July 7
- Prepared solid LB medium (5g LB composition, 2.25g agar powder, 200ml deionized water)
- Prepared liquid LB medium (3.75g LB composition, 150ml deionized water) for colony amplification
- Sterilized media using autoclave (121°C, 20 minutes)
- Set up ultraclean table: UV sterilization for 30 minutes, hand disinfection
- Prepared antibiotic plates: Added antibiotics at 1:1000 ratio (150ml total)
- Initiated host bacterium culture in liquid medium for plasmid preparation
July 8
- Set up PCR reaction system:
- DNA template: 1μl
- Forward/Reverse primers (10μM): 1μl each
- 2× Super Pfx Master Mix: 12.5μl
- ddH₂O: 9.5μl
- PCR program: 98°C 30s initial denaturation; 35 cycles of 98°C 10s, 55°C 30s, 72°C 20s; final extension 72°C 5min
- Prepared 1% agarose gel with TAE buffer and nucleic acid dye
- Ran electrophoresis at 120V for 30 minutes to verify amplification
July 9
- Conducted gel extraction of target DNA bands using Buffer B2 (36× volume) at 50°C
- Measured plasmid concentration via NanoDrop (target: 100200 ng/μl)
- Set up double digestion systems (6 total):
- BamHI: 1μl
- XhoI: 1μl
- 10× Color Buffer: 2μl
- PCR product: 16μl
- Incubated at 37°C for 30 minutes, terminated at 80°C
- Verified digestion via gel electrophoresis
July 10
- Ligation reaction:
- DNA fragment: 10μl
- Vector: 7μl
- T4 DNA Ligase: 1μl
- 10× T4 Ligase Buffer: 2μl
- Incubated at 4°C overnight
- Transformation:
- Thawed TOP10 competent cells on ice
- Added ligation product, ice incubation 30 minutes
- Heat shock at 42°C for 45 seconds
- Added LB medium, incubated at 37°C for 1 hour
- Plated on ampicillincontaining plates, incubated overnight at 37°C
July 11
- Picked 4 colonies per plate for PCR verification
- PCR system:
- 2× Fast Taq PCR Super Mix: 5μl
- Primers: 0.5μl each
- Template: 1μl
- ddH₂O: 3μl
- Program: 94°C 10min; 35 cycles of 94°C 10s, 55°C 30s, 72°C 90s; 72°C 5min
- Ran gel electrophoresis some samples failed due to insufficient template or marker issues
July 14
- Extracted plasmids from overnight culture using adsorption column method
- Prepared samples for sequencing (covering both 5' ends due to ~1200bp fragment length)
- Transformed verified plasmids into E. coli Rosetta for expression
July 15
- Performed plasmid extraction from additional overnight cultures
- Confirmed DNA quality and concentration for subsequent experiments
July 16
- Conducted PCR verification of Rosetta transformants
- Inoculated positive clones into liquid medium for protein expression
July 17
- Diluted culture 1:50, grew to OD600 ≈ 0.6
- Added IPTG at three concentrations (0.1, 0.5, 1.0 mM) at 20°C and 37°C
- Prepared SDSPAGE gel:
- Lower gel (12%): 2.7ml H₂O, 1.0ml 30% AcrBis, 1.25ml Buffer A, 0.05ml 10% APS, 0.005ml TEMED
- Upper gel (5%): 0.67ml H₂O, 0.33ml 30% AcrBis, 1.0ml Buffer B, 0.02ml 10% APS, 0.002ml TEMED
- Stored remaining cultures at 80°C with glycerol
July 18
- Mixed protein samples with 5× loading buffer, heated at 96°C for 10 minutes
- Loaded samples: preinduction, postinduction, supernatant, pellet
- Ran electrophoresis at 120V
- Stained with Coomassie Blue, destained, and imaged
- Results showed target protein expression but required optimization
July 21
- Repeated protein electrophoresis with optimized conditions
- Prepared 50× TAE buffer:
- 242g Tris base, 37.2g Na₂EDTA·2H₂O
- Added 600ml water, 57.1ml acetic acid
- Adjusted pH to 8.3 with NaOH, brought to 1L
- Diluted 50× to 1× for use
July 22
- Analyzed previous protein gel results, selected 0.5mM IPTG as optimal
- Scaled up culture 1:1000 in 200ml and 150ml media
- Added IPTG to final concentration 0.5mM
July 23
- Centrifuged cultures at 5000rpm for 10 minutes
- Washed pellets twice with PB buffer (pH 7.4)
- Sonicated on ice (30 minutes, 40% power, 3s pulse/7s pause)
- Collected supernatant (soluble fraction) and pellet (inclusion bodies)
July 24
- Prepared reaction mixture: 2mM Ltyrosine, 0.2mM CuSO₄ in TrisHCl (pH 7.4)
- Added 290μl reaction mix + 10μl enzyme sample per well in 96well plate
- Incubated at 50°C, measured A475 at 0, 5, 10 minutes
- Calculated enzyme activity: ΔA475/min, where 0.001 = 1U
- Corrected initial calculation errors in reagent concentrations
July 25
- Loaded four samples per construct: preinduction, postinduction, supernatant, pellet
- Ran electrophoresis, stained with Coomassie Blue
- Confirmed target protein mainly in inclusion bodies
- Documented results and stored gels at 4°C
July 28
- Prepared solutions:
- BocLtryptophan methyl ester (0.3g + 40ml water)
- BocLtryptophan (0.28g + 40ml water)
- Copper sulfate (0.2g + 40ml water)
- Tyrosine (0.18g + 40ml water)
- Set up PCR for seamless cloning
- Prepared LB medium and agarose gel for DNA electrophoresis
- Activated bacterial cultures for transformation
July 29
- Diluted activated culture 1:50 in fresh medium
- Added IPTG to 0.5mM when OD600 reached 0.60.8
- Induced overnight at 37°C
- Prepared Phusion PCR system for seamless homologous recombination
July 30
- Centrifuged induced cultures, discarded supernatant
- Washed cells twice with PB buffer (pH 7.4)
- Sonicated for 30 minutes
- Separated supernatant and inclusion bodies by centrifugation
July 31
- Ran SDSPAGE with samples: BM, CIP, L2, L3 (supernatant and pellet)
- Performed gel extraction for L1, L3, CIP, BM, L2:
- Added 600μl Buffer B2, incubated at 50°C
- Centrifuged at 8000×g (30s) and 9000×g (2×30s)
- Added 25μl Elution Buffer, centrifuged 1 minute
- Amplified DNA from extracted samples
August 1
- Centrifuged L1stage cultures, washed with buffer
- Sonicated cells, collected supernatant for enzyme assay
- Measured protein concentration using spectrophotometer
- Combined current samples with previous cultures for comprehensive analysis
August 4
- Prepared dye reaction system (final concentration 200mg/L) at 50°C
- Set up seamless cloning reaction for transformation
- Prepared LB color screening medium (LB + ampicillin + CuSO₄ + Ltyrosine)
- Activated preserved Rosetta E. coli cultures in ampicillin LB medium
August 5
- Measured precipitate enzyme activity in L1, L3, CIP, BM, L2 samples
- Prepared dye concentration series: 70, 100, 140, 150, 160, 180, 200 mg/L
- Established standard curve for adsorption modeling
August 6
- Cut fabric pieces (~0.06g each)
- Added 10ml dye solution per piece, incubated at 37°C
- Measured absorbance at 0, 10, 20, 30, 60, 120, 180 minutes
- Collected data for Langmuir adsorption model:
- qₑ = (C₀ Cₑ)V/m
- Linearized form: 1/qₑ = 1/(qₘₐₓ·Kₗ)·1/Cₑ + 1/qₘₐₓ
- Determined parameters: qₘₐₓ = 16.95 mg/g, Kₗ = 0.0597 L/mg (R² = 0.9727)
August 7
- Transformed Gt6CGT_pET21(a) and Gt6CGTCipA_pET21(a) into Rosetta E. coli
- Applied predictive model for dye dosage:
- C₀ = Cₑ + (m·qₑ)/V
- Where qₑ = (qₘₐₓ·Kₗ·Cₑ)/(1 + Kₗ·Cₑ)
August 8
- Performed colony PCR on transformants
- Verified correct insertion of Gt6CGT and Gt6CGTCipA constructs
- Prepared positive clones for protein expression
August 11
- Inoculated Gt6CGT_pET21(a) and Gt6CGTCipA_pET21(a) Rosetta E. coli
- Grew 2ml overnight cultures for subsequent experiments
August 12
- Diluted overnight culture 1:50 in fresh medium
- Added IPTG to 0.5mM when OD600 reached 0.60.8
- Induced overnight at 37°C
- Reserved 1ml culture for validation
August 13
- Centrifuged cultures, washed pellets with PB buffer
- Sonicated cells, separated supernatant and inclusion bodies
- Ran SDSPAGE with four samples per construct:
- Preinduction, postinduction, supernatant, pellet
- Confirmed protein expression and localization
August 14
- Reaction system:
- PB Buffer (0.2M): 250μl → 50mM final
- MgCl₂ (100mM): 100μl → 10mM final
- UDPglucose (20mM): 50μl → 1mM final
- Enzyme: 500μl
- Water: 90μl
- Added 1naphthol (10μM), incubated at 37°C for 1 minute
- Measured fluorescence: excitation 287nm, emission 335nm, slit width 5nm
- Gt6CGTCipA showed significantly higher activity than Gt6CGT alone (p = 0.0079)
- Demonstrated CipA's scaffold function enhances enzyme activity
↑