May 13rd, 2019
·Kanamycin(100 μg/mL) was added to LB media, and the mixture was moved into culture dishes.
·E. coli containing pET-28a(+) coding (EK)10-TAT-LZE was inoculated into LB liquid medium containing kanamycin, and cultured overnight at 37℃.
May 14th, 2019
·E. coli containing plasmid coding (EK)10-TAT-LZE in liquid medium was inoculated into LB solid medium and cultured overnight at 37℃.
·Plasmid in E. coli was extracted using EasyPure® Plasmid MiniPrep Kit.
·Nanodrop was used to measure the concentration and the purity of DNA extracts.
·Agarose gel electrophoresis.
·LB liquid culture medium was prepared and sterilized.
May 15th, 2019
·Kanamycin(100μg/mL) was added to LB liquid media.
·2 colonies were selected and extended in LB liquid medium with Kanamycin, and cultured overnight at 37℃.
May 16th, 2019
·Plasmid in E. coli was extracted using kit(GO-MMPS-100) based on paramagnetic particle method.
·Nanodrop was used to measure the concentration and the purity of DNA extracts.
·Agarose gel electrophoresis.
·LB solid culture medium was prepared and sterilized.
May 17th, 2019
·LB liquid culture medium was prepared and sterilized.
·LB liquid culture medium was filled into tubes with 5 mL in each tube. 0, 5 μL, 15 μL or 25 μL kanamycin(100 μg/mL) was added to tube respectively, making the concentration of kanamycin in culture medium 0 μg/mL, 5 μg/mL, 15 μg/mL, 25 μg/mL. E. coli containing pET-28a(+) was inoculated into LB liquid media. All these tubes were cultured overnight at 37℃.
·Kanamycin(100μg/mL) was diluted to 1μg/mL and added to the surface of LB solid culture media. E. coli containing pET-28a(+) was inoculated into the LB solid media. The other culture dish with LB solid medium was prepared as blank control. Both of these culture dishes were cultured overnight at 37℃.
May 18th, 2019
·2 colonies were selected and extended in LB liquid medium with Kanamycin, and cultured overnight at 37℃.
·Plasmid in E. coli was extracted using EasyPure® Plasmid MiniPrep Kit.
·Plasmid in E.coli was extracted using kit(GO-MMPS-100).
·Nanodrop was used to measure the concentration and the purity of DNA extracts.
·Agarose gel electrophoresis.
May 19th, 2019
·Plasmid in E. coli(cultured at May 18th) was extracted using EasyPure® Plasmid MiniPrep Kit.
·Nanodrop was used to measure the concentration and the purity of DNA extracts.
May 20th, 2019
·2 colonies were selected and extended in LB liquid medium with Kanamycin, and cultured overnight at 37℃.
May 21st, 2019
·Plasmid in E. coli(cultured at May 20th) was extracted using EasyPure® Plasmid MiniPrep Kit.
·Nanodrop was used to measure the concentration and the purity of DNA extracts.
May 22nd, 2019
·Agarose gel electrophoresis was used to show whether the molecular weight and super helix structure were accorded with the theoretical value.
·LB liquid culture medium was prepared and sterilized.
May 25th, 2019
·E. coli containing plasmid coding LZR-ELP in liquid medium was inoculated into LB solid medium and cultured overnight at 37℃.
May 28th, 2019
·2 colonies were selected and extended in LB liquid medium with Kanamycin, and cultured overnight at 37℃.
May 29th, 2019
·The bacteria liquid was moved from 37℃ incubator to 4℃ refrigerator.
May 30th, 2019
·Plasmid in E.coli was extracted using EasyPure Plasmid® MiniPrep Kit.
·Nanodrop was used to measure the concentration and the purity of DNA extracts.
·Agarose gel electrophoresis:
Results:The concentration of LZR-ELP plasmid is a lot lower than the standard, and existing some protein impurities. Some man-made errors happened in the electrophoresis and two marker strips were seen.
May 31st, 2019
·2 colonies were selected and extended in LB liquid medium with Kanamycin, and cultured overnight at 37℃.
June 1st, 2019
·LB liquid culture medium was prepared and sterilized.
·Nanodrop was used to measure the concentration and the purity of DNA extracts.
Results: The concentration of LZR-ELP plasmid is a lot lower than the standard, and existing some organic phase impurities.
June 4th, 2019
·Cultivation of E.coli:
1. Select 2 colonies containing plasmid coding LZR-ELP and (EK)10-TAT-LZE (for the convenience of recording and reading, the following is abbreviated, e.g., colonies of LZR-ELP and colonies of (EK)10-TAT-LZE) from LB solid culture medium respectively.
2. Extend the colonies in 5 mL LB liquid medium with 10 μg/mL Kanamycin.
3. Culture overnight at 37℃.
·Preparation of LB liquid culture medium (500 mL)
June 5th, 2019
·Extraction and characterization of the plasmid:
1. Extract LZR-ELP plasmid and (EK)10-TAT-LZE plasmid in E.coli using TIANprep® Mini Plasmid Kit.
2. Prepare the gel of agarose gel electrophoresis.
3. Start the electrophoresis at 80 V, 300 mA for 23 min.
·10 μL LZR-ELP plasmid and 10 μL (EK)10-TAT-LZE plasmid were sent to Harbin Ruiboxingke company for sequencing.
June 6th, 2019
·Preparation of LB solid culture medium
·Plasmid transformation:
1. Add 3 μL plasmid coding LZR-ELP into 200 μL AFIQ-BL21 strain E. coli in 1.5 mL EP tube and store it on ice for 30 min. Same operations were taken for (EK)10-TAT-LZE plasmid.
2. Heat-shock at 42℃ for 90 s and add 800 μL LB liquid culture medium into the EP tube on ice. Both tubes were cultured at 37℃ for 40 min.
3. Centrifuge(12000 rpm, 1 min) and abandon 800 μL supernatant.
4. Use 200μL pipette to beat up the precipitate to equality.
5. Coat the bacterial liquid on LB solid culture medium and culture overnight at 37℃.
June 7th, 2019
·Move BL21 containing LZR-ELP plasmid and BL21 containing (EK)10-TAT-LZE plasmid from 37℃ incubator to 4℃ refrigerator.
June 10th, 2019
·2 colonies of LZR-ELP and colonies of (EK)10-TAT-LZE were selected respectively and extended in 10 mL LB liquid medium with 10μL of 100 mM Kanamycin, and cultured overnight at 37℃.
June 11th, 2019
·Protein expression: the absorbance value of bacterial liquid (before induction) at 600 nm: A(LZR-ELP)=0.821, A2=0.861.
July 23rd, 2019
·Select 2 colonies of LZR-ELP and extend them in 10 mL LB liquid medium with 10 μL 100 mM Kanamycin. Culture overnight at 30℃.
·Preparation of LB liquid culture media.
July 24th, 2019
·Preparation of 1 M IPTG:
1. 0.475 g IPTG was dissolved by 1.525 mL deionized water in 5 mL EP tube.
2. Ultrasonic dissolving.
3. Filter with 0.2 μm filter membrane.
·Expression of LZR-ELP and (EK)10-TAT-LZE:
1. Bacterial amplification at 30℃ for 2.5 h.
2. The absorbance value of bacterial liquid (before induction) at 600 nm: A1=0.860, A2=0.930.
3. One tube induction at 37℃ for 6 h and the other induction at 30℃ for 7.5 h.
·2 colonies of LZR-ELP were selected and extended in 10 mL LB liquid medium with 10 μL 100 mM Kanamycin, and cultured overnight at 30℃.
July 25th, 2019
·Preparation of 1 M IPTG:
1. 1.9 g IPTG was dissolved by 6.1 mL deionized water in 10 mL EP tube.
2. Ultrasonic dissolving.
3. Filter with 0.2 μm filter membrane.
·Preparation of 100 mM kanamycin: 1 g kanamycin, dissolved by 10 mL deionized water in 10 ml EP tube. Filter with 0.2 μm filter membrane.
·Protein expression:
1. One tube of bacterial amplify at 30℃ for 2 h and the other amplify at 30℃ for 45 min
2. The absorbance value of bacterial liquid (before induction) at 600 nm: A=0.683
3. Both induce at 37℃ for 6 h.
July 27th, 2019
·SDS-PAGE protein gel electrophoresis.
July 29th, 2019
·Select 2 colonies of LZR-ELP and extend them in 10 mL LB liquid medium with 10 μL 100 mM Kanamycin. Culture overnight at 30℃.
July 30th, 2019
·Protein expression:
1. Bacterial amplification at 30℃ for 3h.
2. The absorbance value of bacterial liquid (before induction) at 600 nm: A1=0.867, A2=0.759.
3. One tube induction at 37℃ for 6 h and the other induction at 30℃ for 7 h.
July 31st, 2019
·Bacteria disruption was performed as follows:
1. Centrifuge 1 mL bacteria liquid (after induction) for 2 minutes at 12000 rpm and abandon the supernatant.
2. Add 200 μL ddH2O in each tubes and disrupt the bacteria with cell cracker.
3. Store 30 μL bacteria liquid (after disruption) in 1.5 mL EP tubes.
4. Centrifuge the rest of the bacteria liquid for 10 min at 15000 rpm.
5. Store 30 μL supernatant in 1.5 mL EP tubes and abandon the rest of the supernatant.
6. Add 30 μL ddH2O and beat the precipitation up.
7. Add 6 μL 5x loading buffer to each EP tube, metal bath at 95℃ for 20 min.
8. Centrifuge for 2 minutes at 12000 rpm.
9. 10 μL supernatant was used for electrophoresis
·SDS-PAGE protein gel electrophoresis.
·Inoculate E.coli containing pET-28a(+) coding LZR-ELP into LB solid medium containing 10 mM kanamycin. Culture overnight at 30℃.
Aug 1st, 2019
·Select 2 colonies of LZR-ELP and extend them in 10 mL LB liquid medium with 10 μL 100 mM Kanamycin. Culture overnight at 30℃.
Aug 2nd, 2019
·Extraction and characterization of LZR-ELP plasmid:
1. Extract LZR-ELP plasmid in E.coli using TIANprep® Mini Plasmid Kit.
2. Agarose gel electrophoresis.
·Preparation of LB liquid culture medium (500 mL * 2)
Aug 3rd, 2019
·Plasmid transformation.
·After transformation, BL21 containing LZR-ELP plasmid was cultured overnight at 30℃.
Aug 4th, 2019
·Select 2 colonies of LZR-ELP and extend them in 10 mL LB liquid medium with 10 μL 100 mM Kanamycin. Culture overnight at 30℃.
Aug 5th, 2019
·Expression of LZR-ELP.
·The absorbance value of bacterial liquid (before induction) at 600 nm: A1=0.799, A2=0.907
·Induction condition: 1) 1 M IPTG, 37℃, 5 h; 2) 1 M IPTG, 30℃, 5 h.
Aug 6th, 2019
·Preparation of buffer for cation exchange chromatography:
·Start buffer(20 mM PBS, pH 7.9)
1. Solution A: 0.2 M NaH2PO4 (0.624g NaH2PO4·2H2O dissolved by 20 mL H2O )
2. Solution B: 0.2 M Na2HPO4 (14.34g Na2HPO4·12H2O dissolved by 200 mL H2O )
3. Mix up 14 mL solution A and 186 mL solution B, and dilution by H2O to 2 L.
·Elution buffer
1. 100 mM NaCl (0.585g NaCl dissolved by 100 mL start buffer)
2. 200 mM NaCl (0.1.17g NaCl dissolved by 100 mL start buffer)
3. 300 mM NaCl (1.755g NaCl dissolved by 100 mL start buffer)
4. 500 mM NaCl (2.925g NaCl dissolved by 100 mL start buffer)
5. 700 mM NaCl (4.095g NaCl dissolved by 100 mL start buffer)
·Store the start buffer and elution buffer at 4℃.
Aug 7th, 2019
·Dissolve 5 μg LZR-ELP co-expression plasmid with 40 μL ddH2O.
·Plasmid transformation.
·After transformation, BL21 containing LZR-ELP co-expression plasmid was cultured overnight at 30℃).
Aug 8th, 2019
·Select 2 colonies of LZR-ELP co-expression plasmid and extend them in 10 mL LB liquid medium with 10 μL 100 mM Kanamycin. Culture overnight at 30℃.
·Pretreatment of cation exchange resin.
Aug 9th, 2019
·Expression of LZR-ELP using co-expression plasmid:
1. the absorbance value of bacterial liquid (before induction) at 600 nm: A1=0.709, A2=0.753
2. Induction condition: 1 M IPTG, 37℃, 5 h.
·Bacteria disruption:
·Supernatant:
1. Add 500 μL ethanol to 500 μL supernatant and stand it at -20℃ for 30 min.
2. Centrifuge for 2 minutes at 12000 rpm.
3. Abandon the supernatant and beat up the precipitation with 30 μL ddH2O.
·Precipitation:
1. Abandon the rest of the supernatant.
2. Add 30 μL ddH2O and beat the precipitation up.
·Add 6 μL 5x loading buffer in each EP tube, metal bath at 95℃ for 20 min.
·Centrifuge for 2 minutes at 12000 rpm.
·10 μL supernatant was used for electrophoresis.
·Before induction: the method of making sample are recorded above.
·SDS-PAGE protein gel electrophoresis to detect LZR-ELP.
Aug 11st, 2019
·SDS-PAGE protein gel electrophoresis to detect LZR-ELP coded by co-expression plasmid.
Aug 15th, 2019
·Select single colonies of LZR-ELP(coded by co-expression plasmid) and extend it in 10 mL LB liquid medium with 10 μL 100 mM Kanamycin. Culture overnight at 30℃.
·Prepare 500 mL LB liquid culture media.
Aug 16th, 2019
·Small aqueous LZR-ELP expression of co-expression plasmid.
·Add LB culture media, bacteria, 100 mg/mL kanamycin and 1 M IPTG according to the table below.
Tube 0 1 2 3 4 5 6
LB/[mL] 9.8
Kanamycin/[μL] 9.8
Bacteria/[μL] 200
IPTG/[μL] 0 5 4 2 5 4 2
Temperature/[℃] 28 20
·Store 1 mL bacteria liquid from tube 0 as before induction sample.
·Store bacteria liquid in tube six 5 ml EP tube.
·The absorbance value of bacterial liquid (before induction) at 600 nm: A=0.682.
·Collect the bacteria in 5 ml EP tube, and dissolved by 2 mL buffer. Disrupt the bacteria and then the supernatant and precipitation were used for SDS-PAGE.
Aug 17th, 2019
·SDS-PAGE protein gel electrophoresis for condition optimization of LZR-ELP co-expression.
Aug 25th, 2019
·SDS-PAGE protein gel electrophoresis to detect co-expression LZR-ELP
Aug 27th, 2019
·Inoculate 10 mL cultures(LB medium containing 10 μL 100 mM Kanamycin) with 2 colonies containing LZR-ELP co-expression plasmid. Incubate the cultures overnight at 30℃.
Aug 28th, 2019
·Small aqueous LZR-ELP expression of co-expression plasmid.
·Add LB culture media, bacterium, 100 mg/mL kanamycin and 1 M IPTG according to the table below.
Tube 0 1 2 3 4 5 6
LB/[mL] 9.8
Kanamycin/[μL] 9.8
Bacteria/[μL] 200
IPTG/[μL] 0 5 4 2 5 4 2
Temperature/[℃] 28 20
·Pretreatment of Ni2+-charged chromatography resine
Aug 29th, 2019
·Bacteria disruption
·Purification by Ni2+-charged chromatography resin
Aug 30th, 2019
·SDS-PAGE protein gel electrophoresis to detect LZR-ELP co-expression(samples: 829(5)+826(1)
).
Sept 1st, 2019
·Pretreatment of dialysis bag
·Dialysis:
1. Using deionized water dissolve the samples(826),which was freeze-dried on Monday .
2. Using deionized water dialyze the protein solution and samples(829) purified by Ni2+-charged chromatography resin overnight.
Sept 2nd, 2019
·Precipitation was observed in dialysis bag.
·Collect these dialyzed protein in six 5 mL EP tubes.
·Centrifuge the six tubes for 20 min at 10000 rpm.
·Supernatant: collect supernatant in 15 mL EP tubes and store them at 4℃.
·Precipitation: using 6 M, 4 M, 2 M guanidine hydrochloride and deionized water dissolve the precipitation respectively and have dialysis overnight.
Sept 3rd, 2019
·Collect proteins after dialysis.
·Centrifuge for 20 min at 10000 rpm and then collect supernatant.
·Freeze-dry the supernatant and protein collected yesterday.
Sept 4th, 2019
·Using 200 μL deionized water dissolve freeze-dried protein.
·Concentration measured by 10μL aliquot was 1.14×10-5 M.
·A small amount of (EK)10-TAT-LZE, which was previously freeze-dried, was dissolved in 200 μL deionized water.
·Concentration was measured by 10μL aliquot but forgotten to write down.
·Establish assembling system:
LZR-ELP 90 μL
(EK)10-TAT-LZE 3 μL
Buffer(0.4 M PBS, pH 7.5) 50 μL
2.4 M NaCl 25 μL
MiliQ (ultra pure water) 32 μL
·Store (EK)10-TAT-LZ10/LZR-ELP system at 4℃ for 15 min and sequentially 37℃ for 1 h.
·Prepare samples for TEM:
·Analyze system(914) by TEM
Sept 5th, 2019
·Inoculate 10 mL cultures(LB medium containing 10 μL 100 mM Kanamycin) with 2 colonies containing LZR-ELP co-expression plasmid. Incubate the cultures overnight at 30℃.
Sept 6th, 2019
·Both tubes of bacteria liquids were induced by 200 μL IPTG, 20℃
·Pretreatment of Ni2+-charged chromatography resin.
Sept 7th, 2019
·Purify by Ni2+-charged chromatography resin and collect interesting protein.
Sept 9th, 2019
·Using 200 μL MiliQ dissolve freeze-dried protein.
·Concentration measured by 10 μL aliquot was 1.71×10-5 μM.
·Establish self-assembly system:
System1 System2 System3
LZR-ELP /[μL] 10 10 10
(EK)10-TAT-LZE /[μL] 2 2 0
Buffer(0.4 M PBS, pH 7.5) /[μL] 5 5 5
2.4 M NaCl /[μL] 2.5 2.5 2.5
MiliQ /[μL] 0.5 0.5 2.5
All /[μL] 20
Incubation temperature /[℃] 4 4 4
Assembly temperature /[℃] 4 37 37
Time /[min] 15 60 60
·Detection by light microscope:
1. Dry sample: Drip 2μL sample on glass slides.
2. Wet sample: Drip 10μL sample on glass slides and cover glass slip.
·Results of samples were dissolve above showing crystals of salt under light microscope and no assembly was found.
Sept 10th, 2019
·Establish self-assembly system:
System1 System2
LZR-ELP /[μL] 10
(EK)10-TAT-LZE /[μL] 2
Buffer(0.4 M PBS, pH 7.5) /[μL] 5
2.4 M NaCl /[μL] 2.5
500 mg/mL Fluorescein /[μL] 2
All /[μL] 21.5
Incubation temperature /[℃] 4 4
Assembly temperature /[℃] 4 37
Time /[min] 15 60
·Dialysis overnight at corresponding assembly temperature.
Sept 11th, 2019
·Detect the system(910) by light microscope and fluorescence microscope.
Sept 12th, 2019
·Detect the system(910) by fluorescence microscope.
Sept 16th, 2019
·Establish self-assembly system:
System1 System2 System3 System4 System7 System8 System9
LZR-ELP /[μL] 20 20 20 20 20 20 20
(EK)10-TAT-LZE /[μL] 4 4 4 4 4 5 5
Buffer(0.4 M PBS, pH 7.5) /[μL] 8 8 0 10 0 10 0
2.4 M NaCl /[μL] 5 5 0 10 0 5 0
Fluorescein /[μL] 3 3 3 4 0 0 0
MiliQ /[μL] 0 0 13 2 16 0 15
All /[μL] 40 50 40
Incubation temperature /[℃] 4
Assembly temperature /[℃] 37 37 37 37 4 37 37
Time /[min] 60
Sept 17th, 2019
·Detect systems(916) by TEM.
·System(916)2 and system(916)4 and system(916)8 were dialyzed.
·System(916)1 and system(916)3 were at ultrafiltration and consequently detected by CLSM.
Sept 18th, 2019
·Preparation of sample(916)1-3 for AFM.
Sept 19th, 2019
·Detect sample(918) by AFM.
Sept 21st, 2019
·Activation of MMP-2 zymogen:
1. Dilute rhMMP-2 to 100 μg/mL in Assay Buffer.
2. Activate rhMMP-2 by adding APMA to a final concentration of 1mM.
3. Incubate at 37 ℃ for 1 h.
·Preparation of MMP-2 Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05%(w/v) Brij 35, pH 7.5.
·Add 100 μL (EK)10-TAT-LZE protein solution (3.2×10-5 M) to 900 μL Assay Buffer, add 10 μL enzyme, mix, and incubate at 37℃ overnight.
Sept 22nd, 2019
·SDS-PAGE protein gel electrophoresis
Result: The dyeing time was too short and the loading concentration was too large to distinguish the strips.
Sept 23rd, 2019
·Expression of protein(induction by 200 μL 1 M IPTG, at 20℃ for 6 h)
·Establish self-assembly system:
System1 System2 System3 System4 System5 System6
LZR-ELP /[μL] 25 25 25 25 25 25
(EK)10-TAT-LZE /[μL] 12.5 12.5 12.5 0 12.5 12.5
Buffer(0.4 M PBS, pH 7.5) /[μL] 25
2.4 M NaCl /[μL] 12.5 25 37.5 25 6.25 12.5
MiliQ /[μL] 25 12.5 0 25 37.25 25
All /[μL] 100
Incubation temperature /[℃] 4
Assembly temperature /[℃] 25 4
Time/[min] 60 15
·Detect systems(923) by DLS.
·Prepare samples(923)for TEM.
Sept 24th, 2019
·Detect systems(923) by TEM.
·Verification of (EK)10-TAT-LZE digestion by MMP-2: Add 50 μL (EK)10-TAT-LZE protein solution (3.2×10-5 M)and 5 μL activated MMP-2 solution to 450 μL Assay Buffer, mix and incubate at 37℃ overnight.
Sept 25th, 2019
·Preparing LZR-ELP, (EK)10-TAT-LZE protein solution on ice, concentration was shown below.
Number LZR-ELP (×10-5 M) (EK)10-TAT-LZE (×10-5 M)
1 1.54 0
2 0 1.88
3 1.54 1.88
4 0.51 0
5 0 0.376
6 0 0.188
7 6.14 0.188
·Detect sample(924) by CD
·SDS-PAGE protein gel electrophoresis
Sept 27th, 2019
·Establish self-assembly system:
The concentration of LZR-ELP deposited solution is 3×10-5 M.
The concentration of (EK)10-TAT-LZE deposited solution is 2.5×10-5 M.
System1 System2 System3 System4
LZR-ELP /[μL] 25
(EK)10-TAT-LZE /[μL] 12.5
Buffer(0.4 M PBS, pH 7.5) /[μL] 25
2.4 M NaCl /[μL] 0 7.25 12.5 25
MiliQ /[μL] 37.5 30.25 25 12.5
All /[μL] 100
Incubation temperature /[℃] 4
Assembly temperature /[℃] 37
Time Culture on the shaking table(37 ℃, 200rpm) overnight
Sept 28th, 2019
·Every sample was diluted to 400 μL in MiliQ, while solution with same concentration of both buffer and NaCl as control.
·Detect OD value at 400 nm.
Sept 29th, 2019
·Establish self-assembly system:
The concentration of LZR-ELP deposited solution was 2.21×10-5 M.
The concentration of (EK)10-TAT-LZE deposited solution was 2.45×10-5 M.
System1 System2 System3 System4 System5
LZR-ELP /[μL] 25
(EK)10-TAT-LZE /[μL] 12.5
Buffer(0.4 M PBS, pH 7.5)/[μL] 25
2.4 M NaCl/[μL] 0 7.3 9.9 12.5 18.8
MiliQ/[μL] 37.5 30.2 27.6 25 18.7
All/[μL] 100
Incubation temperature/[℃] 4
Assembly temperature/[℃] 37
Time Culture on the shaking table(37℃, 200rpm) overnight
Sept 30th, 2019
·Diameters were measured by DLS.
·Secondary structure of building blocks was determined by CD:
Number Sample
1 LZR-ELP 2×10-5 M
2 (EK)10E-5 M
3 LZR-ELP 1×10-5 M
4 LZR-ELP 1×10-5 M
5 LZR-ELP + (EK)10-TAT-LZE 1×10-5 M