"
Team:St Andrews/Lab-book
From 2012.igem.org
Lab Book
Protocols
1. Frozen cells were thawed from -80 °C.
2. Thawed cells (10 μL)were mixed with vector (1 μL), and left on ice for 30 min.
3. LB liquid medium (1 mL) was warmed at 37°C for 30 min.
4. The cell/vector mix was heated at 42 °C for 45 seconds.
5. Warm LB (50 μL) was added to the cell vector mix.
6. Mixture was shaken at 37 °C for 30 min.
7. Mixture was plated onto ampicillin agar plates and left overnight.
8. Recombinant E. Coli was harvested from agar plate by scratching gently with the scraper.
9. The scraper tip was placed into Falcon tubes with LB (10 mL)and ampicillin (1 μL) to transfer the E. Coli into medium.
10. The E. Coli was incubated in a shaker at 37 °C for less than 20 hr.
1. Preparing 1% agarose solution: Agarose powder (1 g) was dissolved in TAE (Tris base, acetic acid and EDTA) buffer (100 mL). This was heated in the microwave until the solution was transparent. Ethidium Bromide (8 μL) was added.
2. The solution was poured into an assembled gel rigand allowed to set.
3. Prepared samples were loaded into the wells(5-20 μL) along with DNA ladder (5 μL).
4. The gel was run at 80 V for 20-50 min.
5. The gel was visualised under UV light
Ratios for sample preparation vary depending on the type of sample:
- Miniprep - use 4 μL sample and 2 μL dye
- Digestion - use 1 μL sample and 2 μL dye
- PCR- use 10μl sample and 1μl dye
When using GoTaq®, simply load 10 μL onto the gel, as dye is already included.
Always make up to 10 μL using water.1. The sample (30μl) was prepared in the following sequnce:
2. The sample was incubated at 37 °C in a water bath. Timings depended on the restriction enzymes brand.
1. Prepare 20 μM primer working stock for both forward and reverse primers
2. Prepare PCR according to polymerase brand:
High-fidelity:
Clontech
KOD
GoTaq
1. Prepare multiple PCR tubes for each plate and make a solution as follows:
to make a 50μL PCR solution
2. Prepare and label corresponding Eppendorf tubes for each PCR tube
3. Using a small pipette tip, scratch one colony off a plate and dip the tip into the PCR tube. Store the tip in the corresponding Eppendorf tubes (for further incubation, if positive).
4. Run PCR using optimal annealing temperature for the required gene, and then take samples of the PCR products for electrophoresis.
6. Select the positive from all samples and incubate the corresponding tips.
Ligation molar ratio calculation:
Compare UV light exposure intensity for quantity in ng (i.e. the concentration of both insert and vector) against ladder position for sequence length (i.e. molar weight ratio of the two) Rule of 3: three times as much insert as vector ensures successful ligation results
1. Prepare 20 μL of ligation solution
- 2 μL 10x buffer
- 1 μL T4 ligase
- ~17 μL consist of vectors and insert, according to calculated ratio
- make up to 20 μL using water
2. Let stand at room temperature for 10-30 min.
3. Mix the ligase-treated vector-insert mixture with 60 μl E. Coli (DH5-α), then proceed the standard transformation protocol and shake for 1 hr; finally add 200 μL LB liquid media.
4. Spread all contents of the shaken mixture onto agar plates, with an additional control of transformed E. Coli with non-inserted linearised vector. Incubate overnight at 37 °C.
1. Using LB as a blank reference, measure the optimal density of 1 mL of sample in the photometer.
2. For induction, the OD should be at 0.5. The sample can be grown further or diluted if necessary.
2. IPTG should be added to a concentration of 1 mM. This figure will vary if protein expression/cell growth is carried out at a lower non-standard temperatures to accomodate for the slower metabolism.
1. Spin down the E. Coli in a centrifuge.
2. Discard the supernatant and add in 1 mL of PBS (phosphate buffer saline) to resuspend the pellet, then tansfer to Eppendorf tubes.
3. Spin down the E. Coli at full speed for 1 min., discard supernatant and then put on ice/in cool room for storage.
4. Prepare lysozyme solution (10 mg/mL) in lysis buffer.
5. Add 200 μL into each pellet and resuspend it; vortex and shaker incubate at 37 °C for 30 min. to 1 h.
6. Sonicate the cells.
7. Take 10 μL total sample and add in 10 μL 2x dye.
8. Spin the original tubes for 1 min. at full speed, remove the supernatant, then resuspend the mass with 200 μL PBS.
9. Take 10 μL membrane sample from the resuspended solution and add in 10 μL 2x dye.
10. Denature the protein samples on the heating block at 95° C or 75°C (for membrane protein).
11. Cool on ice for 1 min., then load the samples to the gel (prepared according to SDS-PAGE standard protocol) and run at 150 V for around 45 min.
1. Making the membrane transfer sandwiches: 2x A2 at the bottom, then 2x A2.
2. Cut out the membrane, GE Healthcare HyBond ECL films, using a glass template; put the membrane onto moist A2 filter papers and press flat using a roll; cut off excessive margins if necessary.
3. Open the gel slides using a wedge; remove the thinner slide and, using the wedge, prize the protein gel from the thicker plate and onto the membrane.
4. Gently press out air bubbles between the gel and membrane.
5. Add on CAT filter paper.
6. Run the machine for 1hr at constant ampere of 40 mA for each gel.
7. If the rpestained markers have transferred, place the CAT filter papers onto a rocker and add stain. If using Ponceau, the stain can be reused.
8. Rinse with water to destain.
21. Pour in 50 mL 5% milk powder and leave on rocker for 2 hours at room temperature; if leaving overnight, rock in cold room using lower speed.
9. Decant blocking milk solution, and then add in 10 mL 1% milk with 5μl of diluted antibodies. Rock for 1 h.
10. Pour off the milk-antibody solution and wash with 0.3% PBS and Tween, three times in 10 min. intervals.
11. Prepare a solution of "Supersignal west Fermto trail" kit (1:1 ratio, 1 mL per membrane).
12. Moisten membrane with visualization solution, then cover for 5 min. Then, dry off excess liquid.
13. Develop the membranes in the dark room.
Project-specific procedures
ω3-Synthesis
1. Spin down the E. Coli in a centrifuge.
2. Discard the supernatant and add in 1 mL of PBS to resuspend the pellet, then transfer to Eppendorf tubes.
3. Spin down the E. Coli again at full speed for 1 min. and discard supernatant.
4. Resuspend the pellet with 100 μL PBS and then transfer to glass tubes.
5. Add organic solution for lysis, chloroform:methanol (1:2, 375 μL).
6. Vortex in the cold room for 1 h and then add 125 μL chloroform and 125 μL water to allow phase separation (lower organic layer contains lipids); let stand for at least 10 min.
7. Spin down using special adapters at 3000 rpm for 5 min.
8. Pipette the bottom layer into a glass sample tube, being careful not to take any milky layer.
9. Dry the organic solvent under nitrogen and store.
1. Per cell type, there will be 3 samples:
- A (negative control)
- B (no added substrate beyond induced 18:1)
- C (addition of substrate PC lipid 18:1)
2. After labelling all screw-cap Eppendorfs, add the following assay components to each B and C tube:
- 20 μL of 40 μM tricine buffer, made up to pH 8 with KOH
- 20 μL of 10 μM MgCl2
- 10 μL ferredoxin
- 10 μL of 5μM NADPH
- 30 μL of catalase
- 10 μL NADPH+
3. Add 100 μL of membrane sample, prepared as in the lipid extration. Use 500 μL lysozyme, and resuspend in tricine buffer. Run a Bradford assay to determine protein content.
4. Vortex at 4°C for 2 min.
5. Leave in water-bath at 37°C.
6. Add 250 μL chloroform and 250 μL water to stop assay reaction and allow for phase separation.
7. Samples will be used to carry out mass spec analysis.
1. Aliquots of the lipd extract were transferred to 2mL glass vessels and dried under nitrogen.
2. Base hydrolysis to release fatty acids were released by base hydrolysis using 500 µL of concentrated ammonia and 5|0% propan-1-ol (1:1), followed by incubation for 5 h at 50 °C.
3. After cooling, the samples are evaporated to dryness with nitrogen and dried twice more from 200ul of methanol : water (1:1) to remove all traces of ammonia.
4. The protonated fatty acids are now extracted by partitioning between 500µL of 20mM HCl and 500uL of ether, the aqueous phase is re-extracted with fresh ether (500µL) and the combined ether phases are dried under nitrogen in a glass tube.
5. The fatty acids are converted to methyl esters (FAME), by adding diazomethane (3x 20uL aliquots) to the dried residue, while on ice. After 30min, the samples were allowed to warm to RT and left to evaporate to dryness in a fume hood.
6. The FAME products are dissolved in 10-20µL dichloromethane and 1-2µL analysed by GC-MS on a Agilent Technologies (GC-6890N, MS detector-5973) with a ZB-5 column (30M x 25mm x 25mm, Phenomenex), with a temperature program of at 70 °C for 10min followed by a gradient to 220 °C at 5 °C /min and held at 220 °C for a further 15min.
7. Mass spectra were acquired from 50-500 amu. The identity of FAMEs was carried out by comparison of the retention time and fragmentation pattern with a Bacterial FAME standard (Supelco).
Metal-binding peptides
Primer annealing was carried out to create short peptides.
10 μL of 200 μM of each primer was put into the PCR machine.
PCR Programme:
1. Spin down the E. coli in a centrifuge.
2. Discard the supernatant and add in 1 mL of PBS (phosphate buffer saline) to re-suspend the pellet. Transfer to Eppendorf tubes.
3. Spin down the E. coli at full speed for 1 min. Discard supernatant and then put on ice for storage.
4. Prepare lysozyme solution (10 mg/mL) in lysis buffer.
5. Add 200 μL lysozyme solution to each pellet and re-suspend. Add 1 µL of benzonase nuclease, dissolve 1 protease inhibitor cocktail pill; vortex and incubate in shaker at 37 °C for 30 min. to 1 h.
6. Sonicate the cells.
7. Spin down sonicated cells at full speed for 1 min in centrifuge.
8. Prepare Nickel beads. Take 100 µL of Ni-NTA slurry per sample. Spin in centrifuge at full speed, remove supernatant. Add 100 µL of PBS per sample, re-suspend and spin down at full speed for 1 min.
9. Remove supernatant; add 100 µL of PBS per sample, re-suspend and transfer to eppendorf tubes 100 µL per sample.Spin down at full speed for 1 minute in centrifuge, remove supernatant. Add supernatant of sonicated cells and re-suspend.
10. Agitate samples for 30 min to 3 h in cold room.Spin down samples at full speed for 1 minute in centrifuge and remove supernatant.
11. Add 100 µL of NINTA elution buffer per sample; agitate samples for 10 to 30 min in cold room.
12. Spin down samples at full speed for 1 min. Take 10 µL of supernatant and add 10µL 2x dye.
13. Denature the protein samples on the heating block at 95° C.
14. Cool on ice for 1 min., then load the samples to the gel (prepared according to SDS-PAGE standard protocol) and run at 150 V for 45 min.
1. Spin down the E. coli in a centrifuge.
2. Discard the supernatant and add in 1 mL of PBS (phosphate buffer saline) to re-suspend the pellet. Transfer to Eppendorf tubes.
3. The E. coli was spun down at full speed for 1 min. The supernatant was then discarded and stored on ice.
4. The lysozyme solution (10 mg/mL) was prepared in lysis buffer.
5. The lysozyme solution (200 μL) was added to each pellet and re-suspend. Benzonase nuclease (1 μL) was added, and one protease inhibitor cocktail pill was dissolved in the mixture. This was then vortexed and incubated in the shaker (37 °C for 30 min to 1 h).
6. The cells were then sonicated.
7. The sonicated cells were then spun down in the centrifuge at full speed for one minute.
8. GST beads were prepared. GST slurry (100 µL) was added per sample and spun down at full speed. The supernatant was then removed. PBS (100 µL) was added per sample, and the mixture re- suspended and spun down at full speed for 1 min.
9. The supernatant was then removed and PBS (100 µL) added per sample, re-suspended and transferred to eppendorf tubes, 100 µL per sample. The mixture re-suspended and spun down at full speed for 1 min and supernatant was then removed. The supernatant of the sonicated cells was then added.
10. 10. The samples were agitated for 30 min to 3 h in the cold room. Samples were spun down at full speed for 1 min. Supernatant was removed.
11. GST beads were washed with PBS, 200 µL per sample. Samples were spun down at full speed and supernatant was removed. Glutathione solution (50 µl, 100 mM) was added . The samples were agitated for 10 to 30 min in the cold room.
12. The samples were spun at full speed for 1 min. 10 µL of supernatant was taken and dye (10 µL, 2x) was added.
13. The protein samples were denatured using the heating block at 95° C.
14. The samples were cooled on ice (1 min) then loaded onto the gel (prepared according to SDS- PAGE standard protocol) and run at 150 V for 45 min.
