Lab Notebook

Literature Review Phase

6.9 ~ 6.20

Twelve days dedicated to wet-lab related literature retrieval and reading.

Sequence Screening

6.21 ~ 6.23

Screen and identify five target sequences for subsequent validation.

Plasmid Construction

6.24 ~ 6.26

Select pET22b(+) as the vector plasmid. Design and construct five novel recombinant plasmids. Commission a company for plasmid synthesis.

Transformation Verification

7.13

Perform plasmid transformation of the plastic-degrading enzyme gene constructs according to the established protocol. Resuspend the bacterial cells evenly and plate onto LB agar plates supplemented with 100 μg/mL ampicillin. Incubate inverted at 37°C overnight.

7.14

LB Plate Overnight Culture Results:

Colonies were observed on all 100 μg/mL Amp LB plates, indicating successful preliminary transformation.

Colony Selection and PCR Setup:

• Select five distinct colonies from each plate and label them 1-30 (across 5 plates x 6 replicates each).
• Prepare the PCR reaction mixture according to the protocol.
• In a 1.5mL microcentrifuge tube, add ddH₂O, forward and reverse primers, and high-fidelity enzyme in equal proportions. Aliquot into PCR tubes.
• Prepare an empty Amp LB plate and evenly label it with numbers 1-30.
• Using a sterile pipette tip, pick individual colonies. First, streak a short line on the corresponding marked area on the plate to preserve the bacterial strain. Subsequently, gently transfer a small amount of bacterial cells from the streak into the PCR tube to serve as the template.

PCR Amplification:

95°C for 3 min ->(94°C for 25s, 55°C for 25s, 72°C for 15s) x 35 cycles ->72°C for 5 min -> 4°C indefinitely

Perform agarose gel electrophoresis on the PCR amplification products. No other non-specific bands were observed. The molecular weights of all five amplified fragments matched the expected sizes.

Excise the DNA bands corresponding to samples 5, 8, 11, 17, and 22 from the agarose gel. Submit these gel slices to the company for Sanger sequencing.

Expression Optimization

7.15

Plasmid Transformation and Expression Optimization:

Culture Preparation for Induction:

Inoculate bacteria from the preserved single colony plates into LB liquid culture medium containing ampicillin (100 μg/mL). Incubate at 37°C on a shaker for 3 hours.

IPTG Induction and Expression Optimization:

Subsequently, add IPTG to final concentrations of 0, 0.05, 0.1, 0.5, and 1 mM for induction. This is to screen for the optimal expression concentration. Incubate the induced bacterial cultures at 25°C on a shaker overnight.

7.16

Bacterial cultures were cryopreserved on July 15th. Sanger sequencing confirmed that the sequences of the recombinant plasmids transformed into E. coli were fully consistent with the design.

Following lysate collection, the initial 4-pNPA enzyme activity assay revealed an optimal enzyme activity profile at 0.1 mM IPTG. However, the PET_03 and PET_04 groups exhibited significantly lower enzyme activity compared to the empty vector control.

7.17

IPTG Induction Culture Preparation and OD Measurement:

Inoculation: Transfer 1 μL of the bacterial culture from July 15th into fresh 15 mL of LB medium supplemented with Ampicillin (100 μg/mL).

Pre-induction Culture: Incubate for 1-2 hours.

Expression was induced using 0.1 mM IPTG (prepared from a 5 mg/mL stock solution, 24 μL added to the culture), followed by incubation at 20°C on a shaker starting at 18:50. However, due to the reuse of the same measurement vessel without proper cleaning when measuring OD values and the practice of returning the measured bacterial liquid to its original tube, contamination was suspected. The following day, the empty LB tube (without bacterial inoculation) appeared turbid, leading to the discard of this set of bacterial cultures.

7.19

A 5 μL aliquot from the cryopreserved glycerol stock of bacterial cells was used to inoculate 15 mL of fresh LB medium supplemented with Ampicillin (100 μg/mL). After 2 hours of incubation, the culture medium remained clear with no significant visible changes. Overnight incubation was then performed.

7.20 ~ 7.22

The bacterial colonies cultured overnight on July 19th were measured for their OD600 using a spectrophotometer, yielding a value above 5.0. As the cell density was too high for immediate use, the cultures were consequently cryopreserved in glycerol stocks.

Following a discussion, it was determined that an operational error had occurred. The correct procedure dictates that glycerol stocks should first be used to inoculate LB agar plates containing ampicillin (100 μg/mL) and incubated overnight. The resulting colonies should then be picked with a pipette tip and used to inoculate 15 mL of fresh LB Amp (100 μg/mL) liquid medium.

After 3 hours of incubation, samples were collected for OD measurement, with the following results:

12 μL of IPTG was added to the bacterial cultures from July 20th, followed by overnight incubation at 20°C on a shaker.

Activity Testing

The bacterial cultures from July 20th were processed according to the protocol. In wells A-F, columns 1-9 of the 96-well plate, 135 μL of sodium phosphate buffer (pH 8.0), 15 μL of crude enzyme solution, and 50 μL of 4-pNPA stock solution were added. For wells G, columns 1-9, 150 μL of sodium phosphate buffer (pH 8.0) and 50 μL of 4-pNPA stock solution were added.

The template is as shown in the table:

Following procedural improvements, secreted proteins from all strains exhibited degradation capabilities towards plastic analogs.

Bacterial cultures from July 20th were used to inoculate LB agar plates supplemented with ampicillin (100 μg/mL) via streaking. The plates were then incubated overnight at 37°C.

7.23 (The first attempt to measure enzyme activity)

PLA Solution Preparation: Dissolve 0.25 g of PLA plastic fragments in 20 mL of dichloromethane. Place the solution on a magnetic stirrer within a fume hood, under ice bath conditions, and in the dark, allowing it to evaporate for 3 hours.

Medium Preparation: Autoclave 500 mL of LB agar medium at 121°C.

Top Layer (5 mL per plate): Mix 87.5 mL of the autoclaved LB agar medium with 42 μL of IPTG stock solution (1 mM; 50 mg/mL) and 100 mL of the prepared PLA solution.

Bottom Layer (15 mL per plate): Combine the remaining 400 mL of LB medium with 1.9 mL of IPTG stock solution (0.1 mM; 5 mg/mL) and 400 μL of Ampicillin stock solution (100 mg/mL).

Then inoculation of bacterial cultures from July 22nd was performed by streaking or spot inoculation onto newly prepared media (in triplicate). The plates were subsequently incubated overnight at 37°C in an oven.

Overnight cultures of bacterial colonies from July 23rd showed no discernible clear zones.

7.24 (The second attempt to measure enzyme activity after the adjustment)

Add 0.25 g of PLA plastic fragments to 20 mL of dichloromethane. Process the mixture on a magnetic stirrer for 1.5 hours. Subsequently, add 100 mL of double-distilled water (ddH₂O) and homogenize for 1.5 hours to facilitate evaporation.

However, after the dichloromethane evaporated, the PLA was unable to form a stable suspension in pure water. Instead, it aggregated into sheet-like structures and floated on the water surface.

Prepare a 2x concentrated LB agar medium by dissolving 20 g of LB agar powder in 250 mL of double-distilled water (ddH₂O). After sterilizing the LB agar medium, add the mixture of PLA and water to the sterile medium. Pour this mixture into two plates. Following solidification of the medium, spot inoculate with PLA-degrading bacteria.

7.25

The overnight cultures from July 24th showed contamination by unwanted microorganisms. Subsequently, 5 mg/mL IPTG was added to the PLA-degrading bacterial colonies, and 100 mg/mL Ampicillin was added to a blank area.

Inoculating bacterial strains from the July 22nd streak plates into 5 mL of LB medium supplemented with 5 µL of ampicillin stock solution and incubated overnight at 37°C.

7.26 ~ 7.28 (The third attempt to measure enzyme activity)

To the overnight cultures from July 25th, 24 µL of IPTG was added, followed by incubation at 20°C with shaking.

Then, preparing the PLA degradation screening plates, 0.25 g of PLA plastic fragments were dissolved in 5 mL of chloroform and stirred on a magnetic stirrer for 1 hour. Subsequently, 100 mL of sterile double-distilled water (ddH₂O) was added, and the mixture was homogenized. This resulting suspension was then combined with 100 mL of 2x concentrated LB medium. Plates were poured at intervals of 15 min, 30 min, 1 h, and 1.5 h from this mixture, with a control group consisting of 100 mL ddH₂O mixed with 100 mL of 2x concentrated LB medium. Inoculation was performed by streak plating or spot inoculation onto the prepared plates with both PLA-degrading bacteria and empty vector control bacteria. All inoculated plates were incubated overnight at 37°C in an oven.

The results for the July 27th inoculation are shown below:

After three days of incubation and observation, no clear zones were detected around the bacterial colonies, indicating a lack of detectable plastic degradation enzyme activity. Therefore, the experiment to verify enzymatic activity on PLA-containing agar plates has been discontinued.

Protein Analysis

7.30

Following the teacher's advice, we revised the experimental plan and decided to attempt using WB for supplementary verification.

Bacterial liquid was processed according to the protocol, commencing with SDS-PAGE electrophoresis at 140 V for 45 minutes using pre-cast gels, with 10 µL of sample loaded per well. Following electrophoresis, protein transfer was performed at 100 V for 1 hour. For antibody incubation, the membrane was initially blocked for 1.5 hours at room temperature with 5% non-fat dry milk in 25 mL of TBST, followed by a single wash with TBST. Primary antibody incubation was conducted overnight at 4°C with shaking in a cold room (lid unsealed) using a 1:1000 dilution in 5 mL of 0.5% non-fat dry milk solution in TBST; this was followed by four TBST washes, each lasting approximately 30 minutes. Subsequently, the membrane was incubated with the secondary antibody at a 1:5000 dilution in 10 mL of TBST for 50 minutes at room temperature, followed by four 10-minute washes with TBST.

The results are as follow:

Left: Protein content in the supernatant. Right: Protein content in the bacterial cells.

8.15

Finally, following the professor's suggestion and adhering to the protocol, the crude enzyme preparation obtained by sonication was added to Oxford cups on agar plates containing a plastic emulsion. The results showed only a faint ring, which, after discussion, was not considered a genuine indicator of plastic degradation.