Protocols

Agarose Gel Preparation

Reagents and Main Equipment

Quantities shown are per 100 mL of Milli-Q.

• 50× TAE 1.0 mL
• Agarose 1.5 g
• LED Stain G 0.5 μL
• Milli-Q (pure water) 100 mL (50 + 50 mL)
• Gel casting tray and plate
• Comb (a comb-like tool that creates wells in the gel for sample loading)

Procedure

1. Add 50 mL of Milli-Q to an Erlenmeyer flask.
2. Add 1.0 mL of 50× TAE.
3. Add 1.5 g of agarose and heat in a microwave until completely dissolved.
4. Add 50 mL of Milli-Q.
5. Add 0.5 μL of LED Stain G.
6. Mix well.
7. (If storing) Wrap the Erlenmeyer flask with aluminum foil to protect from light.
8. Pour the liquid gel into the casting tray (about 80% full), insert the comb, and remove bubbles with a pipette tip.
9. Cover with paper and wait until solidified.

Alternative Version Materials

For 100 mL:

• 0.5× TAE 100 mL (located in the tank next to the ice maker)
• Agarose 1.5 g
• LED Stain G 0.5 μL
• Milli-Q (pure water) 100 mL (50 + 50 mL)
• Gel casting tray and plate
• Comb

For the procedure, use 0.5× TAE instead of Milli-Q.

Agar Media Preparation

Reagents and Main Equipment

Quantities shown are per 100 mL of Milli-Q.

• LB 2.5 g
• Agar 1 g
• Ethanol (EtOH) 1 mL
• Antibiotics (as needed):
  • Chloramphenicol solution (100 mg/mL) 20 μL
  • Ampicillin solution (100 mg/mL) 50 μL
• Dishes (Petri dishes, 10 cm diameter)
• Autoclave, aluminum foil cover

Procedure

1. Prepare chloramphenicol ethanol solution (100 mg/mL):

   • Add 1 mL ethanol to a tube.
   • Add 0.1 g chloramphenicol and mix.

2. Prepare media:

   • Add 100 mL Milli-Q to an Erlenmeyer flask.
   • Add 2.5 g LB and 1 g agar.
   • Add 20 μL of chloramphenicol solution.
   • Cover with aluminum foil and autoclave at 121 °C for 15 min.
   • After cooling to 45 °C, add 50 μL ampicillin solution.
   • Pour into plates and store at 5 °C.

Liquid Media Preparation

Reagents and Equipment

Quantities shown are per 100 mL of Milli-Q.

• LB 2.5 g
• Milli-Q 100 mL
• Antibiotics (as needed):
  • Chloramphenicol (100 mg/mL) 20 μL
  • Ampicillin (100 mg/mL) 50 μL

Procedure

1. Add 100 mL Milli-Q and 2.5 g LB to a flask.
2. Add chloramphenicol solution.
3. Autoclave at 121 °C for 15 min.
4. After cooling to 45 °C, add ampicillin.
5. Store at 5 °C.

PCR

Reagents and Equipment

• Forward primer (100 μM) 0.5 μL
• Reverse primer (100 μM) 0.5 μL
• DNA 1 μL
• Milli-Q 19 μL (9 + 10 μL)
• 2× KOD One (Dye-containing) 12.5 μL
• PCR tube

Procedure

1. Prepare diluted primers (20×):
   Add 9 μL Milli-Q + 0.5 μL each primer → mix.
2. Prepare master mix:
   Add 10 μL Milli-Q + 12.5 μL KOD One → mix.
3. PCR setup:
   Add 22.5 μL master mix + 1.5 μL primer mix + 1 μL DNA → mix.
4. Thermal cycler:
   2-step PCR, extension 5 sec/kb (30–40 cycles).

Electrophoresis

• PCR product 3 μL
• 1 kb DNA Ladder 4 μL (both ends)
• Agarose gel 1 sheet

Run 20–40 minutes → visualize under UV.

Gel Purification

Reagents

• PCR product (Dye-containing) 20 μL
• Dissolve Buffer 400 μL
• DNA Wash Buffer 200 μL
• DNA Elution Buffer 20 μL
• Spin column + collection tube

Procedure

1. Run gel electrophoresis → cut target band.
2. Dissolve in 400 μL buffer at 37 °C.
3. Spin column purification → wash → elute 20 μL.

PCR Product Purification

Reagents

• PCR product 20 μL
• DNA Binding Buffer 40 μL (insert) / 100 μL (plasmid)
• DNA Wash Buffer 200 μL
• DNA Elution Buffer 10 μL

Procedure

1. Mix PCR product + binding buffer.
2. Spin column → wash → elute 10 μL.

Nucleic Acid Concentration Measurement Using NanoDrop

Procedure

1. Launch NanoDrop.
2. Measure with 2 μL sample (after blanking with dH₂O and Tris-HCl buffer).
3. Wipe with Kimwipe after each step.

Overall Object

Create the following fragments and plasmids, and prepare storage plates transformed into BL21(DE3) (DE3).

20250630

Purpose

Prepare LB agar medium, agarose gel, and DNA sample.

Procedure

Prepare agarose gel

200ml of solution using Milli-Q water.

Result

Prepare 6 agarose gels for EP. Prepare 4 agarose gels for gel extraction.

20250701

Purpose

Prepare LB agar medium and DNA sample. Perform PCR for fragment optimization.

Result

PCR

For TtrS, PCR was performed with 2μl of DNA and 9μl of Milli-Q water. The numbers above were written and stored at -20°C. pBluescript SK(-) was substituted with pBlue-shRNA plasmid from last year’s samples.

Discussion

The pBlue-shRNA sample may have failed, so carefully check it with electrophoresis. The TtrS sample concentration is low, so it’s acceptable to use double the amount during PCR.

20250702

Purpose

Prepare LB and LB agar medium. Check the result of PCR carried out yesterday (2025.07.01).

Procedure

Prepare LB medium

I prepared 100ml without antibiotics.

Prepare LB agar medium

I prepared 100ml without antibiotics.

Prepare agarose gel

Prepared 100ml

Load these samples into the wells of the gel.

Result

Prepared 100ml LB medium without antibiotics. Prepared 100ml LB ager medium without antibiotic.

I ran E.P. at 50V for 50 minutes using the gel I made today.

Added 2μl of each PCR product.

Discussion

The gel used in first EP was very thick. That’s why the bands are severely tailing. When you want accurate electrophoresis results, you should use a thinner gel and run it at 50V. Samples 1 and 3 have extra bands appearing, the cause is not immediately apparent.

20250703

Purpose

Prepare the received DNA samples and perform PCR.

Procedure

Load these samples into the wells of the gel.

Result

The wells on both ends contain the ladder.

Discussion

Multiple bands were observed for No. 17, 20, 21, 22, 23.
It would be better to purify these using gel extraction . No band was detected for No. 24. It’s possible that there was a hole in the gel or the sample was forgotten, so it would be better to repeat the electrophoresis along with No. 25.
It would be good to try gel purification for No. 17, 20, 21, 22, 23, 24, 25. PCR product purification should be sufficient for the remaining samples.

20250707

Purpose

Check the result of PCR product No.26.
Purify the PCR products No.2,3-16,18,19,26.

Procedure

Electrophoresis

Loaded PCR product No.26 and DNA ladder then electrophoresis.

PCR product purification

PCR product No.4,8,10,14,16,19: Loaded 40 μL of DNA Binding Buffer.
PCR product No.2,5,6,7,9,11,12,13,15,18,26: Loaded 100 μL of DNA Binding Buffer.
Finally store at -20 ℃.

Result

Electrophoresis

A band was observed．

Discussion

It seems that the desired 1429 bp band of noxE was obtained.

20250708

Purpose

Examine the quality of fragment samples that have been fragment-adjusted.
Re-adjust the failed fragment samples.

Procedure

Electrophoresis

Result

Discussion

Sample24 was not obtained in the first PCR due to omission.
Samples 4, 8, and 10 showed a single band, so PCR purification is fine.
Sample 12 requires gel purification.
For samples 24 and 25, the procedure should be reviewed, including contamination, primers, annealing temperature, and cycle number.
Samples 3, 20, and 23 contained many impurities, resulting in low purity.

20250709

Purpose

Readjust failed samples.

Procedure

Electrophoresis

Result

Electrophoresis

Discussion

The results of NanoDrop were not good entirely.
No.4, 8, 10, and 12 require re-PCR.
The 260/230 ratio frequently deviated substantially from 1, we want to investigate causes.
The values for No.3, 20, and 23 are also not very good.

20250710

Purpose

PCR of failed samples.
Colony PCR of yesterday’s transformation.
Assembly wherever possible.

Procedure

Preparation of gel

Result

Plate with over night

Discussion

Colonies were observed on the transformation plates.
Yesterday, I was not sure about the control Amp plates, but growth was observed and they seem fine, so proceed to large-scale culture (1’,2’,3’: pBl-kill; 4’,5’,6’: pBl-EGF-LARD3).
Since no liquid medium without antibiotics was available, no positive control was included.
pTf16-PrtD-PrtE was missing PrtF, so it will be redone.

20250711

Purpose

Re-run electrophoresis and extraction for samples 4, 8, 10, and 12.
Plasmid extraction from large-scale culture.
Hifi assembly.

Procedure

Result

Electrophoresis

No. 24,25 The wells on ends are ladders.

Discussion

Electrophoresis went well, but during subsequent PCR purification and gel purification, the260/230 ratio was not good, indicating that the buffer was not completely removed.
Since PCR purification tends to give a higher yield and the 260/230 ratio tends to be higher, we would like to proceed with PCR purification as much as possible.
Samples 4, 8, and 10 might have had a higher success rate if PCR purification had been used.
For sample 12, the extension time was longer than intended, so it seems better to shorten it.
If performing gel purification, it may be better to include the wash buffer step twice.

20250714

Purpose

Carry out PCR.
Assemble DNA fragments.
Transform BL21(DE3) and DH5α.

Procedure

Transformation (BL21(DE3))

Use the plasmids extracted last Friday(250711).
(pBlue-EGF-LARD3 and pBlue-TtrR-BsalM1-BsalM2-BsalR)
Carry out recovery culture using SOC medium at 37 ℃for 40 min after transformation.
Use LB-agar medium with 50 μg/mL ampicillin to culture these transformed E.coli BL21(DE3).

Transformation (DH5α)

Use the plasmids assembled today(250714).
(pBlue-6×His-EGF-LARD3, pTf16-TtrS, and Control).
Carry out recovery culture using SOC medium at 37 ℃for 40 min after transformation.
Use LB-agar medium with 50 μg/mL ampicillin for pBlue-6×His-EGF-LARD3 and Control, and use LB-agar medium with 20 μg/mL chloramphenicol for pTf-TtrS.

Result

Electrophoresis

Discussion

We didn’t have enough time for recovery culture, so we changed the time from 60 min to 40 min. This might cause negative effect to cell with in the medium with chloramphenicol.

20250715

Purpose

Confirm whether the plasmid was inserted by transformation performed yesterday.

Procedure

Transformation (DH5α)

Use the plasmids assembled yesterday(250714).
(pBl-xlB-xdh-noxE, pTf16-PrtD-PrtE-PrtF, pTf16-xytB-xytC-xytD-xytE and Control)
Use LB-agar medium with 50 μg/mL ampicillin for pBl-xlB-xdh-noxE and Control, and use LB-agar medium with 20 μg/mL chloramphenicol for (pTf16-PrtD-PrtE-PrtF, pTf16-xytB-xytC-xytD-xytE).

Result

Carry out recovery culture using SOC medium at 37 ℃for 60 min after transformation.

Colony PCR

Electrophoresis

Samples 5-12 were cultured in large volume.

Discussion

No bands appeared in the colony PCR for pbl-TtrR-BsalM1-BsalM2-BsalR; I should have used primer kill-7 instead of kill-5.
Also, the extension time was longer than the appropriate length.
No.12 shows multiple bands and low DNA yield; I need to verify the primers and optimize the PCR protocol.

20250716

Procedure

Transformation (DH5α)

Use the plasmids assembled today(250716).
(pTf16-PrtD_lac-PrtE-PrtF)
Use LB-agar medium with 20 μg/mL chloramphenicol.

Transformation (BL21(DE3))

Use the plasmids minipreped today.
(pTf16-TtrS, pBlue-6xHis-EGF-LARD3 and control)
Use LB-agar medium with 50 μg/mL ampicillin for pBlue-6xHis-EGF-LARD3 and Control, and use LB-agar medium with 20 μg/mL chloramphenicol for pTf16-TtrS.

Result

EP colony PCR

Discussion

For sample 12, the 260/230 ratio was negative. Although not ideal, due to limited time, HiFi was performed.
Colony PCR samples 13, 14, and 15 showed no bands. Possible causes are that KOD One was left at room temperature for a day or human error.
Colonies grew for samples 31, 32, and 33, but no bands appeared in PCR.

20250717

Procedure

Result

EP BL21(DE3)-pBlue-TtrR-BsaIM1-BsaIM2-BsaIR

Discussion

The NanoDrop 260/280 ratio is still abnormal.
After extracting the samples that had not been done by Miniprep and adding them, the values worsened. Since they were from the same strain and carried the same plasmid, I added them to the samples prepared yesterday. Samples handled in separate operations should be stored separately.
One colony from each plasmid was selected and used for transformation.

20250718

Procedure

Result

Electrophoresis

The remaining 3 are PCR samples 4, 5, 6 from the first run.

Discussion

Since the competent cells showed no pellet after 2.5 hours of culture, I suspect the transformation did not work.
Since pBl-xlB-xdh-noxE showed no growth, it may be better to redo the transformation.
For the second electrophoresis, I used a gel that had already started to solidify, which resulted in a messy gel.

20250722

Procedure

Result

Colony PCR

20250723

Purpose

Redo the preparation of low-quality plasmids.
Colony PCR from DH5α plates.
Prepare a large-scale culture of BL21(DE3).
Colony PCR from BL21(DE3) plates.
Using the large-scale BL21(DE3) culture, prepare competent cells → perform transformation.

Result

EP Colony-PCR

Discussion

Colony-PCR

・For 5’ and 6’, the DNA solution was prepared entirely in CP medium, so the bacteria in the DNA solution were dead.
→Colony PCR was redone on Amp plates.
・For 7’ and 8’, one of the two primers was forgotten, so only TtrS is visible.
→Redo colony PCR tomorrow.
・Large-scale culture of 1’ and 3’, which showed bands.
・7’ has not been done yet because no Amp+CP liquid medium was available.

Transformation

・By mistake, I used the remaining large-scale culture before making competent cells for transformation.

20250724

Purpose

Plasmid extraction of samples 1, 2, 4, 5, 6, 8, and 9 and transformation into BL21(DE3).
Redo the transformation and colony PCR that I made a mistake with yesterday.

Result

20250725

Procedure

Result

25’ was the same with 25 because I used the wrong primer.

Discussion

I tried cut-check and partial PCR for xylB and xyt.
Since I am uncertain about the bands for 9, 10, and 11, I would like to perform a cut-check after miniprep.

20250728

Procedure

20250731

Procedure

Result

Electrophoresis

Discussion

The simultaneous transformation of two plasmids is working well.
1 has an issue with noxE, 2 has a suspicious band position, 3 has some samples that show only one band, 5 has no band.
4 and 6 look good.

20250801

Purpose

Check the result of transfection.
Transform BL21(DE3) by pBlue-TtrR-BsaIM1-BsaIM2-BsaIR and pTf16-TtrS.
Make master plate of BL21(DE3) transformed by (pBl-6xHis-EGF-LARD3 & pTf16-lac-PrtDEF) and (pBl-6xHis-EGF-LARD3 & pTf16-J23118-PrtDEF).

Procedure

Transformation.
Transform BL21(DE3) by pTf16-TtrS and pBlue-TtrR-BsaIM1-BsaIM2-BsaIR in one step.
Master plate.
Make master plate of BL21(DE3) transformed by (pBl-6xHis-EGF-LARD3 & pTf16-lac-PrtDEF) and (pBl-6xHis-EGF-LARD3 & pTf16-J23118-PrtDEF).

Result

20250804

Purpose

Check the result of transfection.

Procedure

Transfection

Performed in double quantity.
Add plasmids 20μl each sample.

Result

Both sides are ladder.

All DNA samples within acceptable range.

Discussion

All strains except for the xylitol strain have been completed. Only need to perform large-scale culture and make master plates. Last time, setting the PCR denaturation at 98°C for 5 minutes sometimes failed to produce bands, but increasing it to about 7 minutes resulted in successful bands. It might be worth trying an initial 98°C denaturation time of around 8 minutes.

20250805

Procedure

Result

Discussion

The pBl from yesterday’s transformation did not show any bands.
The pTf16 bands were very faint, but I performed large-scale culture for 1, 2, and 3. It might be better to redo the transformation.
Made master plates. Only the BsaI concentration was low, so it is still being cultured.
Made tetrathionate solution. Tried to make 1 M, but it saturated, so made it at 0.5 M instead.

20250815

Purpose

Perform a wound healing assay in a 6 cm dish according to this protocol. Examine whether the presence or absence of EGF affects wound healing in C2BBe1 cells.

Procedure

Passage cells from the dish.
Culture for 6 hours.
Observe under the microscope.

Result

After 6 hours of culture, cells were observed under the microscope, but there were not enough cells for the wound healing assay.

Discussion

It seems that reaching confluence takes about 3–4 days.
The medium was changed and cells were cultured on the 8/16 and 8/17. The assay will be performed on the 8/18 and checked on the 8/19.

20250818

Purpose

Perform a wound healing assay on the cells passaged on the 8/15. We referred to these documents.

Procedure

Make a scratch in the 6 cm dish using a P200 tip.
Wash with PBS, then add DMEM to one dish and DMEM supplemented with EGF at 50 ng/mL to the other dish.
Observe the wound under the microscope and incubate.

Result

The photos taken with the microscope were turned into a GIF and posted on 20250821.

20250819

Purpose

Check the 6 cm dish.
Perform a wound healing assay in a 24-well dish.
Mass culture for SDS-PAGE and WB.

Procedure

Observation of the 6 cm dish. Make a wound in the confluent C2BBe1 cells in a 24-well dish with a P200 tip. Wash with PBS, then add EGF at the following four concentrations. ① 0 ng/mL ② 5 ng/mL ③ 10 ng/mL ④ 50 ng/mL Three 24-well plates were cultured for each EGF concentration (4 concentrations × 3 plates each). Observe the plate placed in the center under the microscope.

Add LB medium to a 50 mL tube. ① 13 mL of LB medium in 4 tubes. (Amp 100 μg/mL, CP 20 μg/mL) ② 15 mL of LB medium in 4 tubes. (Amp 100 μg/mL) ③ 15 mL of LB medium in 4 tubes. (CP 20 μg/mL) Add 10 μL of the following liquid-cultured bacterial cells to tubes ①–③. ① 250731 BL21(DE3) pTf16-lac-prtDEF, pBl-6xHis-EGF-LARD3 ② 250725 BL21(DE3) pBl-6xHis-EGF-LARD3 ③ 250725 BL21(DE3) pTf16-lac-prtDEF Incubate with shaking at 200 rpm and 37 °C.(O/N)

Result

The photos taken with the microscope were turned into a GIF and posted on 20250821.

Discussion

Cell proliferation was observed in both 6 cm dishes.

SDS PAGE & Western blotting

20250820

Purpose

Induce expression by adding IPTG to the E. coli that were massively cultured on 250819. Observe cell proliferation in the 24-well and 6 cm dishes.

Procedure

Cultured BL21(DE3) containing pBl–TtrR–EGFP, pTf16–TtrS, and control strains.

Result

Strong fluorescence observed under UV.
Culture dense. The photos taken with the microscope were turned into a GIF and posted on 20250821.

20250821

Purpose

Prepare second round of xylitol verification and Kill Switch GFP validation.

Procedure

Prepared xylitol media with IPTG induction (0.5 mM).
OD600 measured every 1–2 hours.

Result

Growth and fluorescence observed;
OD600 increased in xylitol medium.

Results of wound healing assay

Discussion

Possible phosphate precipitation noted;
will repeat experiment under same conditions.

20250822

Purpose

Colony PCR and storage plate preparation. Carry out SDS-PAGE and Western blotting.

Result

Bands confirmed.
Storage plates prepared successfully.

20250825

Purpose

Preparation for verification experiments.
Creation of master plate.

Procedure

Negative control, pBl-xylB-xdh-noxE, and pBl–BsaIR colony PCR
Large-scale culture
・Competent cells only
・Kill strain (with/without tetrathionic acid)
・Negative control

Transform BL21(DE3) with pBl-xylB-xdh-noxE Media preparation ・Tetrathionate 1, 0.5, 0.25, 0.125mM, 2 plates each ・3LB-CP plates ・40mL of LB-only liquid medium ・2LB-Amp plates

Result

Discussion

Since only noxE did not appear in the electrophoresis, we transformed with pBl-xylB-xdh-noxE again.

20250826

Purpose

Xylitol, Kill Switch test. Creation of pBl-xylB-xdh-noxE master plate

Procedure

Xylitol, Kill Switch
Add 10 μL of each bacterial culture to each medium, collect 50 μL in Eppendorf tubes every 30 minutes and measure the respective OD values.

Colony PCR and electrophoresis for noxE. Since no bands were visible, we need to start again from transformation. Creation of master plate for kill strain.

Result

OD: xylitol
OD: Kill Switch
EP: noxE

Discussion

Xylitol: Growth rate is questionable, will continue cultivation & measurement tomorrow.
Kill Switch: Everything grew even though some weren’t supposed to, will check the plates tomorrow as well.
The noxE media surface wasn’t dry enough so colonies didn’t form properly. Also, since no bands were visible in the electrophoresis results, we performed transformation again.

20250827

Purpose

Continuation of xylitol verification experiment.
Creation of noxE master plate.

Procedure

pBl-xylB-xdh-noxE colony PCR, electrophoresis, transformation.
Xylitol verification experiment.

20250828

Purpose

Creation of master plate for bacteria containing only pBl-xyB-xdh-noxE.

Procedure

Colony PCR Transformation Master plate was created using the one stored in the refrigerator.

Result

20250829

Procedure

The master plate had grown so I stored it.

20250901

Purpose

Create media for use in xylitol assimilation verification experiment.

Procedure

Make two of each numbered item1.

1. Weigh 2.3g of MgSO₄ and 2.2g of CaCl₂ (in case of hexahydrates)2.
2. Prepare 10ml of 1M MgSO₄ and 10ml of 1M CaCl₂3.
3. Autoclave M9 salt at 121°C for 15 minutes.4.
4. Weigh 10g of D-glucose and 10g of xylitol, and dissolve them in 50ml of Milli-Q water5.
5. Prepare 2ml of 20.0% D-glucose (w/v) and 50ml of 20% xylitol (w/v)6.
6. Filter sterilize the glucose solution, xylitol solution, MgSO₄aq, and CaCl₂aq7.
7. Prepare solutions according to the table below.

20250903

Purpose

Clone fragments for plasmid formation for Kill Switch verification.
Start night culture for xylitol verification experiment.

Procedure

PCR
Electrophoresis
Gel Extraction

Result

The Nanodrop readings for sample 33 were poor, so we need to redo it.

20250904

Purpose

Xylitol verification experiment.
Kill Switch verification strain preparation (gel extraction).

Procedure

Verification of xylitol-growing strains

Check OD600 by using NanoDrop every 1 or 2 hours.

Result

EP33-1, 33-2 tested (same plasmid, different dates).
OD600 measured by NanoDrop.

Discussion

The concentration is too low, so we need to redo the PCR and extraction.

20250905

Purpose

Xylitol verification experiment.
Preparation of Kill Switch verification strains (gel extraction).

Procedure

Verification of xylitol-growing strains

Check OD600 by using NanoDrop every 1 or 2 hours.

Result

20250908

Purpose

Conduct verification of xylitol-assimilating strains.
Perform transformation of Kill Switch verification strains.

Procedure

Transfection

All created plasmids were transformed into DH5α and cultured on Amp-containing plates.

Verification of xylitol-growing strains

Check OD600 by using NanoDrop every 1 or 2 hours.

20250909

Procedure

Mass Culture

Mass culutred the avive bynvers 2, 4, and 7.

Result

20250910

Procedure

Transformation

All created plasmids were transformed into BL21(DE3) and cultured on Amp-containing plates.

Result

20250911

Purpose

Conduct verification experiments on xylitol.
Create strains for Kill Switch verification experiments.

Procedure

Mass culuture

Performed mass culture of numbers 1 and 2 above.

Preparation for xylitol verification

Culture BL21(DE3) containing pBl-xylB-xdh-noxE and BL21(DE3) containing pBl-xylB-xdh-noxE, pTf16-xytB-xytC-xytD-xytE in 2ml of LB medium overnight.

Result

Discussion

Precipitate was observed in the tube. It had a gritty texture and appears to be phosphate salt. An experiment is needed to identify the exact nature of this precipitate. OD600 increased in both 4 and 5, indicating they can metabolize xylitol. However, because the extent of the increase does not depend on sugar concentration, the experiment will be repeated under the same conditions to check for errors in medium preparation.

20250912

Purpose

Prepare for second round of xylitol-assimilation verification.
Prepare the validation experiment using kill-switch GFP.

Procedure

Storage Plate Creation

Cultured yesterday.

Xylitol verification experiment

Add IPTG to the medium cultured overnight yesterday to a final concentration of 0.5 mM, and incubate for 2 hours.
Prepare the following M9 medium, three tubes each. For 4-3.5-3, prepare six tubes each.

Add 10 µL of the IPTG-induced culture to the above medium as follows.

Kill Switch validation experiment.

To the BL21(DE3) cultures massively grown yesterday containing pBl-TtrR-EGFP and pTf16-TtrS, add 40 μL of 0.5 M potassium tetrathionate solution and continue culturing.

Result

Discussion

Since it was suggested to take three samples under the same conditions, instead of collecting them every hour, I decided to measure the OD600 of the cultures after overnight incubation.

20250915

Result

20250916

Purpose

Conduct the Kill Switch validation experiment (since about 8 plates per sample seem to be needed for CFU measurement).
Prepare for the xylitol verification experiment.
Check whether E. coli can grow in DMEM.

Procedure

Kill Switch verification experiment

Inoculate the pre-cultured cells into media with and without tetrathionate and incubate overnight.
If the strain name of the pre-cultured pBl-Bsa1R is BL21(DE3), use pTf16-TtrS, pBl-TtrR-Bsa1M1-BsaM2-Bsa1R, and pTf16 as controls for the experiment.

Preparation of medium

Prepared more than 24 Amp plates.
Added 5 μL of E. coli suspension to 2 mL of DMEM.

Result

The medium turned yellow and bacterial cells were observed.

20250918

Purpose

Kill Switch verification ① — Measure OD every 30 minutes, calculate doubling time.
Xylitol verification ① — Compare OD600 among strains in glucose/xylitol media.

Procedure

Kill Switch verification experiment 1

Add pTf16-TtrS and pBl-TtrR-Bsa1M1-Bsa1M2-Bsa1R to three LB cultures, measure OD every 30 minutes, and calculate doubling time. You may skip the measurement of first 30 minutes. Depending on the growth rate, continue measurements for about 4–5 hours.

Xylitol verification 1

1. Prepare pre-cultures of WT, pTf16, pBl-xylB-xdh-noxE, and pBl-xylB-xdh-noxE + pTf16-xytBCDE. If the pre-cultures are ready, proceed as follows:
2. Add 10 μL of 100 mM IPTG to the large-scale culture and induce for 3 hours.
3. After centrifuging the above culture and removing the medium, wash with 1 mL of saline solution(Milli-Q water with 0.9 w/v% NaCl) (or M9 medium?).
4. For each strain, add 10 μL to three 10 mL cultures containing 2 g/L glucose and three 10 mL cultures containing 2 g/L xylitol (total of 24 tubes).

The following is the antibiotic compatibility table.

Xylitol verification 2

1. Prepare six Glu1g/L, Xyl1g/L medium by adding 5mL of Glu2g/L and Xyl2g/L(without antibiotics).
2. Add large-scale culture of pTf16 to all tubes and incubate for two nights.
3. Pre-culture large-scale cultures of pBl-xylB-xdh-noxE and pBl-shRNA.
   Probably completed up to this point.
4. Add 10 μL of 100 mM IPTG to pBl-xylB-xdh-noxE and pBl-shRNA cultures and induce for 3 hours.
5. Wash the medium 2 cultures with saline solution (M9 medium), then add 10 μL of pBl-xylB-xdh-noxE to three tubes and 10 μL of pBl-shRNA to the other three tubes, and incubate for two nights.
6. For each tube, dilute the bacterial culture from 1× to 10⁷×, and plate onto 8 CP plates and 8 Amp plates, then incubate overnight.
7. Count the number of colonies

Kill Switch verification 2

1. Prepare nine 2 mL LB cultures for each IPTG concentration (1, 0.5, 0.1, 0 mM): three without antibiotics and six with Amp.
2. Pre-culture WT, pBl-shRNA, and pBl-Bsa1R.
3. Add 2 μL of each combination from the table below to the medium 1 (three tubes per combination), incubate overnight, and measure OD.

20250919

Purpose

Prepare agar plates.

Induce E. coli BL21(DE3) carrying pBl-shRNA and pBl-xylB-xdh-noxE pre-cultured on 20250918 with IPTG, and culture in M9 medium together with BL21(DE3) pTf16.
Measure the OD600 of the samples from Xylitol verification experiment 1.

Procedure

Prepared LB medium containing 20 μg/mL chloramphenicol.

Added 10 μL of 100 mM IPTG to 2 mL cultures of BL21(DE3) pBl-shRNA and pBl-xylB-xdh-noxE and incubated for 3 hours.

Result

OD600 of xylitol verification experiment 1.

20250922

Procedure

Xylitol verification①

OD600 measured by Nanodrop after three nights.

20250923

Result

20250924

Purpose

Perform time-lapse measurements for the EGF wound-healing experiment.

Procedure

Scratch the C2BBe1 cells cultured in DMEM (4.5 g/L glucose, 1% transferrin, 10% FBS) using a yellow pipette tip, then add EGF at 0 ng/mL, 16 ng/mL, 50 ng/mL, 100 ng/mL, 300 ng/mL, and 500 ng/mL. Monitor wound healing by time-lapse imaging every 3 hours for 48 hours.

20250925

Procedure

Transfection

Transform the constructed plasmid into E. coli DH5α.

1. Prepare dilution series from 10¹ to 10⁷ (7 Eppendorf tubes). a. Add 180 μL of M9 salt (wash solution) to each tube. b. Vortex the culture, then transfer 20 μL into the 10× dilution tube and vortex again. c. Vortex the 10× dilution tube, take 20 μL, transfer it into the 10² dilution tube, and vortex. d. Repeat this procedure up to the 10⁷ dilution to complete the dilution series.
2. From these, take 100 μL each of the 10⁴, 10⁵, 10⁶, and 10⁷ dilutions and spread them onto the plates.
3. Count the colonies the next day.

Result

Results from the logarithmic growth phase.

20250926

Procedure

1. Prepare dilution series from 10¹ to 10⁷ (7 Eppendorf tubes).
   a. Add 180 μL of M9 salt (wash solution) to each tube.
   b. Vortex the culture, then transfer 20 μL into the 10× dilution tube and vortex again.
   c. Vortex the 10× dilution tube, take 20 μL, transfer it into the 10² dilution tube, and vortex.
   d. Repeat this procedure up to the 10⁷ dilution to complete the dilution series.
2. From these, take 100 μL each of the 10⁴, 10⁵, 10⁶, and 10⁷ dilutions and spread them onto the plates.
3. Count the colonies the next day.

Sequencing

For plasmids with less than 20 μL pBl-Bsa1R Large-scale culture will be performed to amplify pBl-TtrR-Bsa1M1-Bsa1M2-Bsa1R.

Check whether Bsa1R has successfully cleaved

Introduce the following plasmids into BL21(DE3).

SDS-PAGE and Western blotting

Method

It is almost the same as August 19, but there are a few changes.

• The gel has an acrylamide concentration of 17.5%.
• Ultrasonication was not performed.
• Staining was done with Sypro Ruby instead of CBB-R.

Result

In the Western blot, a band of approximately 20 kDa was visible in lane ②. This band was expected to correspond to the 6×His-EGF-LARD3 fusion protein. However, because similar bands were also detected in the negative controls (lanes ⑩ and ⑫), it could not be conclusively identified as 6×His-EGF-LARD3.

C2BBe1 cell wound healing assay

Results

20250929

Procedure

Check whether Bsa1R has successfully cleaved

Colony PCR of BL21(DE3) transformed with the following plasmids.

If a positive colony is found, culture it on a large scale with 1 mM IPTG.

CFU measurement

1. Prepare dilution series from 10¹ to 10⁷ (7 Eppendorf tubes). a. Add 180 μL of M9 salt (wash solution) to each tube. b. Vortex the culture, then transfer 20 μL into the 10× dilution tube and vortex again. c. Vortex the 10× dilution tube, take 20 μL, transfer it into the 10² dilution tube, and vortex. d. Repeat this procedure up to the 10⁷ dilution to complete the dilution series.
2. From these, take 100 μL each of the 10⁴, 10⁵, 10⁶, and 10⁷ dilutions and spread them onto the plates.
3. Count the colonies the next day.

Check for the plasmid band

Run two plasmids labeled pBl-Bsa1R on an agarose gel. Check that there is only a single band.

Result