Protocols

Bacterial part

1.Transformation
We use officially provided protocols to convert (http://parts.igem.org/Help:Protocols/Transformation)

2.Minipreps
We use TIANprep Mini Plasmid Kit (DP103) for doing small batches of minipreps. (http://www.tiangen.com/asset/imsupload/up0141604001433139285.pdf)

3.Agarose gel electrophoresis
We used the same protocol as openwetware for agarose gel electrophoresis. (https://openwetware.org/wiki/Agarose_gel_electrophoresis)

4.Colony PCR
We used Endy:Colony PCR for verification. (https://openwetware.org/wiki/Endy:Colony_PCR)

5.Plasmid digestion
(1) Instruments
Constant temperature incubator / constant temperature water bath, bench top centrifuge, autoclave, pipette.
(2) Materials
pSB1C3, recombinant plasmid, sterile tip and microcentrifuge tube.
(3) Reagents
Restriction enzymes EcoRI, PstI, digestion reaction buffer.
(4) steps
Take 25 μL of DNA solution, add 3 μL of enzyme digestion buffer, 1 μL of EcoRI and Hind III enzymes (if the plasmid DNA is insufficient, make up 30 μL of sterile water), gently mix and mix, 37 ° C incubator or constant temperature water bath Keep warm for 3 h.

6.DNA reorganization
(1) Instruments
Constant temperature incubator / constant temperature water bath, pipette.
(2) Materials
pSB1C3, target fragment, sterile tip and microcentrifuge tube.
(3) Reagents
T4 DNA ligase, T4 DNA ligase buffer
(4) steps
In a 250 μL PCR reaction tube, add 2 μL of vector fragment DNA, 6 μL of exogenous fragment DNA (GFP-coding gene), 1 μL of 10×T4 DNA ligase Buffer, 1 μ LT4 DNA ligase according to the vector fragment: exogenous fragment = 2:6. After mixing with a suction and suction, it was allowed to act at 16 ° C for 16 h or 4 ° C overnight.

Protein fraction

1.SDS−PAGE detection of expressed protein
(1) Instruments
Vertical electrophoresis tank and matching glass plate, rubber frame, comb, ordinary constant pressure constant current electrophoresis instrument, boiling water bath.
(2) Materials
Sodium dodecyl sulfate (SDS), acrylamide (Acr), N,N'-methylenebisacrylamide (Bis), Tris, glycine (Gly), hydrochloric acid (HCl) , ammonium persulfate (Aps), tetramethylethylenediamine (TEMED), low molecular weight standard protein, pre-stained standard protein (purchased from Bio-lab), bromophenol blue, glycerin, glacial acetic acid, ethanol, sulfhydryl Ethanol, Coomassie Brilliant Blue R250.
(3) Reagents
1.5 mol/L Tris.HCl pH 8.8 (4° storage).
0.5 mol/L Tris.HCl pH 6.8 (4° storage).
10% SDS (room temperature storage)
30% Acr/Bis: 29.2 g Acr + 0.8 g Bis, double distilled water to 100 mL, filtered, stored at 4 °C.
10% Aps (ammonium persulfate) (stored at -20 ° C)
2× loading buffer (room temperature storage) (2 mL 0.5 mol/L Tris.HCl pH 6.8, glycerol 2 mL, 20% (W/V) SDS 2 mL, O.1% bromophenol blue 0.5 mL, 2-β-mercaptoethanol 1.0 mL, double distilled water 2.5 mL, stored at room temperature for use)
5× running buffer (Tris 7.5 g, Gly 36 g, SDS 2.5 g, dissolved in double distilled water, dilute to 500 mL, store at room temperature. Dilute 5 times when used)
Staining solution: 0.2 g Coomassie Brilliant Blue R250 + 84 mL 95% ethanol + 20 mL glacial acetic acid, dilute to 200 mL, and filter.
Decolorizing solution: Medical alcohol: glacial acetic acid: water = 4.5: O.5: 5 (V: V: V)
Preservation solution: 7% glacial acetic acid
(4) the experimental steps
1. Preparation of separation glue
1 Set the rubber plate and install the rubber frame.
2. Prepare the separation gel: 5mL
After mixing, add two glass crevices, and carefully add 1cm distilled water to the rubber surface for about 40 minutes. After the gel is naturally coagulated, tilt the distilled water (add distilled water to the rubber surface to seal the water. The purpose is to keep the rubber surface. Leveling and preventing air from entering, affecting the gel).
Preparation of 2.5% concentrated glue.
After mixing, add to the nip and insert the comb. After coagulation, carefully pull out the comb, and use 100 μL micro-syringe to extract the electrode buffer to rinse the bottom of the loading groove after the comb is pulled out to remove the uncondensed acrylamide.
3. Sample Preparation
The bacterial sample was mixed with 2× loading buffer l:1, and incubated in a 100 ° boiling water bath for 3-5 min, and taken out for use.
4. Electrophoresis
Add 10 μL of standard protein to one well and add the sample to one well (if blotting, pre-stained protein is added). The glass plate gel was placed in an electrophoresis tank, and the electrode liquid was added to the tank, and the power was turned on, and the current was adjusted to lmA/hole. When the sample entered the separation gel, the voltage was adjusted to be constant at 120 V. When bromophenol blue moved to about 0.5 cm from the bottom, the power was turned off and electrophoresis was stopped. Remove the rubber plate from the electrophoresis tank and carefully remove the glue from the glass plate. The concentrated gel was removed, and the separating gel was stained with Coomassie Brilliant Blue staining solution, and the separating gel was also used for blotting.
5. Dyeing
The gel was stained with staining solution for 2 h, decolorized overnight, and the preservation solution was used to preserve the gel.

Liposomal fraction

1.Preparation of artificial liposomes
(1)Preparation of phosphate buffer solution (PBS): Weigh 0.37 g of disodium hydrogen phosphate (NaHPO. 12HO) and 2.0 g of sodium dihydrogen phosphate (NaHPO4.2H), add appropriate amount of distilled water, dissolve and dilute to 1000 m1 (pPH) About 5.7).
(2) 0.9 g of phospholipid and 0.3 g of cholesterol were weighed into a round bottom flask at 50 ml, dissolved in 16 ml of chloroform, and rotary evaporated at 20 ° C to form a film on the wall of the flask.
(3) Another 30 ml of phosphate buffer solution was placed in a small beaker, placed in a 65-70 C water bath, kept warm, and set aside.
(4) Take 30 ml of pre-heated phosphate buffer, add to a small beaker containing phospholipid and cholesterol lipid membrane, and hydrate for 10 min in a 65-70 C water bath. Then place the small beaker on a magnetic stirrer, stir at room temperature for 30~60min. If the volume of the solution is reduced, add water to 30ml and mix well.
The microcapsules were passed through a 0.8 μm microwell. The microcapsules were then passed through a 0.8 μm microwell. The filter was granulated twice, and the morphology of the liposome was observed under an oil microscope. The liposome structure was observed and the particle size of the largest and largest liposome was recorded.

Team:NWU-China/Protocols

Protocols