Contributors
Index
Date: 26/Feb/2025
Objective: To prepare buffer solutions.
Protocol:
| MnCl₂·4H₂O | 5.44 g |
| CaCl₂·H₂O | 1.10 g |
| KCl | 9.325 g |
| MilliQ | up to 480 ml |
The aqueous solution was autoclaved.
The autoclaved solution was designated as Buffer ①.
PIPES (7.55 g) was dissolved in 40 ml MilliQ water.
The pH was adjusted to 6.7 using 5 M KOH.
The volume was adjusted to 50 ml with MilliQ water.
The solution was filter-sterilized in a clean bench.
The filter-sterilized solution was designated as Buffer ②.
Buffer ① and ② were stored separately to be mixed immediately before use.
Result: Buffer preparation was completed successfully.
Date: 25/Mar/2024
Objective: To prepare LB medium.
Protocol: Refer to “LB Medium Preparation.”
Result: The procedure was completed successfully.
Date: 25/Mar/2024
Objective: To isolate JM109 colonies.
Protocol:
A small amount of JM109 competent cells was spread onto solid LB medium without antibiotics.
The plate was incubated overnight at 37°C.
Result: The procedure was completed successfully.
Date: 26/Mar/2024
Objective: To isolate JM109 colonies.
Protocol:
The plate was collected and colony growth was confirmed.
The plate was stored at room temperature.
Result: The procedure was completed successfully.
Date: 26/Mar/2024
Objective: To prepare JM109 competent cells.
Protocol:
A single colony was picked from the plate prepared on 25/Mar/2024 and inoculated into 25 ml liquid LB.
The culture was incubated with shaking at 37°C for 8 hours.
Aliquots of 2 ml, 4 ml, and 10 ml from the pre-culture were added to 250 ml liquid LB in separate 1 L flasks.
The cultures were incubated with shaking at 18°C overnight.
Result: The procedure was completed successfully.
Date: 27/Mar/2024
Objective: To prepare JM109 competent cells.
Protocol: OD₆₀₀ was measured for each culture.
Result: The experiment failed.
Date: 10/Apr/2024
Objective: To prepare LB medium.
Protocol: Refer to “LB Medium Preparation.”
Result: The procedure was completed successfully.
Date: 10/Apr/2024
Objective: To isolate JM109 colonies.
Protocol:
A small amount of JM109 competent cells was spread onto solid LB medium without antibiotics.
The plate was incubated overnight at 37°C.
Result: The procedure was completed successfully.
Date: 11/Apr/2024
Objective: To isolate JM109 colonies.
Protocol:
The plate was collected and colony growth was confirmed.
The plate was stored at room temperature.
Result: The procedure was completed successfully.
Date: 11/Apr/2024
Objective: To prepare JM109 competent cells.
Protocol:
A single colony was picked from the plate prepared on 10/Apr/2024 and inoculated into 25 ml liquid LB.
The culture was incubated with shaking at 37°C for 8 hours.
Aliquots of 200 μl, 400 μl, and 1 ml from the pre-culture were added to 250 ml liquid LB in separate 1 L flasks.
The cultures were incubated with shaking at 18°C overnight.
Result: The procedure was completed successfully.
Date: 12/Apr/2024
Objective: To prepare JM109 competent cells.
Protocol:
OD₆₀₀ was measured for each culture.
The main culture was cooled in ice water for 10 minutes in the flask.
Buffer ① and ② prepared on 26/Feb/2024 were mixed.
The cooled bacterial cells were centrifuged at 2500 g for 10 minutes at 4°C, and the supernatant was discarded.
The pellet was resuspended in 80 ml of pre-chilled Inoue Buffer.
DMSO (1.5 ml) was added and mixed.
The suspension was incubated on ice for 10 minutes.
The sample was aliquoted into 110 μl portions in microcentrifuge tubes.
The tubes were frozen in liquid nitrogen.
The samples were stored at -80°C.
Result: The procedure was completed successfully.
Date: 17/Apr/2024
Objective: To measure the competency of the prepared competent cells.
Protocol:
A 110 μl aliquot of JM109 competent cells was thawed.
pET28a vector solutions at three concentrations (10 pg/μl, 100 pg/μl, 500 pg/μl) were added (1 μl each).
The samples were incubated on ice for 5 minutes.
Heat shock was performed at 42°C for 45 seconds.
The samples were incubated on ice for 2 minutes.
Liquid LB (500 μl, without antibiotics) was added.
The samples were incubated at 37°C for 1 hour.
From the 611 μl of recovery culture, 200 μl was plated.
The plates were incubated overnight at 37°C.
Result: The procedure was completed successfully.
Date: 18/Apr/2024
Objective: To measure the competency of the prepared competent cells.
Protocol:
The plates were collected.
Colonies were counted for each vector concentration: 10 pg/μl: 0 colonies, 100 pg/μl: 56 colonies, 500 pg/μl: TMTC (too many to count).
Competency was calculated using the results from the 100 pg/μl vector concentration.
Result: The procedure was completed successfully. The calculated competency was 1.71 × 10⁶ cfu/μg.
Date: 30/May/2024
Objective: To transform JM109.
Protocol: Refer to “Transformation.” The vector solution was pET28a at a concentration of 124 ng/μl.
Result: The procedure was completed successfully.
Date: 31/May/2024
Objective: To transform JM109.
Protocol: Refer to “Transformation.”
Result: The procedure was completed successfully.
Date: 03/Jun/2024
Objective: To extract vector DNA.
Protocol: Refer to “Vector Extraction 1.”
Result: The procedure was completed successfully.
Date: 04/Jun/2024
Objective: To extract vector DNA.
Protocol: Refer to “Vector Extraction 1.”
Result: The procedure was completed successfully.
Date: 15/Jul/2024
Objective: To perform NcoⅠ digestion and DNA extraction.
Protocol: Restriction enzyme digestion of pET28a was performed according to the Takara Bio NcoⅠ standard protocol. Incubation time was 2 hours.
Result: The procedure was completed successfully.
Date: 17/Jul/2024
Objective: To perform transformation.
Protocol: Refer to “In-Fusion” and “Transformation.” The sample was avGFP.
Result: The procedure was completed successfully. Transformation was successful.
Date: 18/Jul/2024
Objective: To perform transformation.
Protocol: Refer to “Transformation.” The sample was avGFP.
Result: Failed.
Date: 18/Jul/2024
Objective: To prepare an agarose gel.
Protocol:
Agarose (0.5 g) was added to 30 ml TAE buffer.
The mixture was heated in a microwave to dissolve the agarose.
TAE buffer (20 ml) was added and the solution was cooled.
Staining solution (5 μl) was added and mixed.
The solution was poured into a mold and allowed to solidify.
The gel was stored at 4°C.
Result: The procedure was completed successfully.
Date: 18/Jul/2024
Objective: To perform NcoⅠ digestion of pET28a.
Protocol: Restriction enzyme digestion of pET28a was performed according to the Takara Bio NcoⅠ standard protocol. Incubation time was 2 hours.
Result: The procedure was completed successfully.
Date: 19/Jul/2024
Objective: To perform NcoⅠ digestion of pET28a.
Protocol:
Samples and controls were loaded onto a 1% agarose gel.
Electrophoresis was performed at 100 V for 20 minutes.
The gel was visualized under UV light.
The thickest band in the sample lane was excised.
The gel slice was stored at 4°C.
Result: The procedure was completed successfully.
Date: 24/Jul/2024
Objective: To perform PCR.
Protocol:
| PrimeStar | 25 μl |
| Template | 2 μl |
| Primer ① | 1 μl |
| Primer ② | 1 μl |
| MilliQ | 21 μl |
| Total | 50 μl |
Templates were EGFP and avGFP, Primer ① was igem_1_F, Primer ② was igem_1_R.
PCR was performed using a thermal cycler.
10× Loading Buffer (5 μl) was added.
Samples were loaded onto a 1% agarose gel.
Electrophoresis was performed at 100 V for 20 minutes.
Bands appearing at the expected GFP base pair position were excised while visualizing under UV light.
Result: The procedure was completed successfully.
Date: 24/Jul/2024
Objective: To perform gel extraction.
Protocol:
The pET28a NcoⅠ-digested gel excised on 7/19 and the avGFP and EGFP gels excised today were prepared and brought to room temperature.
The experiment was performed according to the Fast Gene Gel/PCR Extraction Kit protocol.
The extracted DNA was stored at 4°C.
Result: The procedure was completed successfully.
Date: 25/Jul/2024
Objective: To perform transformation.
Protocol: Refer to “In-Fusion” and “Transformation.” Samples were avGFP and EGFP.
Result: The procedure was completed successfully.
Date: 26/Jul/2024
Objective: To perform transformation.
Protocol: Refer to “Transformation.” The sample was avGFP.
Result: Failed.
Date: 25/Jul/2024
Objective: To perform transformation.
Protocol: Refer to “In-Fusion” and “Transformation.” Samples were avGFP and EGFP.
Result: The procedure was completed successfully.
Date: 26/Jul/2024
Objective: To perform transformation.
Protocol: Refer to “Transformation.” Samples were avGFP and EGFP.
Result: Failed.
Date: 29/Jul/2024
Objective: To transform JM109.
Protocol: Refer to “Transformation.” The vector solution was pET28a.
Result: The procedure was completed successfully.
Date: 30/Jul/2024
Objective: To transform JM109.
Protocol: Refer to “Transformation.”
Result: The procedure was completed successfully. Transformation was successful.
Date: 31/Jul/2024
Objective: To extract vector DNA.
Protocol: Refer to “Vector Extraction 1.”
Result: The procedure was completed successfully.
Date: 01/Aug/2024
Objective: To extract vector DNA.
Protocol: Refer to “Vector Extraction 1.”
Result: The procedure was completed successfully.
Date: 13/Aug/2024
Objective: To perform NcoⅠ digestion of pET28a.
Protocol: Restriction enzyme digestion of pET28a was performed according to the Takara Bio NcoⅠ standard protocol. Incubation time was 2 hours.
Result: The procedure was completed successfully.
Date: 14/Aug/2024
Objective: To perform NcoⅠ digestion of pET28a.
Protocol:
Samples and controls were loaded onto a 1% agarose gel.
Electrophoresis was performed at 100 V for 20 minutes.
The gel was visualized under UV light.
The thickest band in the sample lane was excised.
The experiment was performed according to the Fast Gene Gel/PCR Extraction Kit protocol.
The sample was stored at 4°C.
Result: The procedure was completed successfully.
Date: 15/Aug/2024
Objective: To perform transformation.
Protocol: Refer to “In-Fusion” and “Transformation.” The sample used was avGFP amplified by PCR on 13/Aug/2024.
Result: The procedure was completed successfully.
Date: 16/Aug/2024
Objective: To perform transformation.
Protocol: Refer to “Transformation.”
Result: Failed.
Date: 03/Sep/2024
Objective: To transform JM109.
Protocol: Refer to “Transformation.” The vector solution was pET28a.
Result: The procedure was completed successfully.
Date: 04/Sep/2024
Objective: To transform JM109.
Protocol: Refer to “Transformation.”
Result: The procedure was completed successfully. Transformation was successful.
Date: 04/Sep/2024
Objective: To extract vector DNA.
Protocol: Refer to “Vector Extraction 1.”
Result: The procedure was completed successfully.
Date: 05/Sep/2024
Objective: To extract vector DNA.
Protocol: Refer to “Vector Extraction 1.”
Result: The procedure was completed successfully.
Date: 06/Sep/2024
Objective: To perform NcoⅠ digestion of pET28a.
Protocol: Restriction enzyme digestion of pET28a was performed according to the Takara Bio NcoⅠ standard protocol. Incubation time was 2 hours.
Result: The procedure was completed successfully.
Date: 07/Sep/2024
Objective: To perform NcoⅠ digestion of pET28a.
Protocol:
Samples and controls were loaded onto a 1% agarose gel.
Electrophoresis was performed at 100 V for 20 minutes.
The gel was visualized under UV light.
The thickest band in the sample lane was excised.
The gel slice was stored at 4°C.
Result: The procedure was completed successfully.
Date: 17/Sep/2024
Objective: To perform transformation.
Protocol: Refer to “In-Fusion” and “Transformation.” The sample used was avGFP amplified by PCR on 13/Aug/2024.
Result: The procedure was completed successfully.
Date: 18/Sep/2024
Objective: To perform transformation.
Protocol: Refer to “Transformation.”
Result: Failed.
Date: 24/Sep/2024
Objective: To isolate colonies.
Protocol:
A small amount of BL21 competent cells was spread onto solid LB medium without antibiotics.
The plate was incubated overnight at 37°C.
Result: The procedure was completed successfully.
Date: 25/Sep/2024
Objective: To isolate colonies.
Protocol:
The plate was collected and colony growth was confirmed.
The plate was stored at room temperature.
Result: The procedure was completed successfully.
Date: 25/Sep/2024
Objective: To prepare competent cells.
Protocol:
A single colony was picked from the plate prepared on 25/Sep/2024 and inoculated into 25 ml liquid LB.
The culture was incubated with shaking at 37°C for 8 hours.
Result: The procedure was completed successfully.
Date: 12/Mar/2025
Objective: To prepare LB medium.
Protocol: Refer to “LB Medium Preparation.”
Result: The procedure was completed successfully.
Date: 12/Mar/2025
Objective: To perform transformation.
Protocol: Refer to “In-Fusion” and “Transformation.” T01_Seq01–04 were used as inserts.
Result: All operations up to this point were completed without issues.
Date: 14/Mar/2025
Objective: To perform transformation.
Protocol: Refer to “Transformation.”
Result: Colonies were confirmed and the plates were stored at room temperature.
Date: 14/Mar/2025
Objective: To perform PCR.
Protocol: The experiment was performed following the same procedure as 24/Jul/2024. The template was pET28a, Primer ① was igem_3_F, and Primer ② was igem_5_R.
Result: All operations up to this point were completed without issues.
Fig. 1. Electrophoresis results
Date: 16/Mar/2025
Objective: To perform transformation.
Protocol: Refer to “In-Fusion” and “Transformation.” T01_Seq01–04 and T02_01–29 were used as inserts.
Result: All operations up to this point were completed without issues.
Date: 14/Mar/2025
Objective: To perform transformation.
Protocol: Refer to “Transformation.”
Result: Colonies were confirmed and successful plates were stored at room temperature.
Date: 17/Mar/2025
Objective: To transform JM109.
Protocol: Refer to “Transformation.” The vector solution was pET28a.
Result: The procedure was completed successfully.
Date: 18/Mar/2025
Objective: To transform JM109.
Protocol: Refer to “Transformation.”
Result: The procedure was completed successfully.
Date: 18/Mar/2025
Objective: To perform NcoⅠ digestion of pET28a.
Protocol:
Two solutions were prepared according to the Takara Bio NcoⅠ standard protocol. One was incubated for 1 hour and the other for 2 hours.
Samples and controls were loaded onto a 1% agarose gel.
Electrophoresis was performed at 100 V for 20 minutes.
The gel was visualized under UV light and photographed.
Result: The 1-hour incubated sample disappeared.
Fig. 2. Electrophoresis results
Date: 18/Mar/2025
Objective: To perform NcoⅠ digestion of pET28a.
Protocol:
Two solutions were prepared according to the Takara Bio NcoⅠ standard protocol. One was incubated for 2 hours and the other at 37°C for 4 hours.
Samples and controls were loaded onto a 1% agarose gel.
Electrophoresis was performed at 100 V for 20 minutes.
The gel was visualized under UV light and photographed.
Result: The procedure was completed successfully.
Fig. 3. Electrophoresis results
Date: 22/Mar/2025
Objective: To perform NcoⅠ digestion of pET28a.
Protocol:
| NcoⅠ | 1 μl |
| 10× K Buffer | 2 μl |
| 0.1% BSA | 2 μl |
| pET28a | 7 μl |
| Nuclease-free water | 8 μl |
The solution was incubated at 37°C for 2 hours.
A control was prepared:
| pET28a | 7 μl |
| Nuclease-free water | 13 μl |
10× Loading Buffer (2 μl) was added to both the control and sample.
Samples and controls were loaded onto a 1% agarose gel.
Electrophoresis was performed at 100 V for 20 minutes.
The gel was visualized under UV light and photographed.
Result: The bands disappeared.
Fig. 4. Electrophoresis results
Date: 22/Mar/2025
Objective: To perform NcoⅠ digestion of pET28a.
Protocol: The experiment was performed following the same procedure as 22/Mar/2025. However, two sample solutions were prepared; one was incubated for 2 hours and the other for 4 hours.
Result: The experiment was repeated using the same procedure and sample collection was performed, but too much time elapsed and the samples were discarded later.
Fig. 5. Electrophoresis results
Date: 22/Mar/2025
Objective: To extract vector DNA.
Protocol: Refer to “Vector Extraction 1.”
Result: The procedure was completed successfully.
Date: 23/Mar/2025
Objective: To extract vector DNA.
Protocol: Refer to “Vector Extraction 1.”
Result: The procedure was completed successfully.
Date: 23/Mar/2025
Objective: To perform NcoⅠ digestion of pET28a.
Protocol: The experiment was performed following the same procedure as 22/Mar/2025. However, the pET28a solution extracted on the same day was used.
Result: The procedure was completed successfully.
Fig. 6. Electrophoresis results
Date: 23/Mar/2025
Objective: To perform NcoⅠ digestion of pET28a.
Protocol: The experiment was performed following the same procedure as 22/Mar/2025. However, nuclease-free water was not used and 15 μl of pET28a was used instead.
Result: Failed.
Fig. 7. Electrophoresis results
Date: 23/Mar/2025
Objective: To perform NcoⅠ digestion of pET28a.
Protocol: The experiment was performed following the same procedure as 23/Mar/2025.
Result: Successful.
Fig. 8. Electrophoresis results
Date: 23/Mar/2025
Objective: To perform NcoⅠ digestion of pET28a.
Protocol: The experiment was performed following the same procedure as 27/Mar/2025. However, the scale was increased 5-fold.
Result: Successful.
Fig. 9. Electrophoresis results
Date: 27/Mar/2025
Objective: To perform transformation.
Protocol: Refer to “In-Fusion” and “Transformation.” T01_Seq03 and T02_01–03 were used as inserts.
Result: All operations up to this point were completed without issues.
Date: 28/Mar/2025
Objective: To perform transformation.
Protocol: Refer to “Transformation.”
Result: Colonies were confirmed and the plates were stored at room temperature.
Date: 28/Mar/2025
Objective: To perform colony PCR.
Protocol: Refer to “Colony PCR” and “Vector Extraction 1.” Colonies from lanes 2, 3, 6, 7, 10, and 11 were used for extraction.
Result: All operations up to this point were completed without issues.
Fig. 10. Electrophoresis results
Date: 29/Mar/2025
Objective: Perform transformation.
Protocol: Refer to “In-Fusion” and “Transformation” protocols. Inserts T01_Seq02, 04, and T02_04~29 were used.
Result: All procedures were completed successfully.
Date: 30/Mar/2025
Objective: Complete transformation procedure.
Protocol: Refer to “Transformation” protocol.
Result: Colonies were confirmed and plates were stored at room temperature.
Date: 30/Mar/2025
Objective: Perform colony PCR.
Protocol: Refer to “Colony PCR” protocol. Colonies from each lane were picked and inoculated onto master plates.
Result: All procedures were completed successfully.
Fig. 11. Electrophoresis results
Fig. 12. Electrophoresis results
Fig. 13. Electrophoresis results
Fig. 14. Electrophoresis results
Date: 31/Mar/2025
Objective: Extract vectors.
Protocol: Refer to “Vector Extraction 1” protocol. Two colonies from each T01 and T02 series that showed growth were used; single colonies were used when only one colony was obtained.
Result: All procedures were completed successfully.
Date: 02/Apr/2025
Objective: Perform transformation.
Protocol: Refer to “In-Fusion” and “Transformation” protocols. Inserts T02_10, 18, 22, 25, and 27 were used.
Result: All procedures were completed successfully.
Date: 03/Apr/2025
Objective: Complete transformation procedure.
Protocol: Refer to “Transformation” protocol.
Result: No colonies were observed; plates were discarded.
Date: 03/Apr/2025
Objective: Extract plasmids.
Protocol: Refer to “Vector Extraction 1” protocol.
Result: All procedures were completed successfully.
Date: 08/Apr/2025
Objective: Prepare competent cells.
Protocol:
SOB medium (×2)
| Component | Amount |
|---|---|
| Tryptone | 2g×2 |
| Yeast extract | 0.5g×2 |
| 5M NaCl | 200μl×2 |
| 2M KCl | 125μl×2 |
| DW | 98~100mL×2 |
| total | 100mL×2 |
The mixture was autoclaved. 1M MgSO₄ (1 mL, autoclaved) ×2 and 1M MgCl₂ (1 mL, autoclaved) ×2 were prepared.
Transformation buffer (TB) stock
| Component | Amount |
|---|---|
| PIPES | 1.5g |
| CaCl₂·2H₂O | 1.1g |
| KCl | 9.3g |
| total | 500mL |
Components were suspended in 400 mL of MilliQ water, and pH was adjusted to 6.7~6.8 using 5M KOH.
MnCl₂·4H₂O (5.45 g) was added, volume was adjusted to 500 mL, and the solution was filter-sterilized.
Result: All procedures were completed successfully.
Date: 08/Apr/2025
Objective: Complete competent cell preparation.
Protocol:
E. coli retrieved from -80℃ were streaked on LB agar medium and cultured overnight at 37℃.
Colonies were transferred to SOB medium.
Cultures were vigorously shaken and incubated at 18~22℃.
When OD₆₀₀ reached 0.4~0.8, culture was terminated and immediately cooled on ice for 10 minutes.
Culture was transferred to multiple 50 mL centrifuge tubes ×2 and centrifuged at 3,000 rpm for 15 minutes at 4℃.
After removing supernatant, pellets were resuspended in a total of 30 mL of ice-cold TB, combined into one 50 mL centrifuge tube, and cooled on ice for 10 minutes.
Centrifuged at 3,000 rpm for 15 minutes at 4℃.
After removing supernatant, pellet was resuspended in 8 mL of ice-cold TB, transferred to a 15 mL centrifuge tube, and 0.6 mL of DMSO (final concentration 7%) was added, followed by cooling on ice for 10 minutes.
Aliquots of 100 μL were dispensed into 1.5 mL tubes and immediately frozen in liquid nitrogen.
Stored at -80℃.
Result: Competent cells were successfully prepared.
Date: 02/Jul/2025
Objective: Transform BL21 strain.
Protocol: Refer to “Transformation” protocol. BL21 strain was used as host E. coli.
Result: Operation was completed successfully.
Date: 03/Jul/2025
Objective: Complete transformation of BL21.
Protocol: Refer to “Transformation” protocol.
Result: Colonies were confirmed.
Date: 03/Jul/2025
Objective: Perform transformation.
Protocol: Refer to “Transformation” protocol. JM109 strain was used as host E. coli.
Result: Operation was completed successfully.
Date: 04/Jul/2025
Objective: Complete transformation procedure.
Protocol: Refer to “Transformation” protocol.
Result: No colonies were observed; plates were discarded.
Date: 09/Jul/2024
Objective: Prepare LB medium.
Protocol: Refer to “LB Medium Preparation” protocol.
Result: Operation was completed successfully.
Date: 10/Jul/2025
Objective: Perform transformation.
Protocol: Refer to “Transformation” protocol. JM109 strain was used as host E. coli. avGFP was used as the insert.
Result: Operation was completed successfully.
Date: 11/Jul/2025
Objective: Complete transformation procedure.
Protocol: Refer to “Transformation” protocol.
Result: Colonies were confirmed.
Date: 11/Jul/2025
Objective: Perform colony PCR.
Protocol: Refer to “Colony PCR” protocol.
Result: PCR was unsuccessful.
Date: 12/Jul/2025
Objective: Perform colony PCR.
Protocol: Refer to “Colony PCR” protocol.
Result: PCR was unsuccessful.
Date: 12/Jul/2025
Objective: Perform transformation.
Protocol: Same procedure as 13/03/2025. However, pET28a was used as the vector, and avGFP along with PCR-amplified avGFP were used as inserts.
Result: All procedures were completed successfully.
Date: 13/Jul/2025
Objective: Examine plates from transformation performed on 13/07/2025.
Protocol: Refer to “Transformation” protocol.
Result: Plates were recovered and colonies were confirmed. Colonies were observed on all plates, but the negative control plate appeared to have more colonies. Therefore, it was decided to verify the competency of JM109.
Date: 13/Jul/2025
Objective: Perform transformation to verify JM109 competency.
Protocol: Same procedure as 26/07/2024. However, 1.5 μL of each insert and 0.5 μL of vector solution were used, with avGFP as the target insert.
Result: All procedures were completed without problems.
Date: 14/Jul/2025
Objective: Examine colony plates from 13/07/2025.
Protocol: Refer to “Transformation” protocol.
Result: One colony was observed for both negative control and avGFP, and zero colonies for positive control. Experiment failed.
Date: 14/Jul/2025
Objective: Perform colony PCR.
Protocol: Same procedure as 28/03/2025. Four colonies from avGFP samples and one from negative control were subjected to PCR.
Result: No bands were detected.
Date: 15/Jul/2025
Objective: Perform transformation.
Protocol: Same procedure as 13/03/2025. However, pET28a was used as the vector, avGFP and negative control as inserts. Competent cells of JM109 provided by a graduate student were used.
Result: All procedures were completed without problems.
Date: 16/Jul/2025
Objective: Examine plates.
Protocol: Refer to “Transformation” protocol.
Result: Colonies were confirmed.
Date: 16/Jul/2025
Objective: Perform colony PCR.
Protocol: Same procedure as 28/03/2025.
Result: Bands were detected but at low concentration. Therefore, colony PCR will be repeated.
Date: 16/Jul/2025
Objective: Perform colony PCR.
Protocol: Same procedure as 28/03/2025. However, the gel was changed to one with smaller wells. 1 μL of colony suspension in MilliQ was added, and MilliQ volume was reduced by 1 μL accordingly.
Result: Experiment was successful.
Date: 16/Jul/2025
Objective: Extract vectors.
Protocol: Same procedure as 31/03/2025.
Result: All procedures were completed without problems.
Date: 17/Jul/2025
Objective: Extract vectors.
Protocol: Same procedure as 01/04/2025.
Result: All procedures were completed without problems.
Date: 18/Jul/2025
Objective: Prepare buffer solution.
Protocol:
The following buffer was prepared by dissolving components in MilliQ water:
Table 15. Buffer composition
| Component | Concentration |
|---|---|
| Tris-HCl | 20mM |
| NaCl | 150mM |
| EDTA | 5mM |
pH was adjusted to 7.8, and total volume was brought to 200 mL.
Result: Buffer was prepared as described in the table above. The completed solution was stored in a medium bottle.
Date: 18/Jul/2025
Objective: Perform transformation.
Protocol: Same procedure as 13/03/2025 from the step of adding competent cells to 0.5 mL vector solution.
However, vector solution extracted on 17/07/2025 was used. BL21 strain was used as competent cells.
Result: All procedures were completed successfully.
Date: 19/Jul/2025
Objective: Examine plates.
Protocol: Refer to “Transformation” protocol.
Result: Colonies were confirmed.
Date: 19/Jul/2025
Objective: Perform pre-culture.
Protocol: One colony each was picked from plates confirmed on 17/07/2025 and inoculated into liquid LB medium.
Result: Pre-culture was completed.
Date: 20/Jul/2025
Objective: Prepare for protein extraction.
Protocol:
50 mL and 200 mL of pre-cultured solution were taken and inoculated into 10 mL of LB medium containing 50 mg/mL kanamycin. These were cultured at 37℃ for 2 hours with shaking.
OD was measured and confirmed to be appropriate. 100 μL of 100 mM IPTG stock solution was added to each test tube, and cultures were incubated overnight at 28℃.
Result: All procedures were completed successfully.
Date: 21/Jul/2025
Objective: Extract proteins.
Protocol:
Samples were collected and transferred to 15 mL centrifuge tubes.
Centrifuged at 1,800 g for 2 minutes, and supernatant was discarded.
Pellets were collected and stored in freezer.
Result: Protein extraction was completed.
Date: 21/Jul/2025
Objective: Perform transformation.
Protocol: Same procedure as 13/03/2025. However, pET28a was used as the vector, T02_01~02 and negative control as inserts. Competent cells of JM109 provided by a graduate student were used.
Result: All procedures were completed without problems.
Date: 22/Jul/2025
Objective: Examine plates.
Protocol: Refer to “Transformation” protocol.
Result: T02_01 and negative control (empty vector) were successful and stored in refrigerator. T02_02 failed; experiment will be repeated at a later date.
Date: 22/Jul/2025
Objective: Perform transformation.
Protocol: Same procedure as 13/03/2025. However, pET28a was used as the vector, and inserts with sufficient remaining volume (T02_21, 22, 24, 27, 29) were selected. Competent cells of JM109 provided by a graduate student were used.
Result: All procedures were completed successfully.
Date: 23/Jul/2025
Objective: Examine plates.
Protocol: Refer to “Transformation” protocol.
Result: Colonies were confirmed on all plates and stored in refrigerator.
Date: 23/Jul/2025
Objective: Perform transformation.
Protocol: Same procedure as 22/07/2025. However, inserts T02_02, 10, 16, 18, and 25 were used.
Result: All procedures were completed successfully.
Date: 24/Jul/2025
Objective: Examine plates.
Protocol: Refer to “Transformation” protocol.
Result: Colonies were confirmed on all plates except T02_10.
Date: 24/Jul/2025
Objective: Prepare agar plates.
Protocol: Previously prepared solid LB medium was melted and poured into plates.
Result: All procedures were completed successfully.
Date: 28/Jul/2025
Objective: Perform colony PCR.
Protocol:
Colonies were touched with pipette tip and suspended in 10 mL of MilliQ. Four colonies were suspended for each insert sequence. One colony from negative control was transferred.
The following PCR reaction mixture was prepared in required amounts:
Table 16. Reaction mixture composition
| Component | Amount |
|---|---|
| Primer (T7 promoter primer) | 0.1μl |
| 2× Gflex PCR Buffer | 2.5μl |
| Primer (T7 terminator primer) | 0.1μl |
| Tks Gflex DNA polymerase | 0.1μl |
| Colony suspension | 2.2μl |
| total | 5μl |
① 94℃ for 10 minutes
② 98℃ for 10 seconds
③ 55℃ for 15 seconds
④ 68℃ for 1 minute
Steps ②~④ were repeated for 35 cycles.
1 μL of Loading Buffer (×10) and 4 μL of MilliQ water were added to samples.
5 μL of marker and entire volume of each sample were applied to 1% agarose gel.
Electrophoresis was performed at 100 V for 30 minutes.
Gel was exposed to UV and photographed.
Two colony suspensions containing insert for each sequence were selected and inoculated into 5 μL of liquid LB containing 50 mg/L kanamycin.
Cultures were incubated at 37℃ overnight with shaking.
Result: All procedures were completed successfully.
Fig. 15. Electrophoresis results
1: Marker 2~5: T02_Seq21 6~9: T02_Seq22 10~13: T02_Seq24 14~17: T02_Seq25
Fig. 16. Electrophoresis results
1: Marker 2~5: T02_Seq27, 6~9: T02_Seq29, 10: empty 11: negative control
Date: 29/Jul/2025
Objective: Perform colony PCR.
Protocol:
Samples were collected.
Culture was centrifuged at 18,000 g.
Supernatant was removed.
Plasmid extraction was performed from E. coli pellet according to Favorprep protocol.
Result: DNA was extracted and stored at 4℃.
Date: 30/Jul/2025
Objective: Perform transformation.
Protocol: Same procedure as 18/07/2025. However, transformation was performed with previously extracted T02_04, 05, 06, 08, and 09.
Result: All procedures were completed successfully.
Date: 31/Jul/2025
Objective: Examine plates.
Protocol: Refer to “Transformation” protocol.
Result: Colonies were observed on all plates and stored in refrigerator.
Date: 31/Jul/2025
Objective: Perform transformation.
Protocol: Same procedure as 18/03/2025. However, vectors T02_11, 12, 13, 14, and 15 extracted previously were used.
Result: All procedures were completed successfully.
Date: 01/Aug/2025
Objective: Examine plates.
Protocol: Refer to “Transformation” protocol.
Result: Colonies were observed on all plates.
Date: 01/Aug/2025
Objective: Perform transformation.
Protocol: Same procedure as 22/07/2025. However, inserts T02_07 and 16 were used.
Result: All procedures were completed successfully.
Date: 01/Aug/2025
Objective: Perform pre-culture.
Protocol:
Colony #4 from T02-16, 21, 4, 25, 29 and colony #3 from T02_22 were picked and inoculated into 5 mL of liquid LB medium containing kanamycin.
Cultures were incubated at 37℃ overnight with shaking.
Result: All procedures were completed successfully.
Date: 01/Aug/2025
Objective: Perform pre-culture.
Protocol:
Colony #1 from plates of T02_04, 05, 06, 08, 09, 11, 12, 13, 14, 15 cultured with BL21 strain were picked and inoculated into 5 mL of liquid LB medium containing kanamycin.
Cultures were incubated at 37℃ overnight with shaking.
Result: All procedures were completed successfully.
Date: 02/Aug/2025
Objective: Perform transformation.
Protocol: Same procedure as 13/03/2025. However, pET28a was used as the vector, T02_07, 16, and negative control as inserts. Competent cells of JM109 provided by a graduate student were used.
Result: Transformation was completed.
Date: 02/Aug/2025
Objective: Induce protein expression.
Protocol: Same procedure as 20/07/2025.
Result: All procedures were completed successfully.
Date: 02/Aug/2025
Objective: Perform miniprep.
Protocol:
JM109 (pET28a T02_16, 21, 24, 25, 29) cultured on 01/08/2025 was centrifuged at 18,000 g. Miniprep was performed following the same procedure as 29/07/2025.
Result: All procedures were completed successfully.
Date: 03/Aug/2025
Objective: Collect protein pellets.
Protocol: Samples cultured the previous day were collected and centrifuged at 18,000 g for 2 minutes. Supernatant was discarded, protein pellets were collected, and transferred to refrigerator.
Result: All procedures were completed successfully.
Date: 04/Aug/2025
Objective: Prepare agar plates.
Protocol: Same procedure as 24/07/2025.
Result: All procedures were completed successfully.
Date: 05/Aug/2025
Objective: Extract and purify proteins.
Protocol:
1 mL of sonication buffer was added to pellet and thoroughly resuspended, then transferred to 1.5 mL tube.
Sample was subjected to sonication for 4 minutes with cycles of 30 seconds sonication and 30 seconds rest.
0.3 mL of 5M NaCl at pH 7.8 and 2.33 mL of ammonium sulfate were added to the supernatant.
1.2 mL of 96% ethanol was immediately mixed with the entire suspension and vigorously shaken for 30 seconds.
Centrifuged at 3,000 g for 7 minutes.
Upper ethanol layer was collected into 15 mL tube.
n-Butanol (one-quarter the volume of ethanol layer) was added and vigorously shaken for 30 seconds.
Centrifuged at 3,000 g for 7 minutes.
Lower layer was collected into 15 mL tube.
100% ammonium sulfate equal to one-quarter the volume of collected layer was added.
Left at room temperature for 2 hours.
Centrifuged at 2,000 g for 5 minutes.
Upper organic layer and lower aqueous layer were removed, and precipitate at interface was collected.
Suspended and dissolved in 1 mL of sonication buffer.
Result: Protein extraction and purification were completed.
Date: 06/Aug/2025
Objective: Prepare primer solution.
Protocol: MilliQ water was added to dried primers iGEM13, 15, 16, 17, and 18 to prepare 100 μM solutions. iGEM7 and 13 were 50 pmol/μL, so they were diluted 50-fold to prepare 1 mM stock (1 μL : 49 μL). iGEM14, 15, 16, 17, 18 (100 μM) were diluted 10-fold, and 1 mM iGEM7 and 13 were diluted 100-fold to prepare 100 μL each of 10 μM primer solutions (iGEM7, 13~18).
Result: Solution preparation was completed.
Date: 06/Aug/2025
Objective: Amplify insert fragments.
Protocol:
The following solution was prepared:
Table 17. Reaction mixture composition
| Component | Amount |
|---|---|
| PrimeSTAR master mix | 25μl |
| Insert fragment | 0.5μl |
| Primer (10μM) ① | 1μl |
| Primer (10μM) ② | 1μl |
| MilliQ | 22.5μl |
| total | 50μl |
Inserts and primers were mixed according to the following correspondence:
Table 18. Insert and primer correspondence
| Insert | Primer ① | Primer ② |
|---|---|---|
| T01_02 | iGEM14 | iGEM7 |
| T01_03 | iGEM15 | iGEM7 |
| T01_04 | iGEM16 | iGEM17 |
| T02_03 | iGEM18 | iGEM7 |
| T02_10 | iGEM13 | iGEM7 |
PCR was performed with the thermal cycler settings as follows.
Result: Insert fragments were successfully amplified.
Date: 06/Aug/2025
Objective: Prepare agarose gel.
Protocol: Same procedure as 18/07/2025.
Result: Agarose gel was prepared.
Date: 06/Aug/2025
Objective: Extract DNA.
Protocol:
5× volume of ① Buffer PB (45 μL × 5 = 225 μL) was added to PCR reaction and mixed.
Transferred to column, centrifuged at 12,000 rpm for 30 seconds, and filtrate was discarded.
700 μL of ② Buffer NW was added, centrifuged at 12,000 rpm for 30 seconds, and filtrate was discarded.
Centrifuged empty at 12,000 rpm for 1 minute to dry.
Transferred to new 1.5 mL tube and 50 μL of ④ Buffer EB was added.
After standing for 1 minute, centrifuged at 12,000 rpm for 1 minute and collected.
Result: DNA extraction was completed.
Date: 06/Aug/2025
Objective: Perform electrophoresis.
Protocol: The following solution was prepared:
Table 19. Reaction mixture composition
| Component | Amount |
|---|---|
| PCR reaction | 5μl |
| 10× Loading | 1μl |
| MilliQ | 4μl |
| total | 10μl |
Electrophoresis was performed at 100 V for 20 minutes.
Result: Electrophoresis was completed.
Fig. 17. Electrophoresis results
1: Marker 2: T01_02 3: T01_03 4: T01_04 5: T02_03 6: T02_10
Date: 06/Aug/2025
Objective: Perform transformation.
Protocol: Same procedure as 13/03/2025. However, pET28a was used as the vector, T01_-04 and negative control as inserts. Competent cells of JM109 provided by a graduate student were used.
Result: Only one colony was confirmed the following day.
Date: 07/Aug/2025
Objective: Prepare primer solutions.
Protocol: All primers iGEM_7, 13, 14, 15, and 18 were adjusted to 10 μM.
Result: Solution preparation was completed.
Date: 07/Aug/2025
Objective: Amplify insert fragments.
Protocol: Same procedure as 06/08/2025. However, inserts T01_02, 03, and T02_10 were used.
Results: Insert amplification was completed.
Date: 07/Aug/2025
Objective: Prepare agarose gel.
Protocol: Same procedure as 06/08/2025.
Results: Agarose gel preparation was completed.
Date: 07/Aug/2025
Objective: Extract DNA.
Protocol: Same procedure as 06/08/2025.
Results: DNA extraction was completed.
Date: 07/Aug/2025
Objective: To perform electrophoresis.
Protocol: The experiment was conducted following the same procedure as on 06/08/2025.
Results: Electrophoresis confirmed amplification in all samples.
Fig. 18. Electrophoresis results
Fig. 18. Electrophoresis results
1: Marker 2: T01_02 3: T01_03 4: T02_10
Date: 07/Aug/2025
Objective: To perform transformation.
Protocol: The experiment was conducted following the same procedure as on 22/07/2025. However, inserts T01_02, 03, 04, and T02_10 were used.
Results: Transformation was completed successfully.
Date: 08/Aug/2025
Objective: To amplify inserts.
Protocol: Amplification of T02_03 and T02_10 was performed following the same procedure as on 06/08/2025. However, since the remaining volume of T02_03 was less than 0.5 μl, all available volume was used.
Results: Insert amplification was completed successfully.
Date: 08/Aug/2025
Objective: To prepare agarose gel.
Protocol: The experiment was conducted following the same procedure as on 06/08/2025.
Results: Agarose gel preparation was completed successfully.
Date: 08/Aug/2025
Objective: To extract inserts.
Protocol: Insert fragments were extracted from PCR reaction mixtures following the same procedure as on 06/08/2025.
Results: Inserts were successfully extracted.
Date: 08/Aug/2025
Objective: To perform electrophoresis.
Protocol: The experiment was conducted following the same procedure as on 06/08/2025.
Results: Electrophoresis was completed successfully.
Fig. 19. Electrophoresis results
Fig. 19. Electrophoresis results
1: Marker 2: T02_03 3: T02_10
Date: 08/Aug/2025
Objective: To perform transformation.
Protocol: The experiment was conducted following the same procedure as on 22/07/2025.
Results: Transformation was completed successfully.
Date: 09/Aug/2025
Objective: To perform colony PCR.
Protocol: The experiment was conducted following the same procedure as on 28/07/2025. However, four insert types were used: T01_02, T01_03, T01_04, and T02_10.
Results: Colony PCR was completed successfully.
Date: 09/Aug/2025
Objective: To reuse previously used agarose gel.
Protocol:
Eight gel sections were placed in an Erlenmeyer flask and TAE buffer (2×8=16 ml) was added.
The agarose gel was melted in a microwave and 17 ml of SYBR Safe was added.
Gel was prepared using a smaller comb.
Results: All procedures were completed without issues.
Date: 09/Aug/2025
Objective: To perform electrophoresis.
Protocol: The experiment was conducted following the same procedure as on 06/08/2025.
Results: All procedures were completed without issues.
Date: 09/Aug/2025
Objective: To perform colony PCR.
Protocol: The experiment was conducted following the same procedure as on 28/07/2025. However, eight colonies of insert type T01_03 were used.
Results: Colony PCR was completed successfully.
Date: 09/Aug/2025
Objective: To perform inoculation.
Protocol: Colonies ⑤ and ⑥ of T01_03 (7.8 μl each) were inoculated into 5 ml of kanamycin-containing medium and cultured overnight with shaking.
Results: Inoculation was completed successfully.
Date: 10/Aug/2025
Objective: To extract vectors.
Protocol:
5 ml of culture was transferred to a 15 ml tube.
The sample was centrifuged at 18,000×g, 4°C for 1 min, and the supernatant was discarded.
250 μl of FAPD1 was added, and the pellet was completely resuspended by pipetting and transferred to a 1.5 ml tube.
250 μl of FAPD2 buffer was added and mixed by inversion 5-10 times until the solution became clear.
150 μl of FAPD3 buffer was added and immediately mixed well to confirm the formation of a small precipitate.
The sample was centrifuged at 18,000×g, 4°C for 10 min, and the supernatant was transferred to a column.
The sample was centrifuged at 11,000×g, 4°C for 30 sec, and the filtrate was discarded.
400 μl of WF buffer was added, centrifuged at 11,000×g, 4°C for 30 sec, and the filtrate was discarded.
700 μl of Wash buffer was added, centrifuged at 11,000×g, 4°C for 30 sec, and the filtrate was discarded.
Without adding buffer, the sample was centrifuged at 18,000×g, 4°C for 4 min, and the filtrate was discarded.
The column was transferred to a new 1.5 ml tube, 75 μl of Elution buffer was applied to the membrane and allowed to stand for 1 min.
The sample was centrifuged at 18,000×g, 4°C for 1 min to collect the vector.
DNA concentration was measured using NanoDrop.
Table 20. DNA Concentration Measurement Results (by Colony)
| Colony Number | Insert | Concentration (ng/μl) |
|---|---|---|
| 1 | T01_02 | 130 |
| 2 | T01_02 | 123 |
| 5 | T01_03 | 202 |
| 6 | T01_03 | 253 |
| 9 | T01_04 | 131 |
| 10 | T01_04 | 182 |
| 13 | T02_10 | 194 |
| 14 | T02_10 | 160 |
Results: DNA concentrations were obtained as shown in the table above.
Date: 11/Aug/2025
Objective: To perform transformation.
Protocol:
The experiment was conducted following the same procedure as on 18/07/2025. However, previously extracted T02_10①, 01②, 02①, 02②, 22①, 22②, and T02_28 were used as vectors.
Results: Transformation was completed successfully.
Date: 12/Aug/2025
Objective: To prepare 2% agarose gel.
Protocol: The experiment was conducted following the same procedure as on 18/07/2025. However, 150 ml of buffer and 3 g of agarose were used.
Results: All procedures were completed without issues.
Date: 12/Aug/2025
Objective: To perform inoculation.
Protocol: Samples cultured the previous day were inoculated into 5 ml of kanamycin-containing medium.
Results: Inoculation was completed successfully.
Date: 13/Aug/2025
Objective: To perform main culture.
Protocol:
200 μl from the pre-culture prepared the previous day was inoculated into 10 ml of kanamycin-containing liquid LB medium.
After 2 hours, OD600 was measured and found to be sufficient, so main culture was initiated.
100 μl of 100 mM IPTG was added to each culture, and incubation was performed overnight at 28°C with shaking.
Results: All procedures were completed without issues.
Date: 14/Aug/2025
Objective: To perform main culture.
Protocol: The entire culture volume was transferred to a 15 ml tube, centrifuged at 18,000×g for 2 min, and the supernatant was discarded.
Results: Main culture was completed successfully.
Date: 21/Aug/2025
Objective: To perform transformation.
Protocol: The experiment was conducted following the same procedure as on 13/03/2025. However, pET28a was used as the vector, BL21 strain as competent cells, and T01_01, 02, 03, 04, T02_01, 04, 07, 08, 10, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 were used as inserts.
Results: Transformation was completed successfully.
Date: 25/Aug/2025
Objective: To perform transformation.
Protocol: Previously extracted vectors of T02_04, 08, 13, 14, 17, 19, 20, 24, 26, 28 were used. For samples with ① and ②, ① was used. Otherwise, the experiment was conducted following the same procedure as on 18/07/2025.
Results: All procedures were completed without issues.
Date: 26/Aug/2025
Objective: To check plates.
Protocol: See “Transformation.”
Results: Plates were collected and colonies were confirmed. Therefore, they were stored in the refrigerator.
Date: 26/Aug/2025
Objective: To perform pre-culture.
Protocol: One colony from each sequence transformed on 25/08/2025 was picked and inoculated into 5 ml of liquid LB medium containing 50 mg/l kanamycin.
Results: All procedures were completed without issues.
Date: 27/Aug/2025
Objective: To perform main culture.
Protocol: The experiment was conducted following the same procedure as on 13/08/2025.
Results: All procedures were completed without issues.
Date: 28/Aug/2025
Objective: To perform main culture.
Protocol: The entire culture volume was transferred to a 15 ml tube and centrifuged at 18,000×g for 2 min.
Results: Main culture was completed successfully.
Date: 29/Aug/2025
Objective: To perform transformation.
Protocol: The experiment was conducted following the same procedure as on 18/07/2025. However, vectors T02_01①, 21②, 25①, and 29② were used.
Results: Main culture was completed successfully.
Date: 30/Aug/2025
Objective: To perform inoculation.
Protocol: Previously cultured samples were inoculated into 5 ml of kanamycin-containing medium.
Results: Inoculation was completed successfully.
Date: 30/Aug/2025
Objective: To perform main culture.
Protocol: The experiment was conducted following the same procedure as on 13/08/2025.
Results: All procedures were completed without issues.
Date: 31/Aug/2025
Objective: To perform main culture.
Protocol: The entire culture volume was transferred to a 15 ml tube, centrifuged at 18,000×g for 2 min, and the supernatant was removed.
Results: Main culture was completed and samples were stored at -4°C.
Date: 02/Sep/2025
Objective: To perform protein extraction and purification.
Protocol: The experiment was performed using avGFP and T02_01 following the same procedure as on 05/08/2025. T02_08 and T02_28 were also processed.
Results: avGFP extraction failed, but the others were successful.
Date: 03/Sep/2025
Objective: To perform SDS-PAGE.
Protocol: See the “SDS-PAGE” protocol. Samples analyzed were avGFP, T02_01, 08, and 28.
Results: All samples showed a single band.
Date: 04/Sep/2025
Objective: To perform transformation.
Protocol: The experiment was conducted following the same procedure as on 13/03/2025. The pET28a plasmid was introduced into JM109 strain for transformation.
Results: Transformation was completed successfully.
Date: 04/Sep/2025
Objective: To prepare a calibration curve.
Protocol:
Three colonies were picked from the E. coli transformed the previous day and dissolved in 450 ml of MilliQ.
100 mg/ml ampicillin was prepared at 16-fold, 32-fold, 64-fold, 128-fold, 256-fold, and 512-fold dilutions.
Two plates with 200 μl of E. coli suspension were prepared.
On each plate, sections were created for 16-fold, 32-fold, and 64-fold diluted ampicillin, and for 128-fold, 256-fold, and 512-fold dilutions, which were then applied.
The two plates were incubated at 37°C.
Results: All procedures were completed without issues.
Date: 06/Sep/2025
Objective: To measure protein concentration.
Protocol: The experiment was conducted following the standard protocol (1 ml use) of the TakaRa Bradford Protein Assay kit. Samples T02_01, 08, 28 (purified on 02/09/2025) were used. T02_08 and 28 were diluted 2-fold before use.
Results: Concentration measurement was completed successfully.
Date: 06/Sep/2025
Objective: To perform transformation.
Protocol: The experiment was conducted following the same procedure as on 18/07/2025. However, vectors T01_02, 03, 04, T02_02, 10, 16, 18, 22, 24, 27 (both ① and ②) were used.
Results: Transformation was completed successfully.
Date: 07/Sep/2025
Objective: To perform inoculation.
Protocol: Samples transformed on 06/Sep/2025 were inoculated into 5 ml of kanamycin-containing medium.
Results: Inoculation was completed successfully.
Date: 08/Sep/2025
Objective: To perform main culture.
Protocol: 200 ml of pre-culture from 07/Sep/2025 was inoculated into 10 ml of kanamycin-containing liquid LB medium. After approximately 2 hours, when OD was sufficient, 100 μl of 100 mM IPTG was added, and overnight culture was performed at 28°C.
Results: Main culture was completed successfully.
Date: 09/Sep/2025
Objective: To perform main culture.
Protocol: Samples from the main culture on 08/Sep/2025 were centrifuged at 18,000×g for 2 min, and the supernatant was discarded.
Results: Inoculation was completed successfully.
Date: 08/Sep/2025
Objective: To prepare a calibration curve.
Protocol: The experiment was conducted following the same procedure as on 04/Sep/2025.
Results: All procedures were completed without issues.
Date: 08/Sep/2025
Objective: To prepare a calibration curve.
Protocol: The experiment was conducted following the same procedure as on 04/Sep/2025.
Results: All procedures were completed without issues.
Date: 10/Sep/2025
Objective: To perform SDS-PAGE.
Protocol:
Table 21. Reaction Solution Composition
| Component | Volume |
|---|---|
| Purified protein solution | 20 μl |
| 5× Western Blotting buffer | 20 μl |
| 1% SDS | 75 μl |
| 2-Mercaptoethanol | 5 μl |
The above solution was heated in a heat block set at 100°C for 10 min.
A pre-cast gel was set in the electrophoresis tank, and running buffer was added.
The comb was removed, and 4 μl of marker and 20 μl of each sample were loaded into the lanes. Samples used were T02_01, 08, and 28.
The gel was removed, washed twice with DW, 5 min each.
CBB staining solution was added to cover the gel and stained for 1 hour.
The staining solution was discarded, and the gel was washed twice with DW, 5 min each.
Results: SDS-PAGE was completed successfully.
Date: 10/Sep/2025
Objective: To perform SDS-PAGE.
Protocol: The experiment was conducted following the same procedure as on 10/Sep/2025. The same samples were used.
Results: SDS-PAGE was completed successfully.
Date: 11/Sep/2025
Objective: To perform pre-culture.
Protocol: E. coli containing avGFP transformed on 10/Sep/2025 was used. The experiment was conducted following the same procedure as on 26/Aug/2025.
Results: Pre-culture was completed successfully.
Date: 12/Sep/2025
Objective: To perform main culture.
Protocol: The experiment was conducted following the same procedure as on 13/Aug/2025. However, avGFP was used as the sample.
Results: Main culture was completed successfully.
Date: 13/Sep/2025
Objective: To perform main culture.
Protocol: The entire culture volume was transferred to a 15 ml tube and centrifuged at 18,000×g for 2 min.
Results: Main culture was completed successfully.
Date: 16/Sep/2025
Objective: To purify proteins.
Protocol: Samples T02_01, 04, 08, 13, 14, 17, 19, 20, 21, 23, 25, 26, 28, 29, and avGFP were used. Otherwise, the experiment was conducted following the same procedure as on 05/Aug/2025.
Results: Main culture was completed successfully.
Date: 17/Sep/2025
Objective: To measure fluorescence and excitation spectra.
Protocol: Samples purified on 16/Aug/2025 were mixed with 2M NaOH at a 1:1 ratio, and absorbance was measured. Fluorescence and excitation spectra of the purified samples were obtained (separate from the mixed samples above).
Results: Measurement of fluorescence and excitation spectra was completed successfully.
Date: 18/Aug/2025
Objective: To prepare buffers.
Protocol: 200 ml of each of the following buffers was prepared.
Table 22. Binding Buffer Composition
| Component | Concentration |
|---|---|
| Tris-HCl (pH 8) | 50 mM |
| NaCl | 300 mM |
| Imidazole | 5 mM |
| Mg(OAc)2 | 20 mM |
| Tween20 | 0.10% |
Table 23. Washing Buffer Composition
| Component | Concentration |
|---|---|
| Tris-HCl | 50 mM |
| NaCl | 300 mM |
| Imidazole | 5 mM |
| Tween20 | 0.10% |
Table 24. Elution Buffer Composition
| Component | Concentration |
|---|---|
| Tris-HCl | 50 mM |
| NaCl | 500 mM |
| Imidazole | 400 mM |
| Tween20 | 0.10% |
Results: Buffer preparation was completed. The prepared buffers were stored in the refrigerator.
Date: 19/Sep/2025
Objective: To perform SDS-PAGE.
Protocol: Samples purified on 06/Sep/2025 were used. Otherwise, the experiment was conducted following the same procedure as on 10/Sep/2025.
Results: SDS-PAGE was completed successfully.
Date: 21/Sep/2025
Objective: To quantify purified proteins.
Protocol: Samples purified on 16/Sep/2025 were used. The experiment was conducted following the standard protocol (200 μl use) of the TakaRa Bradford Protein Assay kit.
Results: Quantification of purified proteins was completed successfully.
Date: 21/Sep/2025
Objective: To perform pre-culture.
Protocol: The experiment was conducted following the same procedure as on 26/Aug/2025.
Results: Pre-culture was completed successfully.
Date: 22/Sep/2025
Objective: To perform transformation.
Protocol: T01 02, 03, 04 and T02 01, 04, 08, 13, 14, 17, 19, 20 were used.
Results: Transformation was completed successfully.
Date: 22/Sep/2025
Objective: To perform main culture.
Protocol: The experiment was conducted following the same procedure as on 13/Aug/2025.
Results: All procedures were completed without issues.
Date: 23/Sep/2025
Objective: To perform main culture.
Protocol: Samples were centrifuged at 18,000×g for 2 min, and the supernatant was discarded.
Results: Main culture was completed successfully.
Date: 24/Sep/2025
Objective: To amplify DNA fragments.
Protocol:
Primer solutions were adjusted to 10 μM.
The following solution was prepared:
Table 25. Reaction Solution Composition
| Component | Volume |
|---|---|
| PrimeSTAR Max Master mix | 25 μl |
| Insert fragment (template) | 1 μl |
| Primer ① (10 mM) | 1 μl |
| Primer ② (10 mM) | 1 μl |
| MilliQ | 22 μl |
| Total | 50 μl |
① 94°C for 2 min
② 98°C for 5 sec
③ 55°C for 5 sec
④ 72°C for 30 sec
⑤ Repeat steps ②-④ for 35 cycles
⑥ Store at 20°C
Results: Amplification was completed successfully.
Date: 25/Sep/2025
Objective: To confirm and purify PCR products.
Protocol: For confirmation, 5 μl of PCR product, 1 μl of 10× loading buffer, and 4 μl of MilliQ were mixed and subjected to electrophoresis for 30 min. Purification was performed following the PCR product protocol of the Takara Genius PCR & Gel Prep Kit.
Results: Confirmation and purification of PCR products were completed successfully.
Fig. 20. Electrophoresis results
Fig. 20. Electrophoresis results
From left: Marker, GFP, PETase, β-lactamase
Date: 25/Sep/2025
Objective: To perform protein synthesis.
Protocol: See “Protein Synthesis by PURExpress” protocol.
Results: Protein synthesis was completed successfully.
Date: 26/Sep/2025
Objective: To repeat the experiment from 24/Sep/2025.
Protocol: The experiment was conducted following the same procedure as on 24/Sep/2025.
Results: DNA fragment amplification was completed successfully.
Date: 26/Sep/2025
Objective: To equilibrate resin.
Protocol:
The resin was mixed for 1-2 min using a 100 μl pipette without pipetting.
The mixture was gently inverted for 1 min.
20 μl of resin solution was aliquoted into an Eppendorf tube.
100 μl of binding buffer was added and gently pipetted.
The sample was centrifuged at 700×g for 2 min, and the supernatant was removed.
The binding buffer addition and supernatant removal steps were repeated 4 times.
Results: Resin equilibration was completed successfully.
Date: 27/Sep/2025
Objective: To confirm and extract DNA fragments.
Protocol: The experiment was conducted following the same procedure as on 24/Sep/2025.
Results: No bands were visible.
Fig. 21. Electrophoresis results
Fig. 22. Electrophoresis results
From left: Marker, PETase, GFP
Date: 28/Sep/2025
Objective: To perform SDS-PAGE.
Protocol: Samples used were total, FT, W1, W2, W3, W4, W5, E1, E2, and E3. Otherwise, the experiment was conducted following the same procedure as on 20/Sep/2025.
Results: DNA fragment amplification was completed successfully.
Date: 28/Sep/2025
Objective: To perform electrophoresis.
Protocol: PCR products from the PCR performed on 27/Sep/2025 were used as samples.
Results: No bands were visible.
Fig. 22. Electrophoresis results
Fig. 22. Electrophoresis results
From left: Marker, PETase
Date: 28/Sep/2025
Objective: To purify PCR products from 27/Sep/2025 for cell-free system.
Protocol: PCR products from 27/Sep/2025 were purified for the cell-free system. The PCR product protocol of the Takara Genius PCR & Gel Prep Kit was followed.
Results: Purification was completed successfully.
Date: 29/Sep/2025
Objective: To express proteins using a cell-free system.
Protocol: See “Cell-free Protein Expression” protocol. PETase was expressed.
Results: All procedures were completed successfully.
Date: 29/Sep/2025
Objective: To equilibrate resin.
Protocol:
The resin was mixed for 1-2 min using a 100 μl pipette without pipetting.
The mixture was gently inverted for 1 min.
20 μl of resin solution was aliquoted into an Eppendorf tube.
100 μl of binding buffer was added and gently pipetted.
The sample was centrifuged at 700×g for 2 min, and the supernatant was removed.
The binding buffer addition and supernatant removal steps were repeated 4 times.
Results: Resin equilibration was completed successfully.
Date: 29/Sep/2025
Objective: To purify proteins using His-tag affinity chromatography.
Protocol: Refer to the “His-tag Purification” protocol. Purification of PETase was performed.
Results: Purification of avGFP was completed.
Date: 30/Sep/2025
Objective: To purify proteins using His-tag affinity chromatography.
Protocol: Refer to the “His-tag Purification” protocol. Purification of avGFP was performed.
Results: Purification of avGFP was completed.
Date: 01/Oct/2025
Objective: To quantify fluorescent protein concentration.
Protocol: Refer to the “Quantification of Fluorescent Protein” protocol. Quantification of avGFP was performed.
Results: Quantification was unsuccessful due to insufficient protein concentration in the solution.
Date: 02/Oct/2025
Objective: To confirm the purity of purified proteins.
Protocol: Refer to the “SDS-PAGE” protocol. SDS-PAGE was performed on proteins purified on 30/Sep/2025.
Results: The procedure was completed without issues.
Date: 02/Oct/2025
Objective: To purify proteins from cultured E. coli cell pellets.
Protocol: Refer to the “Protein Extraction and Purification” protocol. Purification was performed on pellets collected on 24/Sep/2025. However, the final resuspension volume in sonication buffer was modified from 1 mL to 200 μL.
Results: Purification was completed.
Date: 05/Oct/2025
Objective: To quantify the concentration of protein solutions.
Protocol: Refer to the “Bradford Assay” protocol. Samples purified on 02/Oct/2025 were quantified.
Results: Quantification was completed.
Date: 05/Oct/2025
Objective: To perform pre-culture for protein expression.
Protocol: A colony was picked from T01_04 plate (transformed on 22/Sep/2025) and inoculated into 5 mL of liquid LB medium containing kanamycin.
Results: Pre-culture was completed.
Date: 06/Oct/2025
Objective: To perform main culture for protein expression.
Protocol: The experiment was performed following the same procedure as 13/Aug/2025. The T01_04 sample pre-cultured on 05/Oct/2025 was used.
Results: Main culture was completed.
Date: 06/Sep/2025
Objective: To perform main culture.
Protocol: Cells were centrifuged at 18,000 × g for 2 minutes, and the supernatant was discarded.
Results: Main culture was completed.
Date: 05/Oct/2025
Objective: To perform pre-culture for protein expression.
Protocol: Colonies were picked from T01_02, T02_19, 20, 21, 23, and avGFP plates (transformed on 22/Sep/2025) and inoculated into 5 mL of liquid LB medium containing kanamycin.
Results: Pre-culture was completed.
Date: 06/Oct/2025
Objective: To perform main culture for protein expression.
Protocol: The experiment was performed following the same procedure as 13/Aug/2025. Samples T01_02, T02_19, 20, 21, 23, and avGFP pre-cultured on 05/Oct/2025 were used.
Results: Main culture was completed.
Date: 07/Oct/2025
Objective: To perform main culture.
Protocol: Cells were centrifuged at 18,000 × g for 2 minutes, and the supernatant was discarded.
Results: Main culture was completed.
Date: 06/Oct/2025
Objective: To equilibrate the resin.
Protocol:
The resin was gently mixed for 1 to 2 minutes using a 100 μL pipette without pipetting.
The mixture was inverted gently for 1 minute.
20 μL of resin solution was aliquoted into a tube.
100 μL of binding buffer was added and mixed gently by pipetting.
The mixture was centrifuged at 700 × g for 2 minutes, and the supernatant was removed.
Steps 4 and 5 (binding buffer addition and supernatant removal) were repeated 4 times.
Results: Resin equilibration was completed.
Date: 06/Oct/2025
Objective: To express proteins using a cell-free system.
Protocol: Refer to the “Cell-Free Protein Expression” protocol. T03_01 to 05 were expressed.
Results: The procedure was completed.
Date: 06/Oct/2025
Objective: To purify proteins using His-tag affinity chromatography.
Protocol: Refer to the “His-tag Purification” protocol. Purification of T03_01 to 05 was performed.
Results: Purification of T03_01 to 05 was completed.
Date: 07/Oct/2025
Objective: To purify proteins from cultured E. coli cell pellets.
Protocol: Refer to the “Protein Extraction and Purification” protocol. Purification of T01_02, 04, T02_19, 20, 21, 23, and avGFP was performed. However, the final resuspension volume in sonication buffer was modified from 1 mL to 200 μL.
Results: Purification was completed.
Date: 07/Oct/2025
Objective: To quantify the concentration of protein solutions.
Protocol: Refer to the “Bradford Assay” protocol. Samples purified on 07/Oct/2025 were quantified.
Results: Quantification was completed.
Date: 07/Oct/2025
Objective: To quantify the concentration of protein solutions.
Protocol: Refer to the “Bradford Assay” protocol. However, the standard protocol was modified to use 4 μL of each sample and 200 μL of Bradford Dye Reagent. Samples purified on 06/Oct/2025 were quantified.
Results: Quantification was completed.
Date: 07/Oct/2025
Objective: To measure fluorescence and excitation spectra.
Protocol: Fluorescence and excitation spectra were measured for samples purified on 07/Oct/2025.
Results: Measurement of fluorescence and excitation spectra was completed.
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