Notebook

Week 1 · 14 Apr – 20 Apr

• IGEM Bootcamp
  While still finalising the project and plasmid design, the team decided to do a bootcamp for members to practice and be familiar with the laboratory techniques that will be used throughout the project. Preparations include making LB media, plates (LB, LB Cam and LB Spec), doing a plasmid mini prep, and making glycerol stock.
• Separately, our team set up overnight cultures for E.coli amilcp and E.coli rfp to decide on which chromoprotein would be best used for the project. After this, a plasmid mini prep was done and were later sent for sequencing.

Week 3 · 28 Apr – 4 May

• Minimum Inhibitory Concentration (MIC) for Bacillus, E.coli and Yeast
  28/04/25
  Three treatments per organism: MIC Methanol; MIC Ethanol; and pH test
  Two organisms: Escherichia coli DH5α and Saccharomyces cerevisiae.
  E. coli prefers LB media and 37℃. S. cerevisiae prefers YPD media and 30℃.
  Overnight cultures were set up for the following organisms.
• 29/04/25
  OD measurements were taken using 1:10 culture.
  MIC experiments using different media were done in 96 well plates for the two organisms above:
  Ethanol 80% media
  Methanol 20% media
  PH media
  A serial dilution was done for both ethanol and methanol MIC experiments. Plate setups are as shown below. Growth curve measurements were taken using the Agilent Biotek LogPhase 600 Microbiology Microplate Reader. Results were analysed using GraphPad Prism.

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MIC Plate Setups

Methanol concentration
Start with 20% v/v (2×) methanol    note: methanol is 32.04 g per mol

Initial load: 100 µL 20% methanol (col 1); 50 µL media in cols 2–10; 100 µL media in col 11; 50 µL media in col 12.
Next: Transfer 50 µL serial dilution till column 10.
Finally: Add 50 µL bacterial culture. Start loading culture from column 12, skip 11, then 10 → 1 so the same tips can be used.

Ethanol concentration
Start with 80% v/v (2×) ethanol

Initial load: 100 µL 80% ethanol (col 1); 50 µL media in cols 2–10; 100 µL media in col 11; 50 µL media in col 12.
Next: Transfer 50 µL serial dilution till column 10.
Finally: Add 50 µL bacterial culture. Start loading culture from column 12, skip 11, then 10 → 1 so the same tips can be used.

pH test
Start with media of 10 different pH measures

Initial load: 100 µL media into cols 1–12.
Finally: Add bacterial culture.

Week 14 · 14 Jul – 20 Jul

• 14/07/25
  Supelco Formaldehyde Test
  
  A formaldehyde test was done using Supelco Formaldehyde Test. To test for the first time, we created different formaldehyde solutions to match those in the kit’s protocol, and also included 5mg/L and 200mg/L to test for concentrations outside its detection range. We confirmed that the color matching was consistent with the protocol.


• 16/07/25
  Bromothymol Blue (BTB) Formaldehyde Test
  
  A BTB Experiment was done according to ThepChuay Experiment.

Week 15 · 21 Jul – 27 Jul

• 29/07/25
  In addition to the BTB Experiment, the same experiment was done using 1mM Iron.

Week 17 · 4 Aug – 10 Aug

• 08/08/25
  Supelco Formaldehyde Test Strips With Ethanol
  
  This experiment was done using a range of 5mL ethanol concentrations (0%, 5%, 10%, 20%, and 40%) with a range of formaldehyde added (0mg, 40mg, and 100mg).
  
  Supelco Formaldehyde Test Strips With Ethanol
  
  This experiment was repeated without ethanol. However, 1mL of each of the concentrations were used instead of 5mL which was sufficient enough to submerge the test strips. Results show that there is a clear gradient but the colors were all darker than expected. It also showed that the formaldehyde test works in strong 40% ethanol background.


• 11/08/25
  Cell Transformation of DH5a for plasmid amilCP with copper promoter
  
  DH5a was transformed with the amilCP plasmid containing the copper promoter. This was done using the NEB High Efficient Transformation Protocol. Transformants were plated in different dilutions (1x, 10x, and 100x). pUC19 in Amp plate was used as a positive control, while pUC19 in Kan plate as a negative control.
• 12/08/25
  Rususpension of Primers
  
  The following primers were resuspended using TE buffer to a final concentration of 0.1nM.
  mxaA_F
  mxaK_R
  amilCP_R
  amilCP_F
  MDH_R
  MDH_F
  Chim_R
  Chim_R
  
  Primers were diluted in 1:10 to have 10uM as aliquots to be easily used.


• 12/08/25
  PCR for amilCP with copper promoter transformants
  A PCR was done to the amilCP transformants. A mastermix was prepared containing GoTaq Hot Start Green 2x MM buffer, amilCP_F, amilCP_R, and MilliQ. 5 colonies from the 10^-3 diluted transformant plates were picked, along with an amilCP plasmid as a control.


• 14/08/25
  A gel electrophoresis was performed to the PCR products. Results show that bands are visible for all the 6 colonies and the positive control.
  Colonies with correctly assembled plasmid contructs that were verified using PCR were inoculated into LB media with KMSO and incubated at 37C with 200 rpm shaking.


• 14/08/25
  Induction of amilCP transformants with copper promoter using copper chloride
  Cultures were inoculated in LB with 50mg/mL KMSO antibiotic to OD600 = 0.1. These were growth to OD600 = 0.5-0.7 at 37C with 200rpm shaking. The cultures were split into 3mL aliquots. Copper chloride was added to 1uM, 10uM, and 20uM, and then the cultures were placed into shaking incubator at room temperature with 200rpm shaking for 5 hours and checked for color change.


• 15/08/25
  Plasmid Mini Prep of plasmid with amilcp was done according to protocol except for the final step. Once wash 2 was discarded, the samples were centrifuged for another 2 minites to remove residual wash.
  Following the induction of the amilcp gene by cusC, we spun down the samples to check if there was color. After 25hours induction, no color has been produced.

Week 18 · 11 Aug – 17 Aug

• 18/08/25
  Mxa support protein plasmid transformation in 5-alpha and BL21 cells
  Transformation was done according to experimental protocol.
  Competent cells used: C2987 NEB 5-alpha competent E.coli cells & C3010 T7 express lysY competent E.coli cells
  Plasmid: MDH support protein plasmid DNA
  Media: SOC recover media
  After transformation, a serial dilution was done for 10x and 1000x for samples and plated into LB Cam plates and incubated in 37C.


• 19/08/25
  PCR was done according to protocol.
  Primers used: mxaA_F & mxaK_R
  Colonies used: 8 colonies from 18/08/25
  8 reactions were done, including one control (mxA plasmid)
  mxaA_F: Tm = 55.8C
  mxaK_R: Tm = 55.8C
  For 25 cycles

• After PCR, an agarose gel electrophoresis was done. 5uL of samples were loaded onto the wells, and a quickload ladder was used.
• Overnight cultures were also set up for the following samples
  Mxa plasmid E.coli BL21
  Mxa plasmid E.coli Dh5a
  AmilCP plasmid E.coli Dh5a

Week 19 · 18 Aug – 24 Aug

• 21/08/25
  From previous overnight cultures, glycerol stocks were made, followed by a plasmid mini prep.
  Overnight cultures were done for the following:
  Mxa E.coli Dh5a cam
  Mxa E.coli BL21 cam


• 22/08/25
  A plasmid mini prep was done for the MDH accessory proteins (DH5a and BL21).
  SOB and TB mdia were also prepared.

Transformation Buffer (TB) Recipe
Final composition: 10 mM HEPES, 15 mM CaCl₂, 250 mM KCl, 55 mM MnCl₂. Adjust pH to 6.7 with KOH before adding MnCl₂. Do not adjust pH after MnCl₂ is added. Filter sterilise and store at 4 °C.

100 mL Transformation Buffer

250 mL Transformation Buffer

Preparation Protocol

1. In beaker, add ~80% of the final volume of milliQ water.
2. Add HEPES (powder or 1 M stock), dissolve.
3. Add KCl, dissolve.
4. Add CaCl₂, dissolve.
5. Adjust pH to 6.7 with KOH.
6. Dissolve MnCl₂ in a small volume of milliQ water separately (~10% of the final volume).
7. Add MnCl₂ solution gradually to the base solution with stirring.
8. Do not adjust pH after adding MnCl₂ (a drop in pH is expected).
9. Bring to final volume with milliQ water.
10. Filter sterilise.
11. Store at 4 °C (indefinitely stable).

Week 20 · 25 Aug – 31 Aug

• 25/08/25
  Formaldehyde Experiment
  For this experiment, 0.04% of BTB was used which is 0.004g of BTB in 10mL H2O.
  2 concentrations of Hydrogen Peroxide were used (0.1M and 0.005M) alongf with 0.01M of Sodium Hydroxide.
  Iron chloride was also used for this experiment alongside iron sulfate.
  FeSO4 (0.4% and 0.8% final concentration)
  FeCl3 (0.4% and 0.8% final concentration)
  These were done on a 96-well plate. Images of the plate can be found in the result section of the wiki.


• 26/08/25
  Transformation of plasmids
  
  Plasmids used: MDH mxaAK (KanR), MDH (MoxF1), amilCP MxaY, and MDH MoxF1
  Cells used: Dh5a, 10b, abnd T7lysY
  Dh5a was transformed with MDH mxaAK (KanR). 10b was transformed with MDH MoxF1 and amilCP MxaY. T7lysY was transformed with MDH MoxF1
  Transformation protocol was used using SOC media for DH5a and T7lysY cells and using specific 10b media for 10b cells.
  2 LB plates were done for each (1/100 or 1x10^-2 dilution) and 100uL of culture.


• 28/08/25
  Colony PCR was done for plasmids NEB 10b PMDH MoxF1, Dh5a pMDH MxaAK, and NEB 10b pAmilCPMxaycuss.
  PCR protocol was used.
  PCR setup:

• A gel electrophoresis was also done according to protocol.
  Colony 1 from the PCR plasmid came out to be the best and was inoculated into 10mL LB broths with Km antibiotic(10uL).
  Strains were inoculated at 37C.


• 29/08/25
  Glycerol stocks were done in 2 replicates to dh5a pMDHMxaAK, NEB 10b pMDH MoxF1, and NEB 10b pAmilCPMxaYCuss. Following this, a plasmid miniprep was done according to protocol.
  Overnight cultures were set up for NEBa pAmilcp and no plasmid, as well as NEB10beta pamilcpMxaYCusS and no plasmid for induction.

Week 21 · 1 Sep – 7 Sep

• 02/09/25
  Transformation
  NEB T7 express competent cells were transformed with pMDH MoxF1, pMDH MoxF1 + pMca, or PUC19. Transformation was done according to protocol except was heatshocked for only 10 seconds, put on ice for 5 minutes. SOC media was used.
  Streak plates were also done for the transformants.


• 03/09/25
  Induction of chimera sensor
  5mL cultures were set up
  1. NEBa no plasmid
  2. NEB10beta no plasmid
  3. NEBa amilcp
  4. NEB10beta amilcp chimera
  Cultures were incubated at 37C


• 04/09/25
  The OD600 for the overnight cultures were measured. 0.1% (w/v) glucose cultures were inoculated to OD600 and grown at 37C to mid exponential phase.
  Copper chloride induction
  3mL cultures were set up. 3 concentrations were done (1mM, 2mM, and 500uM)
  Methanol induction
  3mL cultures were set up. 4 concentrations were done (0%, 1%, 2.5%, and 5%)
  Cultures used were NEBa amilcp, NEB10beta amilcp chimera, NEBa no plasmid, and NEB1b no plasmid


• 05/09/25
  Transformation
  T7 express cells were transformed accoriding to transformation protocol.
  4 plasmids were used: moxF1, MxaAK, MxaAK + MoxF1 and Puc19.
  These were plated. MoxF1 and MxaAK were diluted in 1x, 1/10x and 1/100x, double trasnformation with 1x and PUc19 in 1x and 1/10x.
  One extra plate was done for each transformation. Concentrated cells were spin down and the pellet was plated labelled as concentrated cells.
  For double plasmids, these were recivered for 60 minutes at 37C. Recovered cells were added to 2mL of LB with Kan+Cam. Additional recovery was done for 60 minutyes at 37C and plated into LB Kan+cam plates.


• 06/09/25
  Cells were spun down. Results are photograpged. Amilcp cells were then resuspended and transferred to 15mL tubes

Week 22 · 8 Sep – 14 Sep

• 08/09/25
  Cultures were inoculated at 10am. OD600 measurements were taken at different time points until it reached 0.6. Glycerol stocks were made after according to protocol and were stored in –80C.
  Control AmilCP Copper were induced using 10uL of 20mM Cu2Cl. No immediate color response during the day and will be further observed the following day.
  Colony from pMDH MoxF1 KanR (05/09/25) was inoculated in 3mL LB +Kan in 15mL falcon tube and incubated in 37C.


• 09/09/25
  Cells were inoculated in SOB 25mL(25uL Kan) with 500uL of overnight culture and incubated at 30C.
  T7x MDH MoxF1: KanR cells were made chemically comptent according to protocol.


• 10/09/25
  Transformation was done to pMDH MoxF1 + pMxa KanR/CamR and Puc19 according to protocol.
  Puc19 was plated with 1/10x dilution. MDH MoxF1 + pMxa KanR/CamR was plated in 1x, 1/10x, and 1/100x dilution. Plates were stored in 37C.
  Overnight cultures for pamilCP and pchimera were done for copper inductions the following day. LB kan was used for cells with plasmid, and LB only for controls (NEB alpha and NEB 10b cells)


• 11/09/25
  Expression of methanol dehydrogenase was done using transformants plates from 10/09/25. Colonies from the 1:10 dilution plate was suspended into 5mL LB.
  Copper induction of amilcp and chimera plasmids:
  1. E.coli 10b pamilcp chimera LB Kan
  2. E.coli NEBa pamilcp LB Kan
  3. E.coli 10b no plasmid
  4. E.coli NEBa no plasmid
  Cells were spun down to pellet and supernatant was discarded. Pellet was resuspended woth 1xTBS (500uL). 1mM CuCl2 was added to E.coli NEBa pamilcp and NEB10alpha. 5mM CuCl2 was added to E.coli 10b and E.coli 10b pamilcp chimera.
  
  Preparation of PQQ (330g/mole)
  In a falcon tube, add 1 tablet of PQQ and add 12mL of MilliQ. Each tablet had 40mg of PQQ [10mM]. Due to starch present, it does not dissolve properly, spin the solution for 10 minutes. Make a sterilised PQQ solution by transferring it using a syringe and 0.2um filter into a new falcon tube.
  
  Protein expression using T7 express
  OD of E.coli T7x pMDHMoxF1 KanR + pMxa was checked and grown to OD=0.4. 3uM of PQQ was added. The cell culture was divided into two conical flask. One is induced with0.4mM IPTG and other is uninduced. Flasks were incubated at 16C overnight.
  
  The following buffers were prepared: 2x binding/wash buffer, His elution buffer, and Buffer modifiers.
  

  	Sodium Phosphate	NaCl	Imidazole	Tween–20
  2× Binding/Wash Buffer	0.69 g	1.75 g	—	10 μL
  His Elution Buffer	0.345 g	0.877 g	1.03 g	5 μL
  1 M NaCl	—	2.92 g	—	—
  0.1 M Imidazole	—	—	0.345 g	—

• 12/09/25
  Methanol Dehydrogenase was extracted and purified from the induced and uninduced cells with 2 replicates each. Cells were spun down and resuspended in 2.5mL of TE buffer, spund down and discreded the TE buffer. It was then resuspended in 2.5mL 1x binding buffer, sonicated for 3 mins on 10 sec pulse to lyse. Protein Isolation and purification were done according to protocol. This was then stored in the –80C.

Week 23 · 15 Sep – 21 Sep

• 15/09/25
  Protein gel was run using cell pellet, supernatant containing lysed cells, and purified protein (induced vs. Uninduced) according to protocol.
  
  Gel running buffer preparation was done. 100mL of 4x Tris/Glycine/SDS and 900mL of RO water were used to make 1L of 1x Tris/Glycine/SDS Buffer.
  Protein concentration for supernatant and purified protein were measured using the nanodrop.
  Loading samples were prepared in PCR tubes for the following (1 set for 10ul and 1 set for 15uL of protein sample)
  Samples:
  Purified protein (induced) 2 replicates
  Purified protein (uninduced) 2 replicates
  Lysates (induced) 1 replicate
  Lysates (uninduced) 1 replicate
  
  For 10uL protein samples, 10uL of 2x loading dye was added. For 15uL protein samples, 15uL of 2x loading dye was added.
  SDS-PAGE was done according to protocol.
  Overnight staining and destraining were done according to Coleman protocol.
  Overnight cultures were done for amilcp, amilcp chimera, and pMDH MoxF1 + Mxa and their controls.


• 16/09/25
  Cells were grown to OD = 0.4
  1. T7x MDH
  2. NEBa amilcp
  3. NEB10beta chimera
  4. NEBa
  5. NEB10beta
  Cells were induced with either (0, 1, 2.5 and 5%) methanol and (0, 0.5, 1mM) copper
  For T7x pMDH MoxF1 + pMxA induced as per 11/09/25. 6uM of PQQ was added to the 50mL culture then split into two flasks. One was for inducing with 0.8mM IPTG.


• 17/09/25
  Purification of protein was done according to protocol. Samples were placed in –80C freezer.
  Samples from 16/09/25 were spun down and photographed results.
  Overnight cultures for pMDH MoxF1 + pMxa t7x was done.


• 18/09/25
  pMDH MoxF1 + pMxa t7x was induced at OD=0.65. 300uL of the 10mM PQQ stock was added to 500mL culture then split culture into 2 lots of 250mL both in 2L flasks, added IPTG 250uL of 1M stock to 250mL culture to induce 1 flask. PQQ = 6uM and IPTG=1mM.
  
  SDS Page was done according to protocol for the following samples. 15uL and 10uL protein samples were loaded.
  1. MDH induced
  2. MDH uninduced
  3. Contaminated MDH induced
  4. Contaminated MDH uninduced
  5. Supernatant induced
  6. Supernatant uninduced
  7. E.coli pellet induced – only for 10uL
  8. E.coli pellet uninduced – only for 10uL
  
  The following were prepared: 2x xT sample buffer, 10% v/v beta-merck to make up the gel running buffer.
  For 5uL protein, 9.5uL of samples were loaded while 20uL was loaded for 10uL protein.
  
  Destaining solution was also made using 10% (v/v) methanol and 10% (v/v) acetic acid.
  Gel was stained in Coomassie blue staining solution for 30 minutes. Stain was removed and added high destain for 30 minutes. After removing high destain solution, pour low destain solution and keep overnight.


• 19/09/25
  1. Testing of PBS affects the formaldehyde test strips, testing the strips at 40mg/L
  Control: 900uL water + 100uL (0.04%) formaldehyde
  Test: 900uL PBS + 100uL (0.04%) formaldehyde
  Added two drops of Fo1
  Result – PBS does affect formaldehyde strips. Color change was inconsistent with reference concentration
  
  2. Repeat of experiment, did one formaldehyde strip just with PBS and water, no formaldehyde added but 2 drops Fo1
  Result – no color change for PBS or water
  
  3. Repeat of experiment
  Control: 900uL water + 100uL formaldehyde (0.04%) + 2 drops pf Fo1
  Test: 900uL + 100uL formaldehyde (0.04%) + 4 drops pf Fo1
  Result – correct color change observed
  
  4. Repeat experiment
  Control: 900uL NaCl (0.9%) + 100uL formaldehyde
  Test: 900uL of sodium chloride (0.9%) + 100uL formaldehyde + 2 drops of Fo1
  
  0.15M =0.9% NaCl used filter sterilised sodium chloride for the experiment “resting cell assay” rather than LBS as PBS interferes with the ph
  Results – 0 formaldehyde for all

Week 24 · 22 Sep – 28 Sep

• 22/09/25
  Uninduced and induced pmdh moxf1 mxa thawed at room temperature, pellet cells and resuspended in 1x binding buffer. These were sonicated for 3 minutes. Dynabeads protocol was followed with 700uL samples to compensate, insteadd of 4 washes, doing 5 wases with 1x binding buffer, 500uL. The resulting supernatant was transferred to fresh centrifuge tubes and stored in –80C freezer.
  Samples in the end: 2x supernatant, 2x pellet and 2x pellet with lower concentration, and 2x his tagged purified.


• 23/09/25
  SDS- gel run for protein (similar procedure from 18/09/25)
  The following samples were used and run in the gel. 20uL was loaded into each well
  1. MDH (in)
  2. MDH (un)
  3. Supernatant (in)
  4. Supernatant (un)
  5. Pellet (in)
  6. Pellet (un)
  Pellet was resuspending in 1x loading dye.
  Gel stain and destain was also done.