Team:Jilin China/Protocol
Plasmid construction
The new plasmid constructed was based on the template, pGH. Two orf and one ori , which were selected from PMB-1 and related to gene duplication, were constructed into the plasmid. And then, a HU promoter was constructed to promote the expression of the gene encoding a histone-like protein. At last, vP3 was added into the plasmid. The construction of the plasmid was outsourced to the company. We improve it by adding the signal peptide (Tmp1 and Sec2) to help the secretion of apoptin protein and TAT to help the entering of apoptin into the cells. We added a linker between TAT and apoptin. We designed three plasmids (Tmp1-TAT-Linker-apoptin,Sec2-TAT-Linker-apoptin,TAT-Linker-apoptin) and have them synthesized by company.
Plasmid was transformed into E.coil
The competence DH5α and plasmid were kept on ice. 2 μl plasmid was put into 50 μl DH5α and kept on ice for 30min. Heat shock was performed the at 42℃ for 90sec. Then put the tube on ice quickly for 2min.Then pour 25μl DH5α that had transformed plasmid on the plate and spread them evenly by spreader. Then seal the petri dish by sealing film and culture it in 37℃ for overnight.
Plasmid was transformed into E.coil
The competence DH5α and plasmid were kept on ice. 2 μl plasmid was put into 50 μl DH5α and kept on ice for 30min. Heat shock was performed the at 42℃ for 90sec. Then put the tube on ice quickly for 2min.Then pour 25μl DH5α that had transformed plasmid on the plate and spread them evenly by spreader. Then seal the petri dish by sealing film and culture it in 37℃ for overnight.
Apoptin expression was induces by IPTG
A monoclonal colony of BL21 that had transformed the plasmid was picked and put into 2ml LB medium with 2μl ampicillin and cultured for 4hours(37℃).The medium was then added into 200ml LB medium with ampicillin and cultured for 2.5h(37℃).After that,pipette some bacteria into 4 EP tubes(Every tubes has 1ml) and treated in centrifuge(7000rmp,10min).The supernatant was abandoned and the sedimentation was kept in -20℃ and marked. Then the left bacteria was added 1ml IPTG(500mmol/l) and cultured for 4h at 37℃(or cultured for overnight at 28℃).Following that,pipette some bacteria into 10EP tubes(Every tubes has 1ml) and treated in centrifuge(7000rmp,10min).The supernatant was abandoned and the sedimentation was sonicated for 20min with 7s work and 30s pulse in ice. After centrifuged(4℃,12000rmp,20min) , the supernatant was collected into 1.5ml EP tubes and were kept in -20℃and marked. The sedimentation were kept in -20℃ and marked.
SDS-PAGE
We had two clean and completely dry the glass plates. Place the short plate on top of a spacer plate. Insert both plates into the casting frame on a flat surface. Be sure that the "legs" of the casting frame are down. Clamp the casting frame and check that the plates were level on the bottom. Then put the casting frame into the casting stand.Combine all reagents except the TEMED for the 15% separating gel. When ready to pour the gel, quickly add the TEMED and mix them and transfer the separating gel solution between the glass plates in the casting chamber to about 3/4 inch below the short plate. Some dH2O was added on top of the gel prior to polymerization to straighten the level of the gel and remove unwanted air bubbles that may be present. After the gel polymerized, the dH2O was removed by absorption with filter paper. Combine all reagents except the TEMED for the 5% stacking gel. When ready to pour the gel, quickly add the TEMED and mix them and transfer the stacking gel solution between the glass plates in the casting chamber. Then insert the well forming comb into the stacking gel quickly. After the the stacking gel polymerized, remove the gel from the casting stand and place it in the electrode assembly with the short plate on the inside.Pour some 1X electrophoresis buffer into the opening of the casting frame between the gel cassettes. Add enough buffer to fill the wells of the gel.Then the comb was gently removed. The polymerized gel between the short plate and spacer plate forms the "gel cassette". Then pipette 5μl marker or sample into the gel well.Then Cover the tank with the lid aligning the electrodes (black or red) appropriately.Connect the electrophoresis tank to the power supply.The sample run at 60mv until the loading reaches the bottom of the stacking gel then changed the voltage to 80mv until the loading reaches the bottom of the separating gel.Turn off the power supply.Put the gel in a box and add some dH2O to boil for 3 times.Then outwell the dH20 and add some dye liquor to the box and boil for 3 times.Following that,outwell the dye liquor and add some dH2O to the box and boil for 3 times.Finally,outwell the dH20 and add some new dH2O to shake overnight.
Western blotting
Every sample were separated on a 15% SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membranes (0.2 um).Membranes were blocked in TBST with 3% bovine serum albumin (BSA) at 37℃ for 2 hours .The primary antibodies were diluted 1: 1000, and incubated with membranes over night at 4℃. Membranes were washed with TBST for 3 times (5 minutes per times), then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at 37℃, washed with TBST for 3 times again. Membranes were incubated with ECL (Tanon Science & Technology) and visualized by Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology)
Activate freeze-drying B.longum(bifi)
The glass tube was wiped by alcohol tampon and was burnt by the fire of alcohol lamp.Then a drop of ddH2O was added to the glass tube.The glass tube was broken by tweezers.Then 300μl PYG medium was added into the tube and freeze-drying bifidobacterium longum(bifi) was dissolved.Pipette 100μl medium that has dissolved the bifi into 15ml fuge tubes.The bifi was put into the anaerobic glove box to culture.
Make the competent Bifi cell
Prepare the PYG medium with 0.5mol/l glucose(has been sterilized).Prepare the washing liquor(10% glycerinum with 0.5mol/l glucose mixture).Prepare the PYG solid medium with AMP.Add 600μl bifi into 15ml medium and culture until the OD up to 0.3.The bifi was washed with washing liquor for 3 times.Then resuspende the bifi with 150ml washing liquor and pipette the liquor into 1.5ml EP tubes(every tube has 50μl) and keep them at -80℃ or use them to do electro transformation.
Electro-transformation
Add 50μl competent bifi cell and 10μl plasmid into the electro transformation cup.Then the plasmid is transformed at 1800V,15μf,200Ω.After that,add 1ml PYG medium to culture it for 1h,then treated them in centrifuge(8000rmp,10min).The most of supernatant was abandoned and the left supernatant resuspended sedimentation and pour them on the plate and spread them evenly by spreader.(The spreader was fired to sterilize before use and after use. )Then seal the petri dish by sealing film and culture it.
Keep the bacteria
Add 400μl 50% glycerinum and 600μl bacteria then keep them at -80℃.
Make the lyophilized powder
Culture the bifi cell until OD up to 0.6.Then treat them in centrifuge(8000rmp,10min) and abandon the supematant(Every tube has 15ml bifi).Prepare the 12% skim milk.Then boil the skim milk at 100℃ for 30min.Add 10ml skim milk into each tube and resuspended the bacteria.Split 1ml the mixture into 1.5ml EP tube.Add the EP tubes into the freeze-dried vial and freeze-dried for 2d.
MTT experiment
The process of cell culture was the same as the process of cell transfection except that only 0.25μl plasmid liquor was added each wall. Then, 10μl MTT reagent was added. After 4 hours of reaction, the liquid was discarded and 150μl dissolution liquor was added. Then, the 96-wall plate was put into the microplate reader (company) and tested under the wavelength of 490nm.
Cell transfection
The nanodrop(company) was used to measure the concentration of the plasmid liquor. 2μg and 5μg plasmid was added separately into the two experimental groups. Firstly, opti-MEM (60:1) and Fugene 6 (3:1) was mixed and stewed for 10 minutes. Then, the plasmid was added into the system and stewed for another 10 minutes. The liquor was added with opti-MEM to 1 ml and it was added into the 6-well plate. After the treatment of pancreatin and centrifuged (1000rmp, 5min), MCF-7 cells was resuspenced with 1ml DMEM medium and added into the six-wall plate.
Wound scratch healing assay
The process of cell culture was the same as the process of cell transfection. After 24 hours of culturing, the yellow tip was used to scratch the cells and PBS was used to wash the plate twice. Then 2ml serum-free media and the apoptin was added. After 24 hours and 48 hours of culturing, Photos were taken and the wounds were measured (in case that the dead cells would influence the result, the medium was changed before taking photos).
Animal Experiment
1. Adjust the concentration of SMMC-7721 cell to 2.0*10^7 ml-1 and make the cell suspension.Inject 0.5ml suspension at the right fore of the mouse by subcutaneous injection and record the time of tumor growth.Record the volume of the tumor everyday.(V=1/2AB2,A=the long diameter of the tumor,B=the short diameter of the tumor)
2.Mice are randomly divided into 5 groups in the 10d.There are 8 mice in each group.
| Number of group | Mice | Injection Material |
|---|---|---|
| 1 | Negative Control(NC) | 50μl PBS |
| 2 | Positive Control(PC) | 50μl Drug |
| 3 | BFC | 50μl BFC |
| 4 | BFS | 50μl BFS |
| 5 | BFT | 50μl BFT |
BFC=The Bifidobacterium Longum
BFS=The Bifidobacterium Longum that transformed the Sec2-TAT-Linker-Apoptin plasmid
BFT=The Bifidobacterium Longum that transformed the TMP1-TAT-Linker-Apoptin plasmid
We adjust the number of active bacteria to the same orders of magntiude.Inject the materials at 10d, 15d, 20d
3. Kill the mice by cervical dislocation in 21d.The tumor mass is weighed and take photo.
