PCR amplified the CPR-P450 fragments and SaSSy-FPPS fragment (just for backup in case the ligation did not work for pSEVA631
* We transformed the pSEVA-SaSSy-FPPS plasmid into E.coli Mach1 at night

# Day 123 - 03/09/2024(PAARTH’S BIRTHDAY- he made Kalyani cry)

---

* We got colonies in the transformed Mach1 cells. We isolated them and transformed them into *P.putida* TA7-EG
* We talked to Rito regarding and he suggested we extract our required band from the gel instead of doing clean-up right after restriction digestion
* We did a single and double restriction digestion of the pSEVA631-SaSSy-FPPS plasmid for 6 hours
* PCR was done for CPR-P450 and SaSSy-FPPS fragments at night

# Day 124 - 04/09/2024

---

* Ran a gel for the PCR amplified fragments and the single and double digested.

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-22-day-123-03-09-2024.jpeg)

1) and 2) PCR-amplified CPR-P450

3)Ladder

4) and 5) PCR-amplified SaSSy-FPPS

6) Double digestion

7) Single digestion* From the gel results, we understood that the fragments have been PCR amplified. Also, there is a high chance the pSEVA631-SaSSy-FPPS has been assembled. We kept the restriction digestion running for 6 hours which may have caused the smear
* We did gel cleanup of both the PCR amplified fragments and got poor concentration. Hence we decided to repeat the PCR amplification but just for the CPR-P450 fragment since we already think the pSEVA631-SaSSy-FPPS plasmid has been assembled
* After the second PCR we got poor nanodrop results again. Hence we decided to put four 20ul reactions for PCR at night

# Day 125 - 05/09/2024

---

* We got colonies for the *P.putida* TA7-EG transformed with pSEVA631-SaSSy-FPPS YAYYYYYYYY!!! Isolation of the plasmid was done and kept for single digestion using EcoRI-HF
* After pooling together the four vials and doing PCR clean-up, we got good nanodrop values. Hence we kept pSEVA241 and the amplified CPR-P450 fragments for restriction digestion using EcoRI-HF and HindIII.
* After 3-4 hours we ran a gel but since the casting tray was not levelled a gel was not uniform and
* formed a huge bump in the middle. Hence we got the following results results \*crying\*

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-22-day-124-04-09-2024.jpeg)

# Day 126 - 06/09/2024

---

* We decided to do restriction digestion again since we had loaded the entire 20ul yesterday for the gel
* We ran the gel and we got confirmation that our pSEVA631-SaSSy-FPPS has been assembled YAYYYYYYYY!!!!!

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-22-day-125-05-09-2024.jpeg)

1. pSEVA241 digested

2. Ladder

3. Digested pSEVA631-SaSSy-FPPS* There is a sharp band in the third lane at around 7kb which is the same size as our assembled plasmid
* Something went wrong while digesting the pSEVA241 plasmid since there is no sign of plasmid in the first lane (not even a smear). Hence we decided to digest the plasmid again
* We made two 20ul reactions for restriction digestion of pSEVA241. For one of the vials, we directly did clean up whereas for the other one, we ran a gel and extracted the vector band from it.
* For ligation we made a 1:1 and 1:3 (vector to insert) reaction and kept it for ligation overnight at 16°C

# Day 127 - 07/09/2024

---

* We transformed the plasmids from yesterday’s ligation into Mach1 competent cells.
* We tried to run a gel for the ligated plasmids but since the concentration was really low we could not see any bands

Week 23

# Day 128 - 08/09/2024

---

* We got colonies for the Mach1 cells transformed with both the 1:3 and 1:1 plasmids
* The colonies were inoculated in the morning and they were isolated at night

# Day 129 - 09/09/2024

---

* We digested the isolated pSEVA241-CPR-P450 plasmids using EcorI for 4 hours

# Day 130 - 10/09/2024

---

* We decided to do colony PCR using a few colonies on the 1:3 ligated plasmids since it had lesser number of colonies
* We ran 6 colony PCR using a PCR master mix. We kept a ligated pSEVA631-SaSSy-FPPS plasmid as a control. After that we ran a gel and there were no bands even for the control indicating that something went wrong during the process. Since we had not previously used the PCR master mix, we do not have a standardised protocol for ourselves, This could be a reason as to why the colony PCR did not work
* Simultaneously we ran a gel for the previously digested pSEVA241-CPR-P450 plasmids. We got a band at 4kb instead of 7kb suggesting that our plasmid has not been ligated (INTERNALLY DYING)
* We inoculated more colonies from the 1:3 and 1:1 and decided to do isolation of both of them separate

# Day 131 - 11/09/2024

---

* We isolated plasmids from the previously inoculated colonies
* We did a double and single digestion of pSEVA241 ligated (1:3 and 1:1)and a double digestion of pSEVA631 ligated
* We had not added enough TAE buffer on the gel before loading and hence the wells got mixed up while loading. So the gel was very difficult to read but we got the following results: The single and double-digested pSEVA241 ligated gave a band at 4kb suggesting it did not get ligated. Due to the mix-up of wells, we could not get a proper results for the double-digested pSEVA631 ligated
* We also kept TPA+EG, TPA and EG media inoculated with *P.putida* TA7-EG for growth curve overnight
* We kept the *P.putida* TA7-EG transformed with pSEVA631-SaSSy-FPPS for induction in both M9 media (TPA+EG) and LB. Both were induced using L-arabinose. It was kept growing for 16 hours at 20°C

# Day 132 - 12/09/2024

---

* There was no growth in the media we kept for the growth curve. We assumed it was because the temperature of the TECAN had not reached 30°C
* We troubleshooted the ligation part. We talked to PhDs and Dr.Nishana and they suggested that there is a high chance that two digested vectors in the reaction mixture may get ligated. Hence they suggested to do dephosphorylation of the vectors after restriction digestion
* We decided to start from the beginning by doing PCR amplification of the CPR-P450 insert. We restriction digested both the insert and the vector backbone pSEVA241. We ran a gel for the vector backbone but there was no sign of DNA in the well. It was completely blank. We weren't able to understand the reason behind this. We assumed the plasmid got degraded so we decided to inoculate new pSEVA241 colonies
* We did a double digestion of pSEVA631 ligated to confirm its ligation

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-23-day-131-1-09-2024.jpeg)

1. Ladder

2. Double Digested pSEVA631-SaSSy-FPPS* From this, we concluded that pSEVA631-SaSSy-FPPS has been ligated
* The induced culture was pelleted and stored at -80°C

# Day 133 - 13/09/2024

---

* There was no growth in the inoculation of pSEVA241 kept last night.
* We decided to temporarily stop using pSEVA241 and use pSEVA631 for CPR-P450 since we know that the pSEVA631 backbone is working
* Hence we kept the pSEVA631 backbone for digestion. We decided to use the CPR-P450 insert which we had digested last night
* We kept for ligation of pSEVA631 and CPR-P450 at 16°C overnight
* The pelleted cells induced using 0.2% arabinose P.putida TA7-EG were sonicated, lysed and stored at -20°C. Glass beads were used to lyse some of the pelleted cells in order to do GCMS

# Day 134 - 14/09/2024

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* We transformed the ligated pSEVA631-CPR-P450 into Mach1 comp cells
* We ran a gel of the ligated mixture

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-23-day-133-13-09-2024.jpeg)

1. Ladder

2. Ligated pSEVA631-CPR-P450* There is a smear till 8kb and our ligated product is around 7.6kb. The band at 4kb must be the insert since had added a 1:3 ratio of vector to insert
* We transformed the ligated product into Mach1 cells
* The lysed cell of *P.putida* TA7-EG transformed with pSEVA631-SaSSy-FPPS was purified using Ni2+-NTA column. SDS-PAGE was conducted and kept for staining overnight.

Week 24

# Day 135 - 15/09/2024(IRIN’S BIRTHDAY - oops we forgot!)

---

* There were colonies for Mach1 transformed with 631-CPR-P450. We isolated the colonies, double-digested the plasmid and ran a gel. However we got a smear indicating that the plasmid most probably got degraded or there was no plasmid at all.
* The SDS gel was kept for destaining. However the purification process did not happen properly. However two bands were seen at 65kDa and 45kDa which corresponds to SaSSy and FPPS proteins. We decided to repeat the experiment with different concentrations of arabinose
* Since our ligation did not work we decided to ligate CPR-P450 to pSEVA424, pSEVA241and pSEVA631 using Gibson assembly

# Day 136 - 16/09/2024

---

* We inoculated pSEVA241/424/631
* We did not get growth in the inoculation. Since the plates we used were quite old, we decided to streak fresh plates using the agar stabs we had received

# Day 137 - 17/09/2024

---

* All the plates had growth except kanamycin plates with pSEVA241.Hence we decided to drop pSEVA241 and focus only on pSEVA424/631
* We inoculated pSEVA424/pSEVA631 and left them growing overnight

# Day 138 - 18/09/2024

---

* We isolated pSEVA424/631 and restriction digested them
* We ran a gel and gel eluted them
* The concentration of pSEVA424/631 we got was very low and was not enough for GIbson assembly
* Hence we decided to do multiple restriction digestion and pool them together

# Day 139 - 19/09/2024

---

* The destaining was complete but we did not get a clear band at our desired size. This may be since the concentration of arabinose used was not enough. We decided to induce the transformed bacteria using multiple concentrations of arabinose
* Not much work was done because of mid-sem exams

# Day 140 - 20/09/2024

---

* We inoculated pSEVA424/631
* Not much work was done because of mid-sem exams

# Day 141 - 21/09/2024

---

* There was no growth in the inoculated pSEVA424 but there was growth in pSEVA631
* pSEVA631 was isolated and stored

Week 25

# Day 142 - 22/09/2024

---

* We conducted multiple restriction digestion of pSEVA631, ran a gel, eluted and pooled them together. We got a higher concentration of the digested plasmid which was enough for Gibson assembly
* We used a previously double-digested pSEVA424 to conduct Gibson assembly
* We made a 20ul Gibson reaction and kept them for a 1 hour incubation

# Day 143 - 23/09/2024

---

* We transformed the Gibson assembled fragments into Mach1 comp cells and let them grow for 8-12 hours
* We got two colonies in the pSEVA631 plates. We inoculated them in LB+Gentamicin and let them grow overnight. There was no growth in pSEVA424 plates

# Day 144 - 24/09/2024

---

* There was growth in the inoculated pSEVA631 Gibson assembled plasmid
* We isolated them, did double restriction digestion, and ran a gel.

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-25-day-143-23-09-2024.jpeg)

1. Ladder
2. Double digested pSEVA631 Gibson assembled* We did not get a band at the desired length. There is a faint band near 8kb. Since the gel did not run fully it is quite difficult to find the exact size
* *P.putida* TA7-EG SaSSy FPPS primary culture was grown in LB

# Day 145 - 25/09/2024

---

* The secondary culture was induced using 0.7% and 1.5% arabinose and was allowed to grow for 16 hours
* We transformed the previously ligated pSEVA631-CPR-P450 and Gibson assembled pSEVA631-CPR-P450 into Mach1 comp cells

# Day 146 - 26/09/2024

---

* The induced cells were pelleted. The cells were lysed using both sonication and glass beads. The glass beads lysed cells were sent for GCMS analysis. The sonicated lysate was further purified using Ni-NTA
* There was no growth in the transformed plates. We still left them to grow in the incubator

# Day 147 - 27/09/2024

---

* SDS-PAGE was run for the purified protein. It was kept for staining overnight
* The GCMS results showed that santalene is present in the cell lysate YAYYAYAYAYAYAYAYYAYAYA. OUR PLASMID IS WORKING

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-25-day-147-26-09-2024-gcms1.jpeg)

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-25-day-147-26-09-2024-gcms2.jpeg)

* The internal santalene standard produced peaks at 204 m/z in the chromatogram. A review of the MS spectrum revealed a distinct pattern, confirming the presence of santalene.

# Day 148 - 28/09/2024

---

* SDS gel was kept for destaining and got the following results

![](https://static.igem.wiki/teams/5181/notebook-updates-photos/week-25-day-148-26-09-2024.jpeg)

* We got colonies in the pSEVA631-CPR-P450 plates we had transformed a few days back. Since it took so long to grow we weren't sure whether it is our transformed bacteria. Regardless we decided to inoculate it and isolate the plasmid

Week 26

# Day 149 - 29/09/2024

---

* We isolated plasmid for the inoculation we kept last night. We ran a gel for the undigested plasmid. We got a smear until 10kb which indicates that the plasmid got degraded :((

# Day 150 - 30/09/2024

---

* Wiki documentation work

# Day 151 - 01/10/2024

---

* Wiki documentation work

# Day 152 - 02/10/2024

---

* Wiki documentation work

***Through all ups and downs, the one constant was “our magic book”. Until next time, signing off***

-Shrushti, Gouri, Irin, Paarth

{% endblock %}

{% extends "layoutother.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

# Engineering Success

## Contents

* [Overview](#overview)
* [Engineering Cycle 1](#Cycle_1)
* [Engineering Cycle 2](#Cycle_2)
* [Engineering Cycle 3](#Cycle_3)
* [Engineering Cycle 4](#Cycle_4)

# Overview

---

As seen in our extensive [Design](./design), our project is intricately woven with engineering principles in biology. From the use and characterisation of modified strains that can take up TPA (Terephthalic Acid) and EG (Ethylene Glycol) to the Assembly and Expression of Gene Constructs associated with the Sandalwood Oil Pathway, our experimental core is firmly embedded within the Design, Build, Test, Learn Framework. Using this framework, we navigate the complexities and challenges associated with our experimental goals.

![](https://static.igem.wiki/teams/5181/engineering/cycle.png)

Primarily, designing through a thorough literature review, we narrowed down the specificity of our  ***P. putida* KT2440** and its ability to sustain on PET monomers. Guided by expert insights and informed by our human practices research, we selected  ***P. putida* TA7-EG** as our primary chassis. We then worked on characterising this strain's performance in different media formulations where TPA and EG served as the sole nutrient sources to understand its capabilities in these conditions fully.

To optimize sandalwood oil production, initially, we evaluated the pCDFDuet system, which was previously studied in *E.coli* and *S. cerevisiae* but encountered low santalene abundance in our  ***P. putida*** chassis. This led us to **redesign our approach**, crafting custom constructs for SaSSy-FPPS and Cytochrome P450-CPR. We assembled our gene fragments using Gibson Assembly for both and integrated these designs into pSEVA631 and pSEVA241 plasmids through either conventional ligation or assembly. This completed plasmid was transferred into our chassis via electroporation. Recognising the unique challenges of expressing P450-CPR in bacteria, we developed multiple strategies, including truncated P450 variants and P450-CPR fusion proteins. Meticulously documented on our parts page, these constructs bring our molecular biology efforts to be open and shared with the global research community

We **harnessed advanced analytical techniques** and **mathematical modelling**  to refine our bioengineering approach in our final engineering cycles. Using GC-MS analysis, we quantified the production of santalol and santalene, our key sandalwood oil components, validating our genetic constructs and design strategies. Simultaneously, through our [Drylab](./model) work, we leveraged the mathematical modelling technique FBA (Flux Balance Analysis) to simulate the biochemical pathways of our modified *P. putida* TA7-EG *in silico* and provided valuable insights into optimal flux distributions, guiding targeted genetic modifications and culture condition adjustments. The FBA results inform our current wet-lab experiment and offer a roadmap for future teams, providing a clear next step for maximising the efficiency of the engineered system.

Iteration/ Engineering Cycle 1

# Strain Making vs Strain Obtaining

### Introduction

This initial phase of the project set the stage for our first engineering cycle, where we grappled with the critical decision of strain development versus obtaining existing one, a process deeply informed by our Integrated Human Practices (iHP) to ensure alignment with real-world applications. We carefully weighed the pros and cons of creating custom strains versus obtaining existing ones. During the ideation, we found a paper in which the ***Pseudomonas putida*** KT2440 could grow on ethylene glycol, one of the PET monomers, through laboratory evolution. Through the literature review, we learned that ***Pseudomonas putida*** KT2440 has a β-ketoadipate pathway, which has an aromatic acid catabolite of protocatechuate (PCA) [1], but the ***P. putida*** can’t convert the TPA into PCA naturally. To convert the TPA into PCA, an enzyme cascade must transform the *tphII* operon from the ***Comamonas sp.***, the multi-subunit enzyme TphAII, which converts TA to 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD), and the enzyme TphBII, which converts DCD to PCA, are encoded in the *Pseudomonas putida* KT2440[2].

**We obtained the wild-type strain from Dr Phale from IIT Bombay to start with our approach. To obtain more insight into this idea, we discussed engineering the strain with Dr Oliver Brandenburg to enable terephthalic acid and ethylene glycol uptake. He reviewed our proposed timeline and concluded that engineering a new organism from scratch alongside developing the santalol pathway was too ambitious within the given timeframe. Instead, he recommended leveraging his existing engineered strains of ***Pseudomonas putida*** KT2440, which could take up both TPA and EG monomers, [TA7-EG strain](https://2024.igem.wiki/iiser-tvm/human-practices).**

### The DBTL CYCLE

**Incorporating this adapted design into our project**, we sought to characterize the growth of TA7-EG strain on terephthalic acid (TPA), ethylene glycol (EG), and a combination of both substrates in minimal M9 media. However, during the course of our experiments, **we encountered several challenges related to the composition of the M9 media and the extended growth time of the strain.**

**One of the primary difficulties we faced was growing the strain on the M9 media**. A major obstacle was the precipitation of calcium salts in the media, caused by both CaCl2 and MgSO4. This issue was further exacerbated by the pH changes resulting from adding TPA and EG to the media. Another challenge was the low solubility of TPA at neutral pH, which required careful optimization to ensure that sufficient amounts of the substrate were available for bacterial growth without significantly altering the media pH and causing calcium salt precipitation.

To address this issue, **we fine-tuned the concentrations of the components of the media and monitored the pH at all times to balance TPA dissolution and ensure no precipitate formation**. We also made sure to add the components just before further experiments to ensure the whole media was prepared each time a new and to avoid pH and precipitate issues. **With all these challenges faced, we were able to successfully grow the *P. putida* TA7-EG strain on the different substrates, one such is shown below**.

![](https://static.igem.wiki/teams/5181/engineering/engg-cycle1-culturetpa.jpg)

Fig.1.: The successful culturing of *P. putida* TA7-EG in M9 media with TPA (shown in image).

**The second challenge we encountered during the characterization of the *P. putida* TA7-EG strain was its slow growth rate on PET monomers**. Upon receiving the strain from Dr Oliver, we observed that the bacteria required 24-36 hours to achieve substantial growth on terephthalic acid (TPA) and ethylene glycol (EG), which was not optimal for real-world applications. **We consulted with our primary PI, Dr Vijay and Dr Yashraj, to address this issue and designed an experimental plan to improve the strain's growth rate**.

**The approach we built involved creating a series of dilutions of the bacteria in fresh media, supplemented with varying glucose concentrations alongside the PET monomers**. Based on our PI's suggestion, we started with a medium containing 5% glucose in addition to TPA and EG and gradually reduced the glucose concentration in subsequent dilutions until the media contained only TPA, EG, or a combination of both. This adaptive evolution strategy aimed to progressively acclimatise the strain to grow solely on the PET monomers.

After following the above plan, **we tested the growth curves of the adapted strain in different media compositions (TPA, EG, and TPA+EG) by measuring the optical density (OD)** using a microplate reader. The results showed that the adapted strain reached the logarithmic growth phase faster than the original TA7-EG strain, which took over 2 days. Notably, the highest growth rate was comparable in the medium containing TPA and EG, except EG, which was slower.

![](https://static.igem.wiki/teams/5181/results/untransformed-growth-curve.jpg)

Fig.2.: Growth Curve of TA7-EG in M9 minimal media, with 20mM of each TPA and EG.

**This result enabled us to learn that the adaptive evolution approach successfully improved the growth characteristics of the *P.putida* TA7-EG strain on PET monomers**, bringing us one step closer to developing an efficient bacterial platform for PET bioconversion.

Iteration/ Engineering Cycle 2

# SaSSy-FPPS Expression Testing

### Introduction

**Our second engineering cycle focused on testing the expression of SaSSy-FPPS enzymes, which catalyse the conversion of IPP/DMAPP into santalene**, the first component of our sandalwood oil ([see our Design Page for more details](https://2024.igem.wiki/iiser-tvm/design)). Dr Bohlmann previously studied the expression of these enzymes in *E. coli* using the pCDFDuet expression vector. In our discussions with him, he mentioned that while his exact construct might not be optimally expressed in our *P.putida* TA7-EG strain and may require further modifications, he kindly agreed to provide us with the pCDFDuet containing SaSSy-FPPS for testing in our chassis.

### The DBTL CYCLE

**To test the initial pCDFDuet construct’s expression in our chassis**. We transformed the pCDFDuet with SaSSy-FPPS into our TA7-EG strain via electroporation. **After successful transformation, we induced the expression of SaSSy and FPPS with 1 mM IPTG and assessed the expression through Coomassie staining of purified protein and chemical analysis using GC-MS**. The SaSSy enzyme, which already had a His-tag upstream from the original construct in the Bohlmann paper[3], allowed us to purify the protein using Ni-NTA columns with His-trap resin. Despite some purification issues, the Coomassie-stained gel revealed a thick band at 65 kDa, corresponding to the expected size of the SaSSy protein.

![](https://static.igem.wiki/teams/5181/engineering/plate-image-ta7.jpg)

Fig.3.: Electroporation of TA7-EG.

![](https://static.igem.wiki/teams/5181/engineering/pcdf-duet-sassy-his-tagged.png)

Fig.4.: Coomassie gel for pCDFDuet showing the band at 65kDa for SaSSy, Lane 1 - Ladder, Lane 2 - Whole cell lysate, Lane 3 - Purified protein.

GC-MS analysis was performed using our institute's Agilent 8890gc system 597 7 GC/MSD with a suitable column. **We observed a peak at an m/z ratio of 204**, which corresponds to the known ratio of santalene as reported in previous literature. **However, the abundance of this compound within the sample was relatively low, confirming Dr Bohlmann's suggestion that the construct might not work optimally in our chassis**.

![](https://static.igem.wiki/teams/5181/engineering/pcdf-duet.png)

![](https://static.igem.wiki/teams/5181/engineering/pcdf-duet-control.png)

Fig.5.: GCMS of pCDFDuet expressed in TA7-EG strain, showing spectrum for 204 m/z corresponding to santalene in the left one; at the right side there is a control with untransformed sample.

To address this issue, **we devised several construct designs that could potentially improve the expression of SaSSy-FPPS in our chassis**. These designs were based on principles such as bicistronic expression and dual expression systems, aiming to optimise the expression of SaSSy-FPPS in different settings. For more details, go to our [Parts page](https://2024.igem.wiki/iiser-tvm/parts).

![](https://static.igem.wiki/teams/5181/registry-parts/part-14-ara-map.png)

Fig.6.: The araBAD\_SaSSy\_FPPS insert.

Due to time constraints and the availability of parts from IDT, we initially worked with our bicistronic SaSSy-FPPS construct under the control of an arabinose promoter, [BBa\_K5181014](https://parts.igem.org/Part%3ABBa_K5181014). **The complete part was received as three separate fragments built using the NEBuilder HiFi DNA Assembly kit**. Following assembly, we PCR-amplified the entire construct to obtain a high yield and to **verify successful assembly and PCR**.

![](https://static.igem.wiki/teams/5181/engineering/sassy-fpps.png)

Fig.7.: PCR of SaSSy\_FPPS confirming the assembly.

After digestion, the gene insert and vector were ligated and transformed into our TA7-EG strain via electroporation. The transformed cells were plated on gentamicin-containing plates. **To test the presence of the plasmid following successful transformation, we isolated the plasmid and performed a confirmatory digest using EcoRI and HindIII**, expecting bands of 3 and 4 kb. As shown in the image below, **both single (EcoRI) and double digests confirmed the presence of our plasmids in the TA7-EG strain**.

![](https://static.igem.wiki/teams/5181/engineering/pseva-631-sassy-fpps-ligated.png)

Fig.8.: Single digested SaSSy\_FPPS.

![](https://static.igem.wiki/teams/5181/engineering/pseva-sassy-fpps-double-digested.png)

Fig.9.: Double Digested SaSSy\_FPPS.

**We also tested the expression of this construct by inducing various concentrations of arabinose** (0.2%, 0.5%, 0.7%, 1%, 1.5% and 2%). We found the best results from the 0.7% and 1.5% inductions. The proteins were purified using the His Trap NI NTA column, and **the expression of these proteins was confirmed by the bands observed in the following Coomassie-stained gel**. To analyse their functionality, **we tested the production of santalene using our designed construct through GCMS**.

![](https://static.igem.wiki/teams/5181/engineering/copy-of-631-sassy-fpps-0-7-coomaise.png)

Fig.10.: Coomassie gel for the SaSSy\_FPPS.

![](https://static.igem.wiki/teams/5181/engineering/631-0-7.png)

![](https://static.igem.wiki/teams/5181/engineering/sassy631-control-0-7-2.png)

Fig.11.: The Left shows the presence of the santalene with a sharp peak at 204 m/z resembling the molecular weight; on the right side of the image, there is an untransformed sample showing no peak at the same retention time for 0.7% induction.

![](https://static.igem.wiki/teams/5181/engineering/631-1-5-ligated.png)

![](https://static.igem.wiki/teams/5181/engineering/631-1-5-control.png)

Fig.12.: A peak of 204 m/z presence strongly suggests the santalene expression in 1.5% arabinose induction in the left one; on the right side, the control shows no peak at the same retention time.

Through these iterative processes, **we learned that our designed SaSSy-FPPS construct (BBa\_K5181014) is functionally expressed in our TA7-EG strain when TPA and EG are the sole carbon sources**. The functionality of the enzymes was confirmed by GC-MS analysis. To our knowledge, this is the first demonstration of santalene production in *P.putida* using PET monomers as the carbon source. **While this is a promising result for us undergrad students, it presents new directions for further testing**.

We still need to investigate the modularity of our parts by changing promoters and RBS sites to find optimal expression conditions. Building upon our current method, we can compare it with our other designed part, BBa\_K5181018, which expresses SaSSy and FPPS under different promoters as part of the pRGPDuo vector and is suitable for separately tuned expression. This can be particularly useful for the independent expression of FPPS, as it is an important step in synthesising other terpenoids.

Iteration/ Engineering Cycle 3

# Assembly and Expression of P450-CPR

### Introduction

For our third and most relevant engineering cycle, **our goal was to design the most suitable constructs for the expression of our P450-CPR enzymes**. The P450-CPR enzymes catalyze the conversion of santalene to santalol, the final major component of sandalwood oil. The expression of these enzymes in prokaryotic systems remains a profound challenge that is still being tackled by many scientists worldwide. **Several challenges are faced in expressing the eukaryotic (plant) membrane-bound cytochrome P450 enzyme** in prokaryotic systems like *Pseudomonas putida*, such as differences in membrane composition, incorrect anchoring of the enzymes, and lack of optimal expression conditions in bacterial hosts.

However, **our answer to this question involved combining experimental and computational efforts, extensively described on our [Design](./design) and [Modelling](./model) page**. To overcome these challenges, we adopted transmembrane domain truncation and fusion with membrane-anchoring proteins to enhance the expression and solubility of membrane-bound P450s in our bacterial system.

### The DBTL CYCLE

**To address the design challenge of expressing *Santalum album* CYP736A167 (P450) in bacteria**, we performed homology studies between this enzyme and P450 CAM, a naturally occurring P450 enzyme in P. putida and saw their core domains match. Following the truncation strategy, we removed the 27 amino acid hydrophobic extension in the N-terminal end of the Santalum album P450, considered to be the transmembrane domain, and similarly truncated the transmembrane domain of the native CPR1, the redox partner of P450. To ensure the proximity of the truncated P450 and CPR1 for FADH shuttling, **we sought inspiration from a self-sufficient bacterial p450 from *Bacillus cereus*. Structural alignment revealed that these P450’s had significant overlaps, and we also identified a 36 amino acid long loop (BC\_linker)** connecting the C-terminal of the oxidase domain to the N-terminal of the reductase domain in pP450 from *Bacillus*. This corresponds to the same area where the transmembrane region of our CPR1 connects to the core enzyme body. **We used this BC\_linker to join the truncated saCYP736A167 and saCPR1, forming our fusion protein.**

![](https://static.igem.wiki/teams/5181/engineering/struct-p450-engg-cyc3.png)

Fig.13.: The alphafold structure of our P450-CPR part with the linker after design.

**To test our design, we built a composite part [BBa\_K5181015](https://parts.igem.org/Part%3ABBa_K5181015) with tet promoter and truncated fusion P450-CPR**, an optimized fusion protein sequence for expression in *P. putida.* This part is also modular with respect to the promoters and RBS sites.

![](https://static.igem.wiki/teams/5181/engineering/fig-13-bba-k5181015-fusion-protein-bc-linker.png)

Fig.14.: The P450-CPR part sequence.

**This part arrived as three separate fragments, which were Gibson assembled** using the NEBuilder DNA HiFi Assembly Kit. After assembly, the whole gene insert was PCR amplified for confirmation and further downstream ligation into a suitable vector.

![](https://static.igem.wiki/teams/5181/engineering/cpr-p450.jpeg)

Fig.15.: 1% agarose gel, showing bands for 4151bp, the expected size of our designed P450-CPR.

**Despite multiple attempts, we encountered difficulties in successfully ligating the vector and insert using both conventional ligation and Gibson assembly methods**. After digesting the vectors and inserts with EcoRI and HindIII, convetional ligation was carried out. At the same time, Gibson assembly was performed using the same kit but following the 2-fragment protocol for the vector backbone and gene. These ligation trials were conducted with various vector backbones, including pSEVA 241, 631, and 424. However, **the transformations either failed to yield colonies after electroporation or resulted in transformed colonies that, when picked and isolated for plasmids, showed only smears when run on a gel**, indicating unsuccessful ligations. More can be found on our [results](https://2024.igem.wiki/iiser-tvm/results) page.

In conclusion, our attempts to assemble the vector backbone with the gene insert through ligation proved challenging. To identify the underlying cause, we sequenced the first and third fragments involved in binding with the vector, focusing on the regions containing restriction sites and overlaps. **The sequencing results revealed a perfect alignment of the first fragment sequence with the expected sequence at 130 base pairs, indicating the correctness of the fragment itself. However, we could not confirm the primer binding site due to its involvement in sequencing.**

![](https://static.igem.wiki/teams/5181/engineering/p450-frag1-seq.png)

Fig.16.: Sequencing chromatogram of the first fragment of [BBa\_K5181015](https://parts.igem.org/Part%3ABBa_K5181015).

![](https://static.igem.wiki/teams/5181/engineering/p450-frag1-align.png)

Fig.17.: Global alignment (Needleman-Wuntsch) of our sequenced and actual ordered fragments.

The sequencing of the third fragment unveiled numerous overlapping peaks throughout the sequenced region, suggesting potential issues related to the sequence, primer binding, or the presence of a homopolymer region. These issues can hinder successful ligation, even if the fragment is amplifying during PCR.

![](https://static.igem.wiki/teams/5181/engineering/p450-frag3-seq.png)

Fig.18.: Sequencing chromatogram of the third fragment [BBa\_K5181015](https://parts.igem.org/Part%3ABBa_K5181015).

Based on these observations, we hypothesise that the fragment may amplify correctly during PCR but fail to ligate properly due to sequence or structural problems. To address these challenges and continue our research, **we propose a two-pronged approach of doing further ligation tests to investigate the ligation efficiency and expressing our designed part [BBa\_K5181017](https://parts.igem.org/Part%3ABBa_K5181017), which incorporates the truncated P450 and CPR genes under the control of tac promoters.**

By acknowledging limitations, proposing testable hypotheses, and outlining a plan for future experiments, we aim to continue our research efforts and make progress towards the successful expression of the P450-CPR in our chassis.

**This, however, did not hinder our efforts in exploring the functions of the designed P450-CPR genes. Our computational team sought to understand how these genes would function if expressed. Through our modeling work, we explored the docking of santalene and its derivatives with our designed P450-CPR genes and investigated their binding effectiveness by analyzing the binding energies.**

As shown in the table below, the results from our docking simulations provide valuable insights into the binding energies of various substrates with the designed P450-CPR constructs. The mean binding energy of rank 1 cluster for α-santalene, a key intermediate in the santalene biosynthesis pathway, was found to be -7.37 kcal/mol. This binding energy suggests a favourable interaction between α-santalene and the P450-CPR enzyme, indicating the potential for effective conversion to the desired product, α-santalol. Similarly, the binding energies for other santalene derivatives, such as β-santalene and epi-β-santalene, were also promising, ranging from -7.64 to -7.41 kcal/mol.

| Sr. No. | Substrate molecule | Mean binding energy of rank 1 cluster in Kcal/mol | Product molecule | Mean binding energy of rank 1 cluster in Kcal/mol | Binding enegy difference in Kcal/mol |
| --- | --- | --- | --- | --- | --- |
| 1 | α-santalene | -7.37 | α-santalol | -7.26 | 0.11 |
| 2 | β-santalene | -7.64 | β-santalol | -7.4 | 0.24 |
| 3 | epi-β-santalene | -7.41 | epi-β-santalol | -7.24 | 0.17 |
| 4 | α-exo-bergamotene | -7.68 | α-exo-bergamotol | -7.24 | 0.44 |

The image below shows one docking pose illustrating the interaction between α-santalene and the P450-CPR enzyme.

![](https://static.igem.wiki/teams/5181/engineering/3-6-interacting-mesh-of-alpha-santalene.png)

Fig.19.: Interacting mesh of P450-CPR with alpha santalene.

**These computational insights provide valuable support for the potential functionality of our designed P450-CPR genes, even as we continue our efforts to overcome the experimental challenges encountered in their expression. By combining experimental and computational approaches, we aim to gain a comprehensive understanding of the P450-CPR system and its role in the conversion of santalene and its derivatives to the desired products**. For more detailed information on our modeling approaches and additional results, please refer to our [Modelling](https://2024.igem.wiki/iiser-tvm/model) page.

Iteration/ Engineering Cycle 4

## FBA and DXS-DXR

Our final engineering cycle focused on optimising the santalene and santalol production pathway by investigating the role of DXS (1-deoxy-D-xylulose-5-phosphate synthase) and DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase), the first two enzymes in the MEP (methylerythritol phosphate) pathway. The MEP pathway is crucial for synthesising IPP (isopentenyl pyrophosphate) and DMAPP (dimethylallyl pyrophosphate), the precursors for santalene production. **Our modelling team analysed the active pathways under our experimental conditions and discovered that DXS and DXR are critical enzymes that must be expressed to increase IPP/DMAPP production**. These findings align with the results previously reported by Dr Jordi[4].

To better understand the metabolic network, **we designed and compared FBA (Flux Balance Analysis) models based on previously published models, namely iJP815, PpuQY1140, and iJN1463, as listed in the table below**. We modified each model by incorporating reactions relevant to our project, such as TPA (terephthalic acid), EG (ethylene glycol) uptake, and the santalol synthesis pathway. **The FBA approach also allowed us to perform in silico tests to determine the optimal TPA and EG uptake rates for maximizing santalol production using Pareto-optimization**. Refer to our [modelling page](https://2024.igem.wiki/iiser-tvm/model) for more details.

| Model name | No. of Metabolites | No. of reactions | No.of genes | Year of publication |
| --- | --- | --- | --- | --- |
| iJP815 | 892 | 948 | 818 | 2008 |
| PpuQY1140 | 1104 | 1171 | 1140 | 2017 |
| iJN1463 | 2160 | 2936 | 1462 | 2019 |

**Based on our modelling results and the advice from Dr Jordi, we obtained plasmids containing DXS and DXS-DXR genes. Dr Jordi suggested that co-expressing DXS-DXR might be the most effective strategy to overexpress the MEP pathway and increase our santalene and santalol yield, which aligns with our modelling predictions**. We successfully revived the bacterial stocks received from Dr Jordi, as shown in the image below.

![](https://static.igem.wiki/teams/5181/engineering/dxs-dxr.jpg)

Fig.20.: Revived DXS, DXS-DXR plasmid bacterial stocks.

**However, due to time constraints and the late arrival of the plasmids, we were unable to co-express the DXS-DXR genes along with our SaSSy-FPPS and P450-CPR genes within the given timeframe. Despite this setback, our modelling efforts provided valuable insights into the potential optimization of the santalene and santalol production pathway.**

This engineering cycle highlights the importance of integrating computational modelling with experimental design to guide metabolic pathway optimization. By identifying critical enzymes and predicting optimal substrate uptake rates, we can make informed decisions to enhance the production of our target compounds. **Although we faced challenges in the experimental implementation, our modelling results lay the foundation for future work on optimizing the MEP pathway and ultimately increasing santalene and santalol yields**.

[**References**](#collapseExample)

1. Harwood, C. S., & Parales, R. E. (1996). The β-Ketoadipate Pathway and the Biology of Self-Identity. Annual Review of Microbiology, 50(1), 553-590. <https://doi.org/10.1146/annurev.micro.50.1.553>
2. Brandenberg, O. F., Schubert, O. T., & Kruglyak, L. (2022). Towards synthetic PETtrophy: Engineering Pseudomonas putida for concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression. Microbial Cell Factories, 21, 119. <https://doi.org/10.1186/s12934-022-01846-w>
3. Jones, C. G., Moniodis, J., Zulak, K. G., Scaffidi, A., Plummer, J. A., Ghisalberti, E. L., Barbour, E. L., & Bohlmann, J. (2011). Sandalwood Fragrance Biosynthesis Involves Sesquiterpene Synthases of Both the Terpene Synthase (TPS)-a and TPS-b Subfamilies, including Santalene Synthases. The Journal of Biological Chemistry, 286(20), 17445–17454. <https://doi.org/10.1074/jbc.M111.231787>
4. Hernandez-Arranz, S., Perez-Gil, J., Marshall-Sabey, D., & Rodriguez-Concepcion, M. (2019). Engineering Pseudomonas putida for isoprenoid production by manipulating endogenous and shunt pathways supplying precursors. Microbial Cell Factories, 18(1), 152. <https://doi.org/10.1186/s12934-019-1204-z>

{% endblock %}

{% extends "layoutother.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

# Experiments

## Contents

* [Materials](#materials)
* [Protocols](#protocols)

# Materials

---

This section provides a comprehensive catalog of the key biological materials including strains, plasmids, primers and antibodies that formed the foundation of our experiments. Each component was carefully selected or engineered to ensure precision and efficiency at every stage of our research, playing a critical role in the success of our project.

### STRAINS

1.*Pseudomonas putida* KT2440: A well-characterised strain derived from the wild-type *Pseudomonas putida* mt-2 with rmo-mod+ genotype.

2.*Pseudomonas putida* TA7: A derivative strain of *Pseudomonas putida* KT2440, engineered for the uptake of terephthalic acid (TPA) as a carbon source.

3.*Pseudomonas putida* TA7-EG: **TA7 EG** is a derivative strain of *Pseudomonas putida* KT2440, engineered for the utilisation of both terephthalic acid (TPA) and ethylene glycol as carbon sources.

We gratefully acknowledge the receipt of these strains from Dr. Oliver Brandenberg, whose contributions have been instrumental to our work in utilising these strains for carbon source engineering.

### PLASMIDS

1. **pCDFDuet**: This plasmid was used to express and produce santalene. It incorporates resistance genes for both streptomycin and spectinomycin, enabling selective pressure during bacterial culture. The expression system is controlled by a lac promoter, which was activated through IPTG induction. This setup allowed for efficient regulation of santalene biosynthesis under controlled experimental conditions.

2. **pSEVA 631\_SaSSy\_FPPS**: The plasmid served as the backbone for the integration of our (Santalene Synthase)SaSSy and Farnesyl pyrophosphate synthase(FPPS) fragments responsible for santalene production. It confers gentamicin resistance and features an araBAD promoter, which was activated through L-arabinose induction.

3. **pSEVA 241\_CPR\_p450**: The plasmid served as the backbone for the integration of our CPR-P450 fragments, which are essential for the biosynthesis of santalol. It is equipped with a kanamycin resistance gene and the expression system is controlled by a tet promoter. The induction was done by anhydrous tetracycline.

4. **pSEVA 424\_DXS\_DXR**: This plasmid was used as backbone for DXS-DXR that increase the flux towards IPP/DMAP. It confers streptomycin and spectinomycin resistance with lac promoter. The induction was done by IPTG.

### PRIMERS

| Sr. no. | Primer information | Primer sequence | 1st round Tm(°C) | Tm(°C) |
| --- | --- | --- | --- | --- |
| 1 | Forward primer for the amplification of araBAD-SaSSy-FPPS | 5’ CGC CTA GGC CGC GGC CGC GCG AAT TCT TAG TGG 3’ | 71.3 | 77.7 |
| 2 | Reverse primers for amplification of araBAD-SaSSy-FPPS | 5’ TTT TCC CAG TCA CGA CGC GGC CGC AAG CTT TTA TGA CAA CTT GAC GGC TAC ATC ATT CAC 3’ | 74.9 | 76.8 |
| 3 | Forward primer for amplification of CPR-P450 | 5’ CGC CTA GGC CGC GGC CGC GCG AAT TCT TAG TGA TG 3’ | 69.2 | 76.8 |
| 4 | Reverse primer for the amplification of CPR-P450 | 5’ TTT TCC CAG TCA CGA CGC GGC CGC AAG CTT TTA AGA CCC 3’ | 70.8 | 75.7 |
| 5 | Forward primer to replace tet promoter with araBAD promoter | 5’ ATATGCATATATCTCCTTCTTAAAAGATC 3’ | 49.5 | 56.9 |
| 6 | Reverse primer to replace tet promoter with araBAD promoter | 5’ CGCAAGCTTTTATGACAAC 3’ | 54.9 | same |

### ANTIBODIES

| ANTIBODY | CATALOG NUMBER/SUPPLIER | DILUTION FOR BLOT |
| --- | --- | --- |
| His Tag (rabbit) | Santa Cruz, sc-804 | 1:1000 (primary), 1:2000 (secondary) |
| His Tag (mouse) | Santa Cruz, sc-8036 | 1:1000 (primary), 1:2000 (secondary) |

# Protocols

---

This section compiles the protocols we followed and refined throughout our laboratory experimentation. This section serves as both a guide and a resource for future experimentation, offering insights into the thought process behind protocol selection and development, as well as a testament to the collaborative effort that drove our success in the laboratory.

MEDIA PREPARATION

## 1. M9 MINIMAL MEDIA PREPARATION

##### 1.1 SOLUTIONS

**1.1.1 M9 media**

1.Measure 64g Na2HPO4.7H2O in a sterilised plastic boat (previously tared) using a digital weighing scale and transfer to a 1L conical flask

2. Weigh out 15g KH2PO4 in the same way and add it to the conical flask.

3. Measure 2.5g NaCl and 5g NH4Cl similarly and transfer to the same conical flask.

4. Make up the 1000mL of the volume by adding MilliQ to the conical flask.

5. Gently close the lid. Autoclave the M9 media bottle at 121 degrees Celsius for 20 minutes.

6. On completion, gently tighten the lid and mix the media if required.

7. Use the required amount and keep it at 4 degrees Celsius until further use.

**1.1.2 MgSO4 solution**

1. Measure 12.325g of MgSO4 in a sterilised plastic boat (previously tared) using a digital weighing scale and transfer it to a 100 ml conical flask.

2. Add 50ml milliQ to the conical flask.

3. Gently close the lid. Now proceed to autoclave the flask at 121 degrees Celsius for 20 minutes.

4. On completion, tighten the lid and mix gently if required.

5. Use the required amount and keep it at 4 degrees Celsius until further use.

**1.1.3 CaCl2 solution**

1. Measure 7.350g CaCl2 in a sterilised plastic boat(previously tared) using a digital weighing scale and transfer to a 100 ml conical flask.

2. Add 50ml milliQ to the conical flask.

3. Gently close the lid. Now proceed to autoclave the flask at 121 degrees Celsius for 20 minutes.

4. On completion, tighten the lid and mix gently if required.

5. Use the required amount and keep it at 4 degrees Celsius until further use.

##### 1.2 TPA+EG LIQUID MEDIA PREPARATION

1. Take a few clean, empty 50 mL Borosil bottles and rinse them with MilliQ.

2.Measure 3mL M9 media using a 1ml pipette and transfer it to the bottle.

3.Measure 30ul of freshly prepared MgSO4 using a 2-20ul pipette and transfer to the same bottle.

4.Measure 1.5ul CaCl2, transfer and mix well.

5. Add the required amount of Ethylene Glycol, depending on the different bottles' concentrations.

6. Make up the remaining 15ml of the volume by adding milliQ to the bottle.

7. Gently close the lid. Autoclave the M9 media bottle at 121 degrees Celsius for 20 minutes. On completion, everything will be dissolved.

8. Tighten the lid.

9. Add the mentioned amount of TPA to the bottles once the temperature is bearable.

10. Now pipette 15ul of previously prepared trace metals and add to each bottle.

11. Pick single colonies of *Pseudomonas putida* TA-7 EG from the freshly streaked agar plate and inoculate to all the EG and TPA+EG media, and pick single colonies from *P. putida* TA-7 and inoculate to the TPA media.

12. Let it grow overnight.

| MEDIA | TPA CONCENTRATION | EG CONCENTRATION |
| --- | --- | --- |
| 5mM TPA | 0.375ml |  |
| 5mM EG |  | 4.2ul |
| 5mM TPA+5mM EG | 0.375ml | 4.2ul |
| 10mM TPA | 0.750ml |  |
| 10mM EG |  | 8.4ul |
| 10mM TPA+10mM EG | 0.750ml | 8.4ul |
| 15mM TPA | 1.125ml |  |
| 15mM EG |  | 12.6ul |
| 15mM TPA+15mM EG | 1.125ml | 12.6ul |
| 20mM TPA | 1.5ml |  |
| 20mm EG |  | 16.8ul |
| 20mM TPA+20mM EG | 1.5ml | 16.8ul |

## 2. GLYCEROL STOCK PREPARATION

1. Inoculate the bacteria into an appropriate liquid medium, preferably LB broth, and let it grow overnight at the required temperature and shaking.

2. Using a sterile pipette, transfer an appropriate amount of 100% glycerol to a sterile cryo vial.

3. Add an equal amount of bacterial culture to the same cryo vial.

4. Gently mix by pipetting up and down.

5. Store the vials in -80°C.

CELL CULTURE

## 1. COMPETENT CELL PREPARATION (*E. coli*)

1. Pick a single colony of *E. coli* from a freshly streaked agar plate and inoculate it into 5 ml of autoclaved LB broth.

2. Let the culture grow overnight at 37 degrees Celsius in a shaker incubator at 250 rpm.

3. Inoculate 3 ml of the overnight culture in 200 ml of autoclaved LB broth.

4. Incubate the secondary culture at 37 degrees Celsius at around 250rpm

5. Monitor the absorbance using a spectrophotometer every 1 hour or 30 minutes till it reaches an O.D of 0.5 at 600nm.

6. Once the OD reaches 0.5, plunge it into an ice bath for 10 minutes.

7. Make sure to pre-chill the falcons, pipette tips and maintain the centrifuge at 4 degree celsius.

8. Transfer the culture(45 ml) into a pre-chilled 50 ml falcon tube under sterile conditions and keep in ice for 30 minutes.

9. Pellet down the culture at 4 degrees Celsius for 10 minutes at 4000 rpm.

10. Carefully discard the supernatant formed.

11. Add 30ml of pre-chilled 0.1M CaCl2 into the falcon, gently flick it around 15 times, and flash freeze for 40 seconds.

12. Again, centrifuge at 4 degrees for 10 minutes at 4000 rpm.

13. Remove 25ml of the supernatant carefully and add 30ml of CaCl2 again.

14. Repeat the CaCl2 wash 5-6 times until all the debris is removed.

15. Carefully measure the volume of the remaining pellet and make it to 400ul using the prepared stock solution.

16. Transfer it into eight vials of 50ul each under sterile conditions.

17. Freeze the aliquots in liquid nitrogen and store them at -80 degrees celsius until further use.

## 2. COMPETENT CELL PREPARATION (*Pseudomonas putida*)

1. Inoculate a single colony of *Pseudomonas putida* into 5 mL of LB medium.

2. Incubate the culture overnight at 30°C with shaking at 200-250 rpm.

3. Add 1 mL of the overnight culture to 100 mL of fresh LB medium.

4. Incubate the diluted culture at 30°C with shaking until the OD₆₀₀ reaches approximately 0.5.

5. Once the culture reaches the desired OD₆₀₀, place the culture on ice for 10-15 minutes to chill and transfer to pre-cooled centrifuge tubes.

6. Pellet down at 3000g for 8 minutes at 4 degrees Celsius.

7. Carefully discard the supernatant.

8. Resuspend the cell pellet in 20ml of sterile 10% ice-cold glycerol.

9. Transfer to 50ml tubes, centrifuge again at 3000g for 8 minutes at 4°C, and discard the supernatant.

10. Repeat this washing step two more times for a total of three washes with ice-cold sterile 10% glycerol.

11. After the final centrifugation, resuspend the cell pellet in 1ml of ice-cold sterile 10% glycerol.

12. Aliquot the electrocompetent cells into sterile, pre-cooled 100ul microcentrifuge tubes.

13. Immediately freeze the aliquots in liquid nitrogen and store the electrocompetent cells at -80°C.

MOLECULAR BIOLOGY

## 1. HEAT SHOCK TRANSFORMATION

1. Take the competent cells from -80°C and thaw the vial in ice for 5-10 mins.

2.Take 1ng/ul of plasmid DNA in a pre-chilled 1.5ml tube and add 50ul of competent cells.Tap mix 3-5 times.

3. Incubate in ice for 30 minutes.

4. Heat shock for 1 minute at 40°C.

5. Immediately transfer back to ice for 5 minutes.

6. Add 1 ml of room temperature LB and incubate at 37 degrees Celsius and 200 rpm for 1 hour.

7. Spin down at 3000 rpm for 5 minutes at 37 degrees Celsius, aspirate 900ul.

8. Resuspend the bacteria in the remaining 100ul LB broth and plate on the selective media.

9. Dry the plates for 2 minutes.

## 2. ELECTROPORATION

1. Keep cells and plasmid on ice before electroporation to maintain cell viability and transformation efficiency.

2.Mix 50-100 µL of electrocompetent cells in a sterile tube with 1-2 µL of plasmid. Gently mix without pipetting excessively.

3.Keep it on ice for 30 seconds.

4.Transfer the cell-plasmid mixture to a pre-chilled electroporation cuvette.

5. Set the electroporator to the appropriate settings. Place the cuvette in the electroporator and pulse.

6. Add 1 mL of recovery medium (pre-heated LB broth) to the cuvette and gently mix to resuspend the cells. Transfer the cells to a sterile tube.

7. Incubate the cells in the recovery medium at 37°C with shaking for 1 hour to allow cells to recover and express antibiotic resistance genes.

8. Spin down at 4000 rpm for 5 minutes.

9. Discard 700 ul of the supernatant and plate the rest of the volume of the electroporated cells onto selective agar plates.

## 3. GIBSON ASSEMBLY

An advanced molecular cloning technique that enables the seamless joining of multiple DNA fragments in a single reaction without the requirement for restriction enzyme sites or traditional DNA ligation. Gibson Assembly was selected over traditional restriction enzyme-based cloning methods for several key reasons that align with our project’s requirements. The following is the protocol we employed for the assembly of our three-fragment plasmid insert, SaSSy-FPPS.

1. Make each fragment's concentration 100 ng/ul by adding the required amount of Nuclease Free Water (NFW) in each vial.

2.Ensure that the DNA fragments to be assembled have overlapping sequences (20-40 bp).

3. Combine the DNA fragments in a reaction tube according to the required volumes.

4. Add the required amount of NFW and make it upto 10 ul.

5. Use 10 ul of 10x Gibson Assembly Master Mix and mix it with the DNA fragments.

6. Incubate the mixture in a thermocycler at 50°C for 60 minutes. The time can vary depending on the complexity and number of fragments.

7. Following incubation, store samples on ice or at –20 °C for subsequent transformation.

8. After the assembly reaction, the assembled product is transformed into electrocompetent cells using standard electroporation protocols.

9. Plate the transformed cells on selective media and incubate overnight at 37°C.

## 4. POLYMERASE CHAIN REACTION

1.Take 2 µL of the assembled fragments for amplification and dilute with 8 µL of nuclease-free water (NFW).

2. In a PCR tube, mix the following components, ensuring that the DNA polymerase is added last.

3. All components should be on ice during the preparation of the PCR reaction mixture.

| COMPONENT | 64°C w/ DMSO | 68°C w/o DMSO |
| --- | --- | --- |
| NFW | 8.4 ul | 10.4 ul |
| BUFFER | 4 ul | 4 ul |
| TEMPLATE | 2.4 ul | 2.4 ul |
| dNTP | 1.6 ul | 1.6 ul |
| FORWARD PRIMER | 0.6 ul | 0.6 ul |
| REVERSE PRIMER | 0.6 ul | 0.6 ul |
| POLYMERASE | 0.4 ul | 0.4 ul |
| DMSO | 2 ul | nil |

4. Set the PCR machine to the required temperatures and cycle conditions, then place the PCR vial in the machine for the appropriate duration.

**CYCLING CONDITIONS**

|  |  |
| --- | --- |
| Initial Denaturation | 5 min (95°C) |
| Denaturation | 30s (95°C) |
| Annealing | 1 min (57°C) |
| Extension | 1 min for 1kb (72°C) |
| Final Extension | 5 min (72°C) |

## 5. RESTRICTION DIGESTION

| REACTION COMPONENT | AMOUNT |
| --- | --- |
| Enzyme Buffer | Upto 1X |
| DNA sample | 2-20 ng |
| Enzyme 1 | 10 units/ul |
| Enzyme 2 | 10 units/ul |
| Nuclease Free Water | Make up to 25 ul |

**5.1 For a 25 ul reaction**

1. Thaw all components (DNA, enzymes, buffer) on ice.

2. Ensure all reagents and tubes are kept on ice during setup to prevent enzyme degradation.

3. In a PCR tube, add water as calculated above.

4. Add the DNA sample to be digested.

5. Add the required amount of restriction buffer.

6. Select the appropriate enzymes required for the digestion of the desired DNA.

7.Add the enzyme(s).

8. Briefly spin the tube to ensure all components are mixed at the bottom of the tube.

9. Incubate the reaction mixture at the temperature recommended for the restriction enzyme (usually 37°C) for 2-3 hours.

10. Verify the digestion by running an aliquot of the reaction mixture on an agarose gel electrophoresis.

## 6. CLONING

1. Digest both the vector DNA and insert DNA with the same restriction enzymes to create compatible ends.

2. Purify the digested products using the PCR cleanup kit.

3. Mix the digested vector and insert in a suitable molar ratio (typically 1:3 vector to insert).

4. Add buffer and DNA ligase (5 ul each for 50 ul reaction).

5. Make it upto 50 ul using NFW and incubate the mixture at 16°C overnight or at room temperature for 1-2 hours.

6. Add the ligation mixture to competent cells and transform using either electroporation or heat shock method.

7. Use colony PCR, restriction digestion, or sequencing to verify the presence and correct orientation of the insert.

**6.1 For SaSSy-FPPS insert and pSEVA 631 vector**

| COMPOUND | 1:3 RATIO | 1:1 RATIO |
| --- | --- | --- |
| NFW | 30.07 ul | 26.94 ul |
| VECTOR | 3 ul | 6.13 ul |
| INSERT | 9.43 ul | 9.43 ul |
| BUFFER | 5 ul | 5 ul |
| T4-LIGASE | 5 ul | 5 ul |

## 7. SEQUENCING

**7.1 Big Dye Reaction (10 ul)**

| COMPONENTS | VOLUME |
| --- | --- |
| Sequencing Buffer | 1.75 ul |
| Sequencing Big Dye Mix | 0.5 ul |
| Primer (10mM) | 1 ul |
| Template | 200 ng |
| MilliQ | Adjust to final volume |

**7.2 Cycling Conditions**

| STAGE | TEMPERATURE | TIME | NO. OF CYCLES |
| --- | --- | --- | --- |
| Initial Denaturation | 96°C | 1 min | 1 |
| Denaturation | 96°C | 10 sec | 25 |
| Annealing | 50°C | 5 sec | 25 |
| Final Extension | 60°C | 4 sec | 25 |
| Hold | 4°C | infinite | 1 |

**7.3 Sequencing Reaction Cleanup**

1. Add 10 µL of the PCR reaction (sample) to each well of the 96-well plate.

2. Add 10 µL of autoclaved Milli-Q water to each well to bring the total volume to 20 µL.

3. Add 5 µL of 125 mM EDTA (pH 8.0) to each well and pipette-mix the contents of each well gently.

4. Add 60 µL of 100% ethanol to the walls of the wells.

5. Seal the plate with aluminum foil or a plastic adhesive seal and invert the plate 4 times to mix thoroughly.

6. Incubate the sealed plate in the dark for 15 minutes at room temperature.

7. Spin the plate at 3000g for 30 minutes at room temperature (25°C) or at 2200g for 45 minutes at room temperature.

8. Carefully remove the seal from the plate and invert the plate onto a Kimwipe to drain excess liquid.

9. With the plate still inverted, spin at 185g for 1 minute at room temperature (~25°C) to remove residual liquid.

10. Add 60 µL of 70% ethanol to the walls of the wells.

11. Seal the plate again with aluminum foil or a plastic adhesive seal.

12. Spin the plate at 1650g for 15 minutes at 4°C.

13. With the plate inverted, spin at 185g for 1 minute at room temperature to remove any remaining liquid.

14. Resuspend the samples in 10 uL HiDi Formamide.

15. Heat the plate at 95°C for 2 minutes to denature the samples.

16. Immediately transfer the plate to ice after snap-chilling to halt any further reactions.

17. Spin the plate to collect all the PCR product to the bottom.

CLEANUP

## 1. PLASMID ISOLATION; High Copy Plasmid (Machery-Nagel Kit)

1. Inoculate 5 ml LB broth in a falcon using appropriate antibody under sterile conditions and let it grow overnight.

2. Pellet down the culture for 5 minutes at 11000g.

3. Discard the supernatant, remove as much liquid as possible.

4.Add 250ul resuspension buffer (A1). Resuspend the cell pellet by vortexing to ensure that no cell clumps remain.

5. Add 250ul lysis buffer (A2) and invert mix 6 to 8 times. Be gentle to avoid shearing of genomic DNA.

6. Incubate at room temperature for up to 5 min until lysate appears.

7. Add 300ul neutralising buffer (A3), and invert the mix 6 to 8 times until the blue sample turns colourless.

8. Centrifuge at room temperature for 5 min at 11000g to precipitate the genomic DNA. Repeat till the supernatant is clear.

9. Place a Plasmid Column in a Collection Tube and decant the supernatant onto the column. Spin down at 11000g for 1 minute. Discard the flow-through and place the column back into the collection tube.

10. Repeat step 9 for the rest of the volume.

11.Add 500 uL of wash buffer (AW) , preheated to 50 degree celsius, and centrifuge for 1 min at 11000g. Discard the flow through

12. Add 600ul wash buffer (A4), centrifuge for 1 min at 11000g, and discard the flowthrough.

13. Centrifuge the column (dry spin) for 2 mins at 11000g, and discard the collection tube.

14. Incubate the column with lid open at 50°C for 1-2 minutes to ensure the complete evaporation of any residual ethanol.

15. Transfer the column to a fresh 1.5ml microcentrifuge tube.

16. Add 50ul of pre-heated elution buffer and incubate for 1 min at room temperature.

17. Spin down at 11000g for 1 minute.

## 2. PCR CLEANUP

1. Add 2 volumes of Binding buffer NTI to 1 volume of PCR product.

2. For sample volumes less than 30 uL make it up to 50uL with molecular grade water and
follow Step 1.

3. Mix thoroughly by pipetting up and down.

4. Place a NucleoSpin® column into a collection tube and load the mixture onto the column.

5. Centrifuge at 11,000 x g for 30 seconds.

6. Discard the flow-through and place the column back into the collection tube.

7. Add 700 µL of Wash buffer NT3 to the column and incubate 1-2 minutes at room temperature.

8. Centrifuge at 11,000 x g for 30 seconds, discard the flow-through and place the column back into the collection tube.

9. Repeat the wash step again and dry spin the column at 11,000 x g for 1 minute to remove any residual ethanol from the wash buffer.

10. Incubate the column at 50°C for 1-2 minutes to ensure the complete evaporation of any residual ethanol.

11. Place the NucleoSpin® column into a clean 1.5 mL microcentrifuge tube.

12. Add 15-30 µL of pre-heated elution buffer NE (or nuclease-free water) directly onto the center of the membrane.

13. Incubate at room temperature for 1-2 minutes.

14. Centrifuge at 11,000 x g for 1 minute to elute the DNA.

15. Store the purified DNA at -20°C for long-term storage.

DNA & PROTEIN EXPRESSION ANALYSIS

## 1. AGAROSE GEL ELECTROPHORESIS

**1.1 To prepare 1% agarose gel**

1. Add 0.7 g of agarose powder to 70 mL of 1X TAE buffer.

2. Heat the mixture in the microwave for 1 minute or until the agarose is fully dissolved.

3. Allow the agarose solution to cool for 5 minutes.

4. Carefully add 5 µL of ethidium bromide to the gel. Avoid any skin contact.

5. Pour the cooled agarose solution into the casting tray. Ensure it covers the base of the tray evenly.

6. Place the comb and allow it to set for 20 minutes.

7. Once the gel is set, gently remove the comb to create wells.

8. Mix DNA samples with loading dye (usually a 1:5 ratio), and load the DNA samples and the DNA ladder into the gel wells.

9. Place the gel in the electrophoresis tank and add enough 1X TAE buffer to cover the gel.

10. Set the voltage to 80V and run the gel until the samples are sufficiently resolved.

**1.2 To visualise the gel**

1. Place the gel on a UV transilluminator to visualise the DNA bands.

2. Use appropriate protective measures when working with UV light.

## 2. SDS PAGE

**2.1 Prepare the gel**

| COMPONENT | 12% RESOLVING GEL | 5% STACKING GEL |
| --- | --- | --- |
| Distilled Water | 3.3 ml | 2.75 ml |
| 30% Acrylamide-Bis | 4 ml | 0.65 ml |
| Tris HCl | 2.5 ml (1.5M, pH8.8) | 0.5 ml(0.5M, pH 6.8) |
| 10% SDS | 0.1 ml | 0.1 ml |
| 10% APS(Freshly made) | 0.1 ml | 40 ul |
| TEMED | 30 ul | 4 ml |

**2.2 Casting the gel**

1. Set the SDS apparatus and make sure there is no leakage.

2. Mix the components mentioned above in the required amounts to prepare 12% resolving gel.

3. Add APS and TEMED at the end to initiate polymerisation.

4. Pour the resolving gel solution between glass plates, layer with butanol and let it polymerise for 20-30 minutes.

5. Mix the components to make a 5% stacking solution.

6. Pour the stacking gel solution over the resolving gel.

7. Insert the comb to create wells.

8. Allow the stacking gel to polymerise for 20-30 minutes.

9. Remove the comb once it solidifies.

**2.3 Running the gel**

1. Place the gel in the electrophoresis apparatus.

2. Fill the inner and outer chambers with 1X running buffer.

3. Load the prepared protein samples and protein ladder into the wells.

4. Attach the apparatus to the power supply and run the gel at 100V until the dye front reaches the bottom of the gel.

## 3. WESTERN BLOT

1. After SDS-PAGE, carefully remove the gel from the electrophoresis apparatus and soak it in the transfer buffer for 10 minutes to equilibrate.

2. Activate the PVDF membrane by soaking it in 100% methanol for 30 seconds, then rinse with milliQ and equilibrate in transfer buffer for 10 minutes.

3. Assemble the transfer sandwich: sponge, filter paper, gel, membrane, filter paper, sponge.

4. Make sure to wet the sponge and filter paper before placing the gel and ensure no air bubbles are trapped between the gel and the membrane.

5. Place the setup inside the transfer apparatus and fill it with a transfer buffer.

6. Transfer the proteins at 100V for 1-2 hours at 4°C.

7. Once the transfer is complete, remove the membrane and briefly rinse it in TBS-T.

8. Place the membrane in a blocking buffer for 1 hour at room temperature with gentle shaking to prevent non-specific binding.

9. Add primary antibodies diluted in the preffered ratio in the blocking buffer and seal the membrane in a plastic cover.

10. Keep it overnight at 4 degree celsius with gentle rotation.

11. After incubation, decant the primary antibody and wash the blot with 1X TBST shaking for two times 5 minutes each. Then wash the membrane with 1X TBS with shaking for 10 minutes once.

12. Add secondary antibody, diluted in the preffered ratio, in blocking buffer and seal the membrane in a plastic cover.

13. Keep it for 1-hour incubation at room temperature with gentle rotation.

14. After incubation, decant the secondary antibody and wash the blot with 1X TBST shaking for two times 5 minutes each. Then wash the membrane with 1X TBS with shaking for 10 minutes once.

CHEMICAL ANALYSIS

## 1. GCMS (Gas Chromatography-Mass Spectrometry)

Gas Chromatography-Mass Spectrometry (GC-MS) is an analytical technique that combines the features of gas chromatography (GC) and mass spectrometry (MS) to identify and quantify compounds in complex mixtures. GC separates volatile compounds based on their retention times as they travel through a column, while MS provides detailed information on the molecular structure by ionising and fragmenting the compounds and analysing their mass-to-charge ratios.
We performed GC-MS to analyse and confirm the production of our desired compounds santalene and santalol, providing both qualitative and quantitative insights essential for our metabolic engineering work.

**1.1 Cell Lysis**

1. Harvest the bacterial culture by centrifugation and remove the supernatant.

2. Transfer the pellets to a falcon.

3. Add acid-washed glass beads to the falcon. (Acid-washing typically involves treating the beads with a strong acid and thoroughly washing with distilled water. Ensure they are completely dry before use.)

4. Add 2 ml of hexane to it.

5. Vortex the tube at high speed for 1-3 minutes and let it set.

6. Carefully decant the hexane layer containing the soluble cellular components without disturbing the glass beads and debris pellet into a clean GC-MS vial.

7. Set up in the GC-MS apparatus.

{% endblock %}

{% extends "layoutother.html" %}
{% block title %}Results{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

# Results

## Contents

* [Overview](#overview)
* [Growth Curve](#Growth_Curve)
* [Santalene Synthesis](#Santalene_Synthesis)
* [Santalol Synthesis](#Santalol_Synthesis)
* [Conclusion](#Conclusion)

# Overview

---

Our research project focuses on developing engineered *Pseudomonas putida* strains capable of producing high-value sesquiterpenes, specifically santalene and santalol isomers. We initiated our wet-lab experiments by analyzing the growth curves of transformed and untransformed P. putida strains grown in media supplemented with varying concentrations of terephthalic acid and ethylene glycol. These experiments revealed optimal growth conditions, paving the way for our next steps.

The main objective of our project is the design of biobricks for santalene production. Initially, the pCDF-Duet vector expressed SaSSy\_FPPS genes involved in sesquiterpene synthesis. However, to enhance the efficiency of the pathway, we transitioned to pSEVA631\_araBAD\_SaSSy\_FPPS (BBa\_K5181014)plasmid, which facilitated improved production of santalene.

In the final phase of our project, we advanced the design of the CPR-P450 fusion protein, which is responsible for converting santalene into santalol. The expression of these eukaryotic transmembrane proteins is challenging in our prokaryotic chassis, and hence, we ideated and used computationally determined design strategies, such as truncation of the transmembrane domain and fusion protein (link to  [design](https://2024.igem.wiki/iiser-tvm/design) and [modelling](https://2024.igem.wiki/iiser-tvm/model)  page).

We assembled the truncated fusion candidate but couldn't finish due to time and experimental hurdles. However, this step represents another milestone in achieving P450 expression, thereby santalol production. We have shown through our P450-CPR docking studies that it can indeed function as a multi-substrate enzyme and provided docking results for the high abundance of alpha and beta santalol over the rest of the isomers.

# Growth Curve

---

We set our goal to determine whether *Pseudomonas putida* TA7-EG could grow using terephthalic acid (TPA) and ethylene glycol (EG) as sole carbon sources which is a crucial step towards optimising the growth conditions. As per the suggestion given by [Dr. Brandenberg](https://2024.igem.wiki/iiser-tvm/human-practices), we prepared minimal M9 media containing TPA and EG in varying concentrations - 5 mM, 10 mM, 15 mM, and 20 mM. After many attempts at optimising the growth conditions of the bacteria on different concentrations of TPA and EG, we concluded that 20 mM TPA + 20 mM EG provided the optimal growth conditions for P. putida TA7-EG.
While significant growth was observed within 36 hours from inoculation, this was not practical for real-world applications. We adopted a strategy to supplement the media with 5% glucose. We reduced its concentration using gradual serial dilutions, transitioning from 5% glucose supplementation and TPA + EG to reach glucose-free conditions and optimise the growth of the bacteria solely on TPA and EG. This enabled the bacteria to adapt to TPA and EG and utilise them as their sole carbon source within 10-12 hours.

While significant growth was observed within 36 hours from inoculation, this was not practical for real-world applications. We adopted a strategy to supplement the media with 5% glucose. We reduced its concentration using gradual serial dilutions, transitioning from 5% glucose supplementation and TPA + EG to reach glucose-free conditions and optimise the growth of the bacteria solely on [TPA and EG](https://2024.igem.wiki/iiser-tvm/engineering). This enabled the bacteria to adapt to TPA and EG and utilise them as their sole carbon sources and grow within 10-12 hours.

**Aim**

To optimise the growth of *Pseudomonas putida* TA7-EG in terephthalic acid (TPA) and ethylene glycol (EG) as its sole carbon sources.

**Results**

* The analysis of the bacterial growth confirms the growth of wild type *Pseudomonas putida* TA7-EG in terephthalic acid and ethylene glycol media and displays the potential of *P. putida* to grow and utilise PET monomers TPA and EG as its sole carbon sources.
* *P. putida* TA7-EG exhibits growth 12 hours post inoculation.

![](https://static.igem.wiki/teams/5181/results/untransformed-growth-curve.jpg)

*Fig.1 : Growth curve at 600 nm of *P.putida* TA7-EG*

**Aim**
To optimise the growth of transformed *Pseudomonas putida* TA7-EG with the ligated plasmid (pSEVA 631\_SaSSy\_FPPS) in terephthalic acid and ethylene glycol as its carbon source.

**Results**

* Initially, we observed slower growth, possibly due to the stress induced by transformation - the bacteria took up to 36 hrs to reach the log phase compared to the wild type, which reached the log phase faster.
* We found that the bacterial growth improved significantly upon performing [serial dilutions](https://2024.igem.wiki/iiser-tvm/human-practices), and we successfully reduced the time required for the bacteria to grow to 24 hours by refining the dilution process.
* These adjustments showed positive improvements, but we can continue future experiments to optimize growth conditions like pH additional media supplements to increase product yield, and better growth.

![](https://static.igem.wiki/teams/5181/results/transformed-final.jpg)

*Fig.2 : Growth curve at 600 nm of transformed P.putida TA7-EG pSEVA631\_SaSSy\_FPPS*

# Santalene Synthesis

---

Santalene synthesis using pCDFDuet-SaSSy\_FPPS

Enzymes [FPPS](https://parts.igem.org/Part%3ABBa_K5181005) and [SaSSy](https://parts.igem.org/Part%3ABBa_K5181004) are required for the production of santalene.

Our primary objective was to successfully synthesise santalene in *Pseudomonas putida* TA7-EG by expressing pCDFDuet-SaSSy\_FPPS, a breakthrough achievement for a budding research team like ours. We contacted [Dr Bohlmann](https://2024.igem.wiki/iiser-tvm/human-practices), who had previously expressed SaSSy using the pCDFDuet expression vector in *E.coli* to achieve santalene synthesis. We decided to test the expression of pCDFDuet-SaSSy\_FPPS in our chassis, *P.putida* - bacterial system.

Transformation of pCDFDuet\_SaSSy\_FPPS into *P.putida*

**Aim**

To transform the Mach-1 *E.coli* electro-competent cells with pCDFDuet\_SaSSy\_FPPS plasmid.

**Experiment**

The pCDFDuet\_SaSSy\_FPPS plasmids were electroporated into the Mach-1 *E.coli* strain for amplification.

**Results**

* Colonies were observed in the petri dish containing Mach-1 *E.coli* cells transformed with pCDFDuet\_SaSSy\_FPPS, confirming the successful transformation of the plasmids into the bacteria.
* Plasmids were isolated from the bacteria and amplified to prepare stocks for future use.

![](https://static.igem.wiki/teams/5181/results/pcdf-mach1.png)

*Fig. 3 :pCDFDuet\_SaSSy\_FPPS transformation in Mach-1 *E.coli* cells on a plate containing streptomycin.*

**Aim**

To transform the isolated pCDFDuet\_SaSSy\_FPPS plasmids into *P. putida* TA7-EG electrocompetent cells.

**Experiment**

The plasmids were isolated using the Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit and further transformed into the engineered *P. putida* TA7-EG strain.

**Results**

Petri plates containing streptomycin show that the transformation of the pCDFDuet\_SaSSy\_FPPS plasmids in the P.putida TA7-EG cells was successful.

![](https://static.igem.wiki/teams/5181/results/pcdf-ta7eg.png)

*Fig. 4 :pCDFDUET P.putida TA7-EG in Streptomycin plate*

Expression of pCDFDuet\_SaSSy\_FPPS

**Aim**

To check the expression of His-tagged SaSSy protein in *Pseudomonas putida* TA7-EG .

**Experiments**

We grew our *Pseudomonas putida* TA7-EG transformed with pCDFDuet\_SaSSy\_FPPS in minimal M9 media supplemented with terephthalic acid (TPA) and ethylene glycol (EG) as the sole carbon source. 1mM of IPTG was used to induce the plasmid. The His-tagged SaSSy enzyme was purified using Ni-NTA chromatography technique with HisTrap resin. The purified protein was later analysed with SDS-PAGE gel and visualised using Coomassie staining.

**Results**

SDS-PAGE of the purified protein revealed a band at 65 KDa corresponding to the expected molecular weight of the Santalene Synthase (SaSSy) protein. The band observed confirms that SaSSy was expressed successfully in the engineered *Pseudomonas putida* TA7-EG strain.

![](https://static.igem.wiki/teams/5181/results/pcdf-duet-sassy-his-tagged.jpeg)

*Fig. 5 :Thermo protein ladder, Lane 2 is whole cell lysate, and lane 3 is His-tagged SaSSy at 65kDa.*

**Aim**

**Chemical Analysis:**

To detect the presence of santalene using GC-MS analysis to provide functional evidence of protein expression.

**Results**

**GCMS Analysis of Santalene Quantification**

The GC chromatogram shows the intensities of detected peaks at their respective retention times, while the mass spectra depicted relative intensities versus m/z (Mass to Charge) values. Santalene showed a characteristic peak at 204 m/z in the chromatogram (as previously shown by [Dr Bohlmann](https://doi.org/10.1074/jbc.m111.231787)). Analysis of the MS spectrum confirmed the presence of **santalene**, which had an abundance of **1.14% in the sample**. In contrast, the GC-MS analysis of the **wild-type *Pseudomonas putida* TA7-EG strain showed no santalene peak** at the corresponding retention time.

The absence of a peak corresponding to santalene in the wild type *Pseudomonas putida* also showed that pCDFDuet\_SaSSy\_FPPS expression was necessary to observe santalene production.

Below are the GC-MS results for the transformed pCDFDuet\_SaSSy\_FPPS in *Pseudomonas putida*:

![](https://static.igem.wiki/teams/5181/results/pcdf-duet.jpeg)

*Fig. 6 : Displays the 204 m/z peak in *Pseudomonas putida* pCDFDUET*

Results for the untransformed wild type *Pseudomonas putida*:

![](https://static.igem.wiki/teams/5181/results/pcdf-duet-control.jpeg)

*Fig. 7 :Absence of peak in the wild type Pseudomonas putida*

The abundance of santalene observed was lower than expected, showing limited effectiveness of pCDFDuet\_SaSSy\_FPPS in our system. To overcome this issue simultaneously we designed a suitable plasmid with an appropriate promoter, RBS, and SaSSy genes codon optimised for *P.putida*. For more details refer to the part design [BBa\_K5181014](https://parts.igem.org/Part%3ABBa_K5181014).

### **Santalene synthesis using araBAD\_SaSSy\_FPPS ([BBa\_K5181014](https://parts.igem.org/Part%3ABBa_K5181014)) in *Pseudomonas putida* TA7-EG**

We designed a system of **bicistronic inserts** with appropriate promoters, RBS, inter-ribosomal entry site (IRES) and spacer sequences, which can be ligated and cloned with the pSEVA backbones for stable expression.

This experiment was designed to express the insert araBAD\_SaSSy\_FPPS ([BBa\_K5181014](https://parts.igem.org/Part%3ABBa_K5181014)) in *Pseudomonas putida* TA7-EG growing on the PET monomers TPA and EG.

![](https://static.igem.wiki/teams/5181/registry-parts/part-14-ara-map.png)

*Fig. 8: [araBAD\_SaSSy\_FPPS](https://parts.igem.org/Part%3ABBa_K5181014)*

**Aim**

To assemble and amplify the araBAD\_SaSSy\_FPPS fragments.

**Experiment**

We assembled the three fragments to get a complete insert having araBAD promoter, RBS and [SaSSy](https://parts.igem.org/Part%3ABBa_K5181004) and [FPPS](https://parts.igem.org/Part%3ABBa_K5181005) genes using NEBuilder® HiFi DNA assembly. After the assembly, we performed bridge PCR using specific forward and reverse primers designed by our team.

**Results**

The gel results verified that the Gibson assembly was successful, confirming proper ligation of the araBAD\_SaSSy\_FPPS fragments. The size of our Gibson assembled insert was expected to be **4082 bp**. Our results confirmed successful fragment assembly, as shown in the gel image. A 1 Kb ladder was used to check the size of bands.

![](https://static.igem.wiki/teams/5181/results/sassy-fpps-labelled.jpeg)

Fig.9 : *araBAD\_SaSSy\_FPPS\_ showing bands at 4082 bp (all lanes have the same fragment sample)*

**Aim**

Restriction digestion and ligation of PCR-amplified araBAD\_SaSSy\_FPPS insert with the pSEVA631 vector backbone.

**Experiment**

The plasmid backbone pSEVA631 was isolated from the LB culture using the Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit. The plasmid and the insert (araBAD\_SaSSY\_FPPS insert) were restriction-digested using the EcoRI-HF and Hind III endonucleases.

| Name of Fragment | Expected size (bp) |
| --- | --- |
| pSEVA 631 (backbone) | 3519 |
| araBAD\_SaSSy\_FPPS (insert) | 4082 |

**Results**

Upon running the gel, the 56 bp long MCS (Multiple Cloning Site) regions of the pSEVA631 backbone ran out of the gel, and the backbone reflected a size of **3159bp**. Later, the gel was cut and cleaned using the Macherey-Nagel™ NucleoSpin™ Gel and PCR Clean-up Kit.

![](https://static.igem.wiki/teams/5181/results/631-digested.png)

*Fig.10 : The thick band between 3-4Kb shows the backbone corresponding to 3519 b.p*

**Aim**

To ligate the insert araBAD\_SaSSy\_FPPS with the backbone pSEVA631

**Experiment**

1. The PCR amplified samples of araBAD\_SaSSy\_FPPS were now cleaned up using the Macherey-Nagel™ NucleoSpin™ PCR Clean-up kit. Further, the insert was isolated after the PCR clean-up, and the pSEVA631 backbone was ligated using **NEB T4 DNA Ligase**.
2. Single and double digestion (EcoRI and HindIII) of the ligated plasmid (pSEVA631\_araBAD\_SaSSy\_FPPS) was performed to verify the ligation, followed by gel electrophoresis.

**Results**

* The 1% agarose gel electrophoresis confirmed the successful ligation of the insert into the backbone, ensuring proper assembly of the construct.
* In the case of single digestion, a thick band was found at 7Kb, corresponding to the size of our ligated plasmid (7005 bp), confirming the ligation process.
* Similarly, a single and double digestion of our ligated plasmid was performed to confirm ligation.

![](https://static.igem.wiki/teams/5181/results/pseva-sassy-fpps-double-digested.png)

Fig.11 : *The thick bands between 3-4Kb show

successful double digestion.*

![](https://static.igem.wiki/teams/5181/results/pseva-631-sassy-fpps-ligated.jpeg)

Fig.12 : *The thick band at 7Kb shows

successful single digestion.*

Transformation of ligated pSEVA631\_araBAD\_SaSSy\_FPPS into P.putida TA7-EG

**Aim**

To transform the ligated araBAD\_SaSSy\_FPPS\_pSEVA631 plasmid into *Pseudomonas Putida* TA7-EG.

**Experiment**

The ligated plasmids were subsequently transformed into the Mach1 *E. coli* electro-competent cells to amplify the plasmids. The amplified plasmids were isolated using Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit. Finally, the plasmids were electroporated into *Pseudomonas Putida* TA7-EG electro-competent cells for transformation.

**Results**

![](https://static.igem.wiki/teams/5181/results/631-sassy-mach.png)

Fig.13: *successful transformation of MACH-1 *E.coli* cells in Gentamycin plates*

![](https://static.igem.wiki/teams/5181/results/631-sassy-ta7eg.png)

Fig.14:  *successful transformation of *P.putida* TA7-EG cells in Gentamycin plates*

Expression of pSEVA631\_araBAD\_SaSSy\_FPPS into *P.putida* TA7-EG

**Aim**

To perform the expression of pSEVA631\_araBAD\_SaSSy\_FPPS in *P. putida* TA7-EG.

**Experiment**

We transformed the ligated pSEVA631\_araBAD\_SaSSy\_FPPS plasmid into *P.putida* TA7-EG in the minimum M9 media containing 20 mM TPA and 20 mM EG. The culture was induced with an optimised arabinose concentration of 0.7% and allowed to grow for 16 hours at 20°C. The bacteria was later pelleted and lysed using sonication.

Further, the Ni-NTA column was performed to purify the SaSSy and FPPS protein fragment, both of which were tagged with His tag. We performed SDS-PAGE, followed by Coomassie staining.

**Results**

We got a band at **65 KDa** and **45 KDa** corresponding to SaSSy and FPPS, respectively, confirming the expression of our desired proteins successfully.

![](https://static.igem.wiki/teams/5181/results/631-cpr-0-7-coomaise.jpeg)

Fig.15 : *on destaining, the bands were seen at 65KDa and 45KDa*

### Quantitative Analysis of Santelene through GC-MS

The GC chromatogram displayed intensities of the detected peaks at their respective retention times, while the mass spectra showed relative intensities versus m/z. The santalene standard produced peaks corresponding to 204 m/z in the chromatogram. A review of the mass spectrometry spectrum of the sample transformed with araBAD\_SaSSy\_FPPS\_pSEVA631 plasmid revealed a distinct pattern, confirming santalene, which quantified a **10.25%** abundance. It showed an **abundance ten times greater than that of pCDF-Duet.** In contrast, the GC-MS output for the **wild type *P. putida*** strain **did not exhibit any peak at 204 m/z.**.The wild-type *P. putida* TA7-EG cells and the transformed *P. putida* TA7-EG showed different peaks at the 19-minute retention time, verifying that the 204 peak corresponds to the santalene m/z ratio, which is expressed by the pSEVA631\_araBAD\_SaSSy\_FPPS plasmid incorporated into our chassis.

Consistent with our hypothesis, the modified *P. putida* strains expressing the SaSSy and FPPS proteins with the pSEVA631 backbone exhibited **successful santalene synthesis,** supporting our approach of achieving the **first-ever santalene production in *Pseudomonas putida* TA7-EG**

**Results**

![](https://static.igem.wiki/teams/5181/results/631-0-7.png)

Fig.16 :  *of sample induced with 0.7% arabinose in  *P.putida* TA7-EG transformed cells.*

![](https://static.igem.wiki/teams/5181/results/631-07-control.png)

Fig.17 :  *GCMS of Wild-type *Pseudomonas putida* TA7-EG cells*

# Santalol Synthesis

---

### [CPR-P450](https://parts.igem.org/Part%3ABBa_K5181015)

Expression of Cytochrome P450 Monoxygenase and Cytochrome Reductase

Building on our previous success with the expression of Santalene synthase (SaSSy) and Farnesyl pyrophosphate synthase (FPPS) in *Pseudomonas putida* TA7-EG, utilising PET-derived terephthalic acid and ethylene glycol as feedstock, we next aimed to incorporate the Cytochrome P450 Monooxygenase (CYP450) and Cytochrome Reductase system (CPR1). These enzymes, sourced from the *Santalum album*, catalyse the conversion of santalene to santalol. However, the expression of eukaryotic membrane-bound proteins in a prokaryotic host remains a big challenge due to the differences in membrane composition, enzyme anchoring, and varying post-translational modifications.

In this project phase, we focused on the expression of the CPR-P450 fusion protein in *P. putida* TA7-EG. Refer to [Project design](./design) and part [BBa\_K5181015](https://parts.igem.org/Part%3ABBa_K5181015) for a detailed approach to designing a fusion protein.

![](https://static.igem.wiki/teams/5181/engineering/fig-13-bba-k5181015-fusion-protein-bc-linker.png)

Figure. 18 : *CPR-P450 Fusion Protein used for the production of Santalol. Linker protein BC\_linker (BBa\_K5181016)*

**Aim**

To assemble CPR-P450 fragments using Gibson assembly.

**Results**

We performed Gibson assembly with three fragments synthesised by GenScript. A complete insert consisting of tet Promoter\_RBS\_P450-CPR fusion protein was assembled using NEBuilder® HiFi DNA assembly. Following the assembly, we performed bridge PCR using [specific primers](https://2024.igem.wiki/iiser-tvm/parts) to confirm assembly and amplify the insert. We loaded the sample in 1% agarose gel and performed electrophoresis.

![](https://static.igem.wiki/teams/5181/engineering/cpr-p450.jpeg)

Fig.19 : *Lane 1 Origin 1Kb Ladder, Lane 2 64°C with DMSO (no band), Lane 3,4,5 gradient PCR was performed to amplify the product of CPR-P450 at 67°C with DMSO, 67°C w/o DMSO, 68°C w/o DMSO, respectively.*

We observed bands around 4 Kb, which closely matched our expected assembled gene insert size of 4151 bp, confirming the success of the Gibson assembly. The detailed protocols for PCR amplification and Gibson assembly can be found on the [protocols page](https://2024.igem.wiki/iiser-tvm/experiments).

**Aim**

To ligate the tet\_CPR\_P450 insert into the vector backbone pSEVA241.

**Experiment**

1. The pSEVA241 vector backbone was isolated from *E.coli* Mach1 using Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit.
2. The PCR amplified CPR-P450 was PCR cleaned using Macherey-Nagel™ NucleoSpin™ PCR Clean-up XS kit.
3. Both the insert and the vector backbone were restriction-digested using the endonucleases EcoRI-HF and HindIII. To remove the MCS region of the vector, the digested backbone was run on 1% agarose gel and later eluted from the gel.

| Name of Fragment | Expected size (bp) |
| --- | --- |
| pSEVA 241 (backbone) | 3252 |
| CPR-P450 ( insert) | 4125 |

Both the digested CPR-P450 fragment and the pSEVA241 backbone were ligated using **NEB T4 DNA Ligase** in a 1: 3 vector-to-insert ratio.

**Results**

* To confirm our ligation, we started off by transforming the ligated product into *E.coli* Mach-1. We grew them on Kanamycin plates, and the colonies formed were inoculated using Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit to isolate the plasmid. Single digestion of the plasmid was done using EcoRI-HF. The digestion was run on 1% agarose gel.
* After analysing the gel results, we realised the presence of a smear instead of a thick band, indicating the **degradation of the plasmid**. We repeated the experiment multiple times after this, only to get the same result. In order to confirm the issue we switched to ligate the insert into pSEVA631 backbone.

**Aim**

Ligation of pSEVA631 backbone with tet\_CPR\_P450 insert.

**Experiment**

1. The pSEVA631 vector backbone was isolated from *E.coli* Mach1 using Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit.
2. The PCR amplified tet\_CPR\_P450 insert was PCR cleaned up using Macherey-Nagel™ NucleoSpin™ PCR Clean-up XS Kit.
3. Both the insert and the vector backbone were restriction digested using the endonucleases EcoRI-HF and HindIII. In order to remove the MCS region of the vector, the digested backbone was run on 1% agarose gel and further eluted from the gel.

| Name of Fragment | Expected size (bp) |
| --- | --- |
| pSEVA 631 (backbone) | 3519 |
| CPR-P450 ( insert) | 4125 |

The restriction digested tet\_CPR\_P450 insert, and the pSEVA631 backbone were ligated using **NEB T4 DNA Ligase** in a 1:3 vector-to-insert ratio. Gibson assembly was also used in an attempt to ligate the vector and backbone following a 2-fragment protocol.

**Results**

To confirm the ligation, we started off by running a gel for the ligated product.

![](https://static.igem.wiki/teams/5181/results/cprp450-ligated-smear.png)

Fig.20 : *Lane 1 NEB 1Kb Ladder, Lane 2 Ligated product (pSEVA631-CPR-P450)*

From the gel results, a smear can be seen from 10 Kb to 8 Kb, where the expected band is 7 Kb hence this result indicates that the ligated plasmid might have been degraded. Regardless, we decided to transform the ligated product into *E.coli* Mach1 competent cells. We grew them on Gentamicin plates, and the colonies formed were inoculated, and the plasmid was isolated using Miniprep. Single digestion of the plasmid was done using EcoRI-HF. The digestion was run on 1% agarose gel. Upon analysis of the gel electrophoresis, we noticed that there was a smear instead of a thick band at the required size.

**Based on a detailed structural analysis of our P450-CPR fusion protein,**

* These proteins are stable under catalytic conditions. As per Expasy Protparam, the protein might be stable >10 hr in E.coli under in-vivo conditions. This opens new avenues for experimentation and establishing the expression of this fusion protein inserted into our chassis *P. Putida*.
* Evaluation of transmembrane tendencies of the fusion protein revealed that both domains have less tendency to get embedded in the membrane and hence are more likely to work as native prokaryotic P450. This analysis enhances our understanding of the workings of our bio-bricks and paves the way for promising results in the near future.
([Refer](https://2024.igem.wiki/iiser-tvm/model) to molecular docking and structural studies)

# Conclusion

---

Results are beyond our expectations. We were successfully able to express the SaSSy-FPPS and synthesise santalene, a fragrant sesquiterpene responsible for the fragrance of sandalwood oil and the key precursor for santalol, which is the major component of sandalwood oil. We analytically detected santalene through GS-MS. Our main aim was synthesising santalene from PET, and we succeeded. Our second ambition was to oxidise (or convert) santalene to santalol, which was a lot more challenging as the expression of P450s in *Pseudomonas putida*, a prokaryote, is challenging.

While we were not able to express the designed P450 and CPR fusion protein in *Pseudomonas putida* due to time constraints, we successfully built a probable fusion protein and determined its structure via alpha fold and its catalytic domain via molecular docking with santalene and santalol isomers. Ultimately, we’ve shown that within a limited timeframe a small team of students can successfully engineer a microbe to produce a distinct scent.

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