Experiments

The lab procedures for our project include a series of standard molecular biology and microbiology experiments designed to support our research objectives. These protocols cover media preparation, bacterial transformation, plasmid isolation, PCR amplification, gel electrophoresis, protein analysis, and chitinase activity assays. Together, these methods enabled the growth and maintenance of bacterial cultures, the introduction and verification of genetic constructs, and the expression and characterization of target proteins necessary for the birth of Chitinator et al.. Each step of the lab process has been organized into a separate card in the overview panel, allowing you to easily browse and select the specific procedure you wish to read and learn more about.

Details about the experimental process can be also found on our Lab Book Page

• Weigh the required amount of LB Broth powder (according to manufacturer’s instructions)
• Dissolve the powder in distilled water up to the desired volume
• Sterilize by autoclaving at 120 °C for 15 minutes
• Allow to cool
• Store in bottles at 4 °C until use

• Weigh the required amount of LB Agar powder (according to manufacturer’s instructions)
• Dissolve the powder in distilled water up to the desired volume
• Sterilize by autoclaving at 120 °C for 15 minutes
• Allow to cool
• Store in bottles at 4 °C until use

• LB agar plates supplemented with antibiotic were inoculated with 50 μL of bacterial stock and incubated at 37 °C
• Inoculation of a single colony in 5 mL LB broth. Incubation at 37 °C
• Addition of 1 mL culture to 100 mL LB broth (1:100 dilution). Incubation until OD₆₀₀ 0.4–0.6
• On ice for 15 min for growth arrest
• Division of culture into two 50 mL Falcon tubes. Centrifugation at 3000 × g for 15 min at 4 °C
• Removal of supernatant. Resuspension of pellets in 50 mL ice-cold 50 mM CaCl₂ for each pellet
• Gentle inversion of Falcon tubes. Placement on ice every 10 inversions to prevent temperature drop
• Incubation on ice for 20 min
• Centrifugation at 3000 × g for 5 min at 4 °C. Resuspension of pellets in 4 mL 50 mM CaCl₂ with 15% glycerol in the same way
• Transfer of 200 μL from the Falcon tubes into fresh Eppendorf tubes, under flame
• Rapid transfer of each Eppendorf tube into a bath of absolute ethanol pre-chilled at –80 °C
• Verification of complete freezing of all tubes; disposal of incompletely frozen samples. Storage at –80 °C
• Alternative procedure: immersion of Eppendorf tubes in liquid nitrogen within a dry vessel in the absence of absolute ethanol

• Mixing of 100 μL competent cells with 100 ng plasmid DNA
• Incubation on ice for 30 min
• Heat shock at 42 °C for 1.5 min
• Incubation on ice for 5 min
• Addition of 400 μL LB pre-warmed at 37 °C. Incubation at 37 °C with shaking for 1 h
• Centrifugation at 3000 × g for 10 min. Removal of 200 μL supernatant to increase cell concentration
• Plating on LB agar plates supplemented with appropriate antibiotic. Incubation for 16–18 h at 37 °C

PCR Reaction Setup (Total volume: 95 μL)

Polymerase used: NEB Taq 2× standard buffer master mix

• Forward primer (10 μM): 0.5 μL was added → 3.5 μL in scaled reactions.
• Reverse primer (10 μM): 0.5 μL was added → 3.5 μL in scaled reactions.
• Polymerase (Master Mix): 19.5 μL was included → 80 μL in scaled reactions.
• Plasmid DNA: For each 25 μL reaction, 2.5 μL plasmid DNA was added.
• Nuclease-free water: 9 μL was added → 63 μL in scaled reactions.

(Volumes were adjusted proportionally for multiple reactions.)

According to NEB, the recommended plasmid DNA amount was 1 pg – 10 ng. The plasmid stock concentration was 2000 ng/μL (2 μg/μL). So for each 25 μL reaction, 2.5 μL plasmid DNA was added.

Primer Dilution (from IDT, 100 μM stock)

• Forward primer: 1 μL of primer diluted in 9 μL nuclease-free H₂O → 10 μM working stock.
• Reverse primer: 1 μL of primer diluted in 9 μL nuclease-free H₂O → 10 μM working stock.

Due to the high variability of the primers, a temperature gradient was performed. Gradient annealing temperatures applied: 61°C, 62°C, 63°C, 64°C, 65°C, 66°C.

Thermocycling Conditions (30 cycles)

• Initial denaturation: Samples were heated at 94°C for 30 sec.
• Denaturation: 94°C for 15–30 sec.
• Annealing: 45–68°C for 15–60 sec.
• Extension: 68°C for 1 min/kb.
• Final extension: 68°C for 5 min.
• Hold: 4°C.

• Mixture of 100 μL PCR product and 200 μL Buffer NTI
• Mixture transferred onto NucleoSpin spin column
• Centrifuged at 11,000 × g for 30 sec
• Flow-through discarded
• 700 μL of Buffer NT3 (supplemented with 100% ethanol) added to the column
• Centrifuged at 11,000 × g for 30 sec
• The previous step was repeated once more
• Flow-through discarded
• Centrifugation of column without any added liquid at 11,000 × g for 1 min
• Incubation at 70°C for 5 minutes on the thermoblock
• Flow-through discarded
• Transfer of the column into a 1.5 mL Eppendorf
• 25 μL of elution buffer NE were added
• Incubation at room temperature for 1 min
• Centrifugation at 11,000 × g for 1 min
• Step repeated two more times
• Fresh preheated buffer added at 70 °C and incubated for 5 min
• Centrifugation at 11,000 × g for 1 min
• Eluted DNA stored at −20 °C

• Weighing of 0.5 g agarose
• Dissolution in 50 mL TAE buffer
• Heating in microwave until complete dissolution
• Addition of 2.5 μL EtBr when the solution has cooled down
• Pouring into casting tray and placement of comb
• Solidification at room temperature
• Mixing of 5 μL sample from each Eppendorf with 1 μL loading dye
• Loading onto gel
• Electrophoresis at 90 V for 30–40 min

• Cells collected by centrifugation at 11,000 × g, 10 μL LB-BL21
• Supernatant discarded
• Addition of resuspension buffer A1 and complete resuspension of cell pellet
• Addition of lysis buffer A2
• Gentle inversion 6–8 times, not vortexing
• Incubation at room temperature for 5 min
• Addition of neutralization buffer A, gentle inversion 6–8 times, avoiding vortexing
• Centrifugation at 11,000 × g for 10 min to clarify lysate
• Transfer of cleared supernatant onto NucleoSpin spin column placed in a collection tube
• Centrifugation at 11,000 × g for 1 min
• Flow-through discarded, spin column placed into a new collection tube
• Addition of 500 μL wash buffer AW1 to the spin column
• Centrifugation at 11,000 × g for 1 min
• Flow-through discarded
• Addition of 600 μL wash buffer AW2 to the spin column
• Centrifugation at 11,000 × g for 1 min
• Centrifugation repeated at 11,000 × g for 2 min to remove residual ethanol
• Spin column placed into a clean 1.5 mL Eppendorf tube
• Addition of 50 μL preheated elution buffer AE to the center of the membrane
• Incubation at 70 °C in a thermal block for 2 min
• Centrifugation at 11,000 × g for 1 min to elute plasmid DNA
• Nanodrop measurement

Preparation of lysis solutions:

• NaCl: 10 mM, 330 μL
• Tris HCl: 1 mM, 66 μL
• EDTA: 100 mM, 2000 μL
• Triton X-100: 0.5 v/v, 500 μL
• H2O: 79 mL

Division of 100 μL bacterial culture into:

• Two 50 mL Falcon tubes containing 40 mL culture each
• Two 15 mL Falcon tubes containing 10 mL culture each

Calculation of required lysis buffer:

• For 2 mL culture: 6 mL lysis buffer needed
• For 10 mL culture: 150 μL lysis buffer needed

Bacterial Lysis:

• Centrifugation of Falcon tubes without lysis buffer
• Discarding of supernatant and addition of LB medium to the pellet
• Gentle vortexing to resuspend the pellet
• Incubation at room temperature for 30 min
• Placement on ice for 10 min
• Sonication 3 times for 30 sec at 70% amplitude; 2 min rest on ice between each cycle
• Centrifugation at 15,000 × g for 30 min at 4 °C
• Separation of supernatant from the pellet

• Preparation of protein samples: proteins mixed with SDS sample buffer (containing SDS, β-mercaptoethanol, glycerol, bromophenol blue, and Tris-HCl)
• Heating of samples at 95 °C for 5 min to denature proteins
• Assembly of polyacrylamide gel in electrophoresis apparatus
• Loading of samples and protein ladder into wells
• Addition of running buffer to gel tank
• Electrophoresis at constant voltage (e.g., 120 V) until dye front reaches bottom of gel
• Removal of gel from apparatus
• Staining of gel with Coomassie Brilliant Blue or other protein stain
• Destaining to visualize protein bands

• Weigh 20 g chitin powder into a glass beaker
• Add 350 mL cold concentrated HCl slowly while stirring
• Incubate at 4 °C for 24 h to allow partial depolymerization
• Filter the suspension through glass wool into 2 L pre-cooled ethanol (–20 °C) with vigorous stirring
• Centrifuge at 10,000 × g, 20 min, 4 °C
• Wash the resulting pellet repeatedly with dH₂O until the pH becomes neutral
• Lyophilize (freeze-dry) the washed colloidal chitin, or store it as a suspension at –20 °C

• Resuspend colloidal chitin in dH₂O to 1% (w/v)
• Homogenize by passing 5× through a hand homogenizer or by sonication until a milky suspension forms
• Prepare LB agar (1.6% agar) and autoclave
• Mix equal volumes of molten LB agar (cooled to ~50 °C) with colloidal chitin suspension to obtain final 0.5% colloidal chitin agar
• Pour plates (~20 mL per Petri dish) and allow them to solidify
• Store plates at 4 °C until use

• Well diffusion assay: Punch wells into agar, load with culture supernatant or cell lysate
• Wait for at least 48 hours