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.

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

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.

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

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

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.

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.

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.

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.

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

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

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

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.

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

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.

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

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.

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

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

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

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



• 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

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



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




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

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




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



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)



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

Day 114 - 25/08/2024

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



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



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




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

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




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




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.




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*

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!!!!!




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

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




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

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




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.

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

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.



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










• 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



• 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

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