Team:NTNU-Trondheim/Protocols

For purifying our pcr product from an agarose gel, we use the QIAquick PCR Purification kit.

To perform the Gibson assembly we jused BioLabs inc. Gibson Assembly^TM Cloning Kit.

Note that this protocole has been revised many times during the different attempts to isolate vesicles.
1. Start a culture by adding 2μL of cell-culture or one colony in 5 mL of LB. Incubate the cell culture for 7-8 hours at 37°C.
2. Inoculate 2.5 mL of the overnight starter culture in each of (up to six) 500 mL-flasks containing 250 mL of LB.
3. Grow cultures at the desired temperature for the desired time and agitation. About 12-14 hours at 37°C for untransformed ''E.coli'' and 16 hours transformed ''E.coli''.
4. Determine cell-number by finding the optical density (OD) at 600 nm. NB! If the OD₆₀₀ is over 1, dilute the solution with LB-medium so it is under 1 (note how much you diluted). You can also plate out dilutions to determine colony forming units (CFU). Make sure that the plate you count has in between 30-300 colonies, more or less than this range gives unaccurate results.
5. To obtain vesicle-containing, cell-free supernatant for non-encapsulated strains, pour 240 mL of the growth culture into 250 mL bottles using a graduated cylinder and centrifuge at 9,715 × g (using the SLA-1500 rotor at 8000 rpm) for 10 min.
6. Collect the supernatant in a sterile container, and filter it through a 0.45 μm Durapore PVDF Millipore vakuum filter. Filter the supernantants into new sterile 250 mL centrifugation bottles.
7. Centrifuge the filtered supernatant at 31 916 × g for 2.5 h (using the SLA-1500 rotor at 14 500 rpm) NB! The vesicle pallet may not be visible, mark on the tube were the pallet will form.
8. Be ready for when the centrifugation start to deacclererate and dont let it sit in the rotor for long. NB! Keep your eyes on the pallet! If it disappears, stop decanting and redo step 8. Collect the supernantant in a sterile container in the case of that recentrifugation is necessary.
9. Resuspend the vesicle-containing pellet in the residual supernatant remaining in the centrifuge bottle after decanting (approximately 5.5 mL so that the 10.4 mL tubes are at least half full) or re-suspend in 5.5 mL fresh DPBSS. Transfer to ultracentrifuge tube and centrifuge in ultracentrifuge at 100,000 × g for 1 h (using the type 70.1 ti rotor at 38 247 rpm). NB! Make sure that the centrifuge de-acceleration function stands on “No break” (Not “fast” or “slow”) and be ready for decanting the vesicle pallet when the centrifuge is finished (as in step 8).
10. Decant the supernatant and resuspend the pellet in 500 μL DPBSS (add more if the pallet is too big and note down how much more DPBSS you added). The resuspended pellet is the vesicle preparation. To freeze the sample at -80°C add OptiPrep 60% so that the final sample contains 45% OptiPrep.
11. Filter-sterilize the vesicles using a 0.45 μm centrifugal filter or a syringe filter. Check for sterility by plating on LB agar (optional). Re-sterilize using 0.45 μm filters if needed.
12. To quantitate the vesicle yield in terms of mg vesicle protein/CFU, use a protein concentration determination assay to determine the total protein concentration in the vesicle preparation and divide the number by the CFU obtained from dilution plating of the culture at the time of harvest. In some cases, flagella and other non-vesicle proteins contaminate the preparation; however, further purification steps (e.g., using density gradient purification) prevent quantitative recovery. Therefore, vesicle yields often can be best compared using the quantity of vesicle-specific protein or lipid in the pelleted cell-free supernatant preparations. Here, an aliquot of the vesicle preparation is run on SDS-PAGE, stained for protein using Ruby or Coomassie, and either the total protein in each sample or the major outer membrane proteins (e.g., Omps F/C and A for E. coli ) in each sample are determined by densitometry. Subsequently, the densitometry value is divided by the CFU and this vesicle yield compared between strains or treatments. Finally, vesicle yield can also be determined based on lipid content using a lipophilic fluorescent dye. For FM4-64-based measurements, 10 μL vesicle preparation is diluted in 189 μ L of DPBSS, and 7 μL FM4-64 (1mg/mL) is added. Fluorescence (RFU) is measured with an excitation of 515 nm and emission of 640 nm using a spectro fluorometer. Subsequently, the RFU value is divided by the CFU or/and measured OD and this vesicle yield value compared between strains or treatments.

1. Add OptiPrep (60% stock) to the resuspended vesicles at a ratio of 1:3 (by volume) to adjust the vesicle preparation to 45% OptiPrep (v:v) (NB! OptiPrep is viscous, use disposable plastic pipettes near a flame and draw approximately 1mL more then necessary).
2. Pipette (max 2 mL) of vesicles in 45% OptiPrep to the bottom of a 10.4-mL Ultraclear centrifuge tube.
3. Chake the Optiprep dilution before use. Carefully layer 2 mL of each OptiPrep dilutions in descending order on the top of the preceding layer (Place the tip of a plastic pipette to the inside of the tube below the rim and slowly add each OptiPrep solution in a slow, steady stream. There should be a distinct boundary between each layer).
4. Centrifuge the tube at 292,700 × g (using the type 70.1 ti rotor at 65435 rpm) in an ultracentrifuge for 3h to overnight. NB! The ultracentrifuge tubes we are using need to be at least half full.
5. Collect sequential fractions by placing the tip of a 1 mL pipette at the top of the gradient and carefully removing 1 mL (use a new tip for each fraction).
6. Analyze a portion (20 μL) of each fraction using 15% SDS-PAGE and visualize the proteins in the gel using Coomassie or Ruby protein staining. Identify the vesicle fractions by the presence of outer membrane proteins (OmpF/C and OmpA for E. coli). Freeze down the fraction if needed at -80°C
7. Pool the selected vesicle-containing fractions, and add them to a centrifuge bottle with at least tenfold the sample volume using DPBSS.
8. Centrifuge at 38,400 × g for at least 3 h to remove the OptiPrep.
9. Resuspend the vesicle pellet in the smallest volume of DPBSS, and filter-sterilize the vesicles using 0.45μm-PVDF Millipore centrifuge filters (Sometimes, the vesicle suspension will be too concentrated to pass through the filter and may be retained on the filter. In these cases, a visual pellet will be visible on the filter. Resuspend the pellet in sterile DPBSS and re-sterilize the vesicles by filtering the suspension using more centrifugal filters.).
10. Confirm sterility by plating an aliquot of vesicles on LB agar plates. Filter again through 0.45μm-PVDF Millipore centrifuge filters, if necessary.

Materials:
* A PCR fragment of the insert was generated with primer containing a 5’-end 20 bp overlap with the vector fragment.
* The linear vector fragment was either generated by digestion of by PCR amplification and gel purification.
Prodedure:
* The vector and insert were mixed in a 1:4 molar ratio, with at least 0.5 ng/ μL vector.
* Add the reaction mixture:
**1 μL NEBuffer 2,
** 1 μL BSA and
** 0.2 μL/0.6 U T4 polymerase (New England Biolabs)
* In total the reaction volume of 10 μL.
* Incubate the reaction mixture at room temperature for 2.5 min.
* Then incubate the mixture for 10 min. on ice.
* At the end, 5-10 μL of the product use for E. coli or DH5α transformation.

* Measure the DNA concentration (ng/ml) of each assembly piece (with Nanodrop)
* Add 100 ng of the linearized vector backbone and equimolar amounts of the other assembly pieces to a 25ml total
volume assembly reaction mixture:
Reaction Mixture
***100 ng linearized vector backbone
***each additional assembly piece (to equimolar with backbone)
***5ml 5X HF Phusion Reaction Buffer
***1ml 10 mM dNTPs
***0.75ml DMSO
***0.5m 2U/ml Phusion Polymerase
*** dH20(Total volum should be 25ml)
Perform the assembly reaction in a thermocycler:
** 3min at 98°C 1cycle
** 30sec at 98°C
** 30sec at 55°C 1 to 15 cycle(s) ** length(kb) x15 sec at 72°C
** 10min at 72°C 1 cycle
***The total length of the assembled product (in kb)
***The number of repeated cycles should exceed the number of assembly pieces
* Transform 5 ml of the assembly reaction into 100 ml of competent E. coli and/or run a diagnostic agarose gel to check for successful assembly.
Adapted from Quan 2009.

Materials:
* Your two part samples (Miniprepped DNA)
* Linearized plasmid backbone (with a different resistance to the plasmid backbones containing your part samples)
* EcoRI, XbaI, SpeI, PstI Enzymes
* NEB Buffer 2
* BSA
* dH20
Procedure:
I) Digest Reaction
* adjust to 20 ul dH20
* 2.5 ul NEB Buffer 2
* o.5 ul BSA
* DNA and Exzymes
** Digest Part A with EcoRI and SpeI (0.5 ul of each)
** Digest Part B with XbaI and PstI (0.5 ul of each)
** Digest linearized plasmid backbone with EcoRI and PstI (0.5 ul of each)
***Optional: digesting with DpnI will eliminate any template plasmid DNA
II) Ligation
* Combine 1 ul of each restriction digest reaction with 1 ul of ligase in a 25 ul reaction.
III) Transformation
* Transform the ligation product.
* If the input parts are good, almost all colonies will be correct.
* If desired analyze the transformation with single colony PCR followed by agarose gel electrophoresis (In large scale assembly, this step is often omitted).
* Miniprep clones
* Do the agarose gel electrophoresis
* Sequence the clone that generated a band of the appropriate size.