Team:Macquarie Australia/Experiments
Experiments & Protocols
This page is for descriptions of the experiments, research and protocols we used in our iGEM project.
What should this page contain?
- Protocols
- Experiments
- Documentation of the development of our project
Inspiration
Protocols
Making competent cells
1.
Using a sterile
plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate.
Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22oC,
200-250rpm.
2.
A600 should be
0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~
0.5.
3. Remove the flask from the incubator and place on ice
for 10 minutes.
4. Transfer the culture to a 15mL centrifuge tube and
spin at 2500 x g for 10 min at 4oC
5. Pour off and discard the supernatant, and immediately
place the tube on ice.
6. Resuspend your cells in 1mL of ice-cold TB buffer,
make sure there are no clumps of cells left, but also treat your cells gently
and keep them cold.
7. Add ice-cold TB buffer to bring volume up to 1/5th of
the original culture volume (~30mL in this case). Mix the tube by gently
inverting 3 times.
8. Incubate the tube on ice for 10 minutes.
9. Centrifuge at 2,500 x g for 7
minutes at 4oC, discard the supernatant.
10.
Gently resuspend
the cells in ~1/20th of the original culture volume of ice-cold TB buffer.
NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. e.g. if the culture OD600 was 0.1 then resuspend in 1/100th
of original volume.
11.
Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension,
keeping the remainder on ice in the 15mL tube.
12.
Add 70µl of DMSO
to the 930µl of cell suspension. Mix gently by swirling, and place on ice.
13.
Aliquot 100µl of
the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the
tubes with: Date – Strain. Snap freeze with
liquid nitrogen.
14.
Repeat step 11-13
for the rest of your cell suspension in step 10. Store cells at -80oC
TB BUFFER
● Ingredients: 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g KCl.
● Methods: All components (except for MnCl2-4H2O) were mixed and
dissolved in 500 mL of water and pH adjusted to 6.7
with KOH. Then, MnCl2-4H2O, was dissolved in 300 mL
of water, mixed and solution adjusted to 1 L. Sterilisation via filtration
followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C.
EDTA BUFFER
● Ingredients: 37.22g EDTA solid, 180 mL
of water and pH adjusted to 8.0 using 10M NaOH.
● Methods: Components were combined then pH adjusted.
TAE BUFFER
● Ingredients: 121.2g Tris base (dissolved
in water) with 28.55mL of glacial acetic acid & 50mL 0.5M EDTA (pH 8.0).
● Methods: A total volume of 500 mL
was made up as a 50x stock solution using all components
Making agarose gel
Preparing the Gel
1. Mix 1g of agarose powder with 100ml of 1x TAE buffer
and heat for 1minute or until all agarose is dissolved.
2.
Wait until it has
cooled (not set), and add 1ul of GelRed into the
mixture.
3.
Pour the solution
into a cast with an appropriate comb.
4.
Leave to set.
Running the Gel
1. Mix 1ul of 1kbp DNA ladder with 6ul of loading dye (bromophenol blue) and 4ul of 1x TAE buffer (total 6ul) and
load onto first well
2.
Mix 5ul of PCR
products with 1ul of loading dye and load onto wells.
3.
Run gel at 90V
for 45minutes approximately
4.
Photograph gels
under UV light
Composite part ligation
The assembly of composite parts from
two existing BioBricks i.e. BioBrick
plasmid A (parent vector) and BioBrick plasmid B
(gene to be inserted) was performed through a restriction digest/ligation
protocol.
1.
200ng of each BioBrick plasmid was digested as follows: Plasmid BioBrick A with NEB restriction enzymes SpeI
and PstI; plasmid BioBrick B with XbaI and PstI, according to supplier protocol. (1hr @37oC,
then 20mins @80oC).
2.
1µL of Fast alkaline phosphatase (Thermo
Scientific) was added to reaction tube of plasmid BioBrick
A to dephosphorylate the gene fragments and prevent
re-ligation of the parent vector. Reaction tubes were incubated at 37oC
for 60 mins, followed by a deactivation step at 80oC for 20 mins.
3.
Ligation was then
performed with an insert to vector molar ratio of 3:1. 1µL of T4 ligase (NEB)
was added to the mix for ligation, according to supplier protocol. Ligation
reactions were performed at 37oC for 60 mins and kept on ice for
transformation into chemical competent cells.
MANUAL TRYPSIN IN-GEL DIGESTION PROTOCOL FOR COOMASSIE STAINED GELS
1. Coommassie de-stain gels: by washing briefly with 200µL NH4HCO3
(Solution A) to make sure gel pieces are at the correct pH.
2.
Add 200µL 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) into each
well. Vortex to mix and incubate for 10 minutes. Remove liquid.
3.
Repeat step 2.
Gel pieces should be clear at this stage. If they are still blue, repeat as
necessary until color is gone.
4.
Wash for 5 min
with 50µL of 100% Acetonitrile (Solution C) to
dehydrate gel pieces. Vortex during incubation.
5.
Remove Acetonitrile, then let air-dry for
10 min. The gel pieces should be noticeably shrunken and probably white.
6.
Reduction and
Alkylation: Cover gel pieces with 50µL 10mM DTT in 50mM NH4HCO3 (Solution D).
Let proteins reduced for 45-60 min in 37oC incubator.
7.
Remove DTT solution
and add 50µL of 55mM iodoacetamide in 50mM NH4HCO3
(Solution E). Incubate for 45 min in dark place at room temperature.
8.
Remove
iodoacetamide, discard.
9.
Wash gel pieces
with 200ul of NH4HCO3 (Solution A) for 5 min with vortexing.
before adding 100ul of 100% Acetonitrile
(Solution C).
10.
Remove
liquid after 5 min, discard.
11.
Wash gel pieces
with 50ul 100mM NH4HCO3 (Solution A) for 10 min, then twice with 200ul 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) for 10min
12.
Dehydrate with
100 ul 100% Acetonitrile
(Solution C) for 5 min as above.
13.
Remove remaining
liquid and let the gel dried.
14.
Trypsin Digestion: Prepare trypsin
mix to final concentration of 13ng/µL in 50mM Ammonium bicarbonate.
15.
Add 30µL (or more
if required) of mixed trypsin solution to cover the
gel pieces.
16.
Allow 30 min for
gel rehydration at 4oC (on ice).
17.
Digest overnight
at 37oC.
18.
Peptide
Extraction: Transfer the digest solution supernatant (if any) into clean 0.65ml
Eppendorf tubes.
19.
To the gel
pieces, add 50 µL of 50% acetonitrile / 2% formic acid,
incubate 30 min. Spin, remove supernatant and combine with initial digest
solution supernatant. Please note that total volume may vary depending on the
gel sizes.
20.
Vortex the
extracted digests, speed vac to reduce volume to 10 µL. If the remaining volume
is less than 10ul, use 2% formic acid to bring the volume up to 10 µL.
21.
Spin at 14,000
rpm for 30 min to remove any micro particulates.
22.
Transfer the
supernatant to a fresh 0.65ml eppendorf tube for
storage at 4oC fridge OR directly into PCR plate for Mass spec for
analysis.
Reagents
● Buffer 100mM Ammonium
Bicarbonate
○ ADD 0.78 g ammonium bicarbonate to 100ml ddH20. Fill
first medium size reservoir.
● Reduction: - Dithiothreitol (DTT)
○ Add 15 mL of 100 mM ammonium bicarbonate to 23.1 mg of DTT to give a solution
of 10 mM DTT.
● Alkylation - Iodoacetamide(DTT)
○ Add 15 mL of 100 mM ammonium bicarbonate to 153 mg of iodoacetamide
to give a solution of 55 mM iodoacetamide
- 15 mL of each is enough for up to 96 samples.
● Peptide Extraction Solution - 2% Formic acid/ 50% Acetonitrile solution
○ Add 2000µL of formic acid and 50 mL
of acetonitrile to 75 mL of
ddH2O. 15 mL is enough for up to 96 samples.
● Dehydration - Acetonitrile
○ A full square reservoir is plenty for up to 96 samples
with dehydration and/or Coomassie destaining.
● Trypsin
○ Trypsin is prepared by adding 200µL of Resuspension
Buffer into one vial containing 20ug of Proteomics grade trypsin.
○ Add 1.2ml of 50mM NH4HCO3 into each trypsin vial (final concentration of Trypsin
is 16ng/µL). Mix thoroughly.
○ This will provide enough Trypsin
for 46 digestions.
Mass Spec
1.
Samples need to
be spotted on the 384 spot MALDI plate: Odd rows are used for samples, and even
rows are for calibration standards. It is important that one sample spot in the
odd row is coupled by a calibration spot in the even row.
2.
Samples are
cleaned up with C18 Zip tips before spotting onto the plate.
3.
Start with a
fresh Ziptip, using a 10 µL pipette, and slowly
withdraw and dispense to waste 10 µL 90% (v/v) acetonitrile
/ 0.1 % TFA three times. Do not throw away the tip!
4.
Slowly withdraw
and dispense to waste 10 µL 0.1 % TFA five times to equilibrate the C18
material in aqueous buffer.
5.
Slowly withdraw
and dispense your first sample (10 µL) ten times. After this step the peptides
from your digest are bound onto the C18 resin in the tip.
6.
Withdraw and
discard to waste 10 µL of 0.1 % TFA five times. This washes away unwanted salts
and contaminants. Make sure you expel all the liquid on the last wash.
7. Withdraw 1 µL of matrix solution. This contains 70% acetonitrile so it is used to elute peptides in the presence of
matrix. (4 mg / mL CHCA
[α-cyano-4-hydroxycynnamic acid], 70 % MeCN, 0.1
% TFA).
8. Spot the eluted peptide sample in
matrix solution directly onto the MALDI target plate in an odd row, trying not
to touch the surface of the plate with the tip. Record the location of your
samples.
9. New C18 tip is needed for
each sample.
10. BSA digest at 1pmol/µL is used as control. This does not required ziptipping.
11. Next, prepare calibration
standards (Pepmix and Matrix solutions at 1:1 ratio),
and spot 1ul of this onto spots in even row next to your samples. These are
your near-point calibration standards.
12.Allow the plate to
air-dry.
13. Plates can be stored in 4oC
fridge and in the dark for up to a week, ready for analysis.
14. Samples are analyzed using the 4800 Plus MALDI Tof-Tof
Analyzer.
LB AGAR PLATE PREPARATION
1. Add 15g Bacto agar to 1000mL
of LB media and autoclave (121oC for 15mins).
2.
Add 1000uL of Chloroamphenicol (25mg/mL), Ampicillin (50mg/mL) or Kanamycin (30mg/mL) and mix well before plating out and
setting the agar.
LB MEDIA
● Ingredients: Tryptone 10g, Yeast extract
5g, NaCl 10g, Milli-Q water
to 1000mL.
● Methods: Dissolved 10g tryptone, 5g
yeast extract and 10g NaCl in 800mL Milli-Q water, making use of a magnetic stirrer. Once
dissolved, bring volume up to 1 L using Milli-Q
water. Autoclave 1L of the solution (121oC, 15 min, standard liquid
cycle).
SOC MEDIA (FOR COMPETENT CELLS)
● Ingredients: 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl 5M NaCl,
417µL 3M KCl, 1.205g 20mM Mg2SO4, 805g 20mM D-glucose
& 500mL Milli-Q water.
● Methods: The adjusted quantities were combined in a 1 L
measuring column with constant stirring and then placed in the autoclave for
sterilisation (121oC, 15 min, standard liquid cycle).
NANODROP for THERMO SCIENTIFIC NanoDrop 2000 Spectrophotometer
1. Pipette 1-2µl of blanking buffer onto the bottom
pedestal, lower the arm and click the BLANK button. The blank solution is the
same buffer that the DNA of interest is dissolved in.
2.
Wipe away the
blank. Pipette 1-2µl of DNA sample and hit MEASURE
3.
Wipe away the
last sample to prepare for the next sample, and repeat.
PCR Mixture: General recipe for PCR is as follows:
● 4 µL of 5x phusion buffer.
● 0.4 µL dNTPs.
● 0.6 µL of DMSO.
● 0.2 µL of polymerase.
● 11.8 µL of water.
To this mixture, add:
● 1 µL of forward primer.
● 1 µL of reverse primer.
● 1 µL of template.
● Total volume = 20 µL
PCR settings: A general program for PCR is as follows:
● Initial denaturation at 98oC
for 30seconds.
Followed by 30 repeats of:
● Denaturation – 98oC for 10seconds.
● Annealing – 60oC for 10seconds.
● Extension – 72oC for 2minutes.
● Final extension – 72oC for 10minutes.
Plasmid Prep
1.
Centrifuge
@13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL Eppendorf’s
2.
Discard
supernatant and add 1.9 mL of culture and centrifuge
again @13,200 rpm for 10 min
3.
Re-suspend pelleted cells in P1 Buffer (250µl)
4.
Add 250µL of P2
buffer & invert 4-6 times (turns homogenous blue)
5.
Add 350µL of N3
& mix by inverting (turns colourless)
6.
Centrifuge @
10min 13,000 rpm to obtain a pellet
7.
Transfer
supernatant in QIA Prep Spin Column by pipetting
8.
Centrifuge 30-60
sec - discard flow through
9.
Wash QIA Prep
Spin Column with 0.5mL of PB
10.
Centrifuge for
30-60 seconds - discard flow through
11.
Wash spin column
by adding 0.75 mL PE buffer
12.
Centrifuge for
30-60 seconds - discard flow through and centrifuge for a further 1min
13.
Place QIAPrep Column in clean Eppendorf
14.
Elute DNA add
50µl of water, stand for 1min, centrifuge for 1min
15.
QIA prep - Spin miniprep buffer
SDS-PAGE
1.
Re-suspend pelleted bacterial cells in 200µL of Milli-Q-water
2.
Transfer 50 µL of
suspension into new Eppendorf tubes and combine with
50ul of 2xTruSep sample buffer.
3.
Shear the cells using a Hamilton
syringe.
4.
Centrifuge the
preparation for 3minutes @ 13,000 rpm.
5.
Load 20 µL of the
supernatant into gel.
6.
Conduct
electrophoresis at a constant voltage (200V) for 1 hour.
7.
Coommassie Stain for ~30 minutes.
Heat Shock
1.
Obtain competent
cells from -80oC.
2.
Defrost gently on
ice. 100µl is sufficient for 2 transformations.
3.
Add 1-10µl of
plasmid DNA/ ligation mix to each tube. Incubate on ice for 5 min.
4.
Put the tubes in
the 42oC water bath for 30 seconds, then
back on ice for 2 min.
5.
Add 200µl of SOC
media to each tube, and incubate in the 37oC shaker for 10 min or up
to 1h (10min for purified plasmid or 1h for ligation mix).
6.
For each tube of
cells, spread 50µl onto one LB plate with appropriate antibiotic, and 500µl
onto a second plate, using aseptic technique. Place your plate upside-down in
the 37oC incubator.
Induction of Operons
1. Re-transform restriction-digest screened plasmids
according to the heat shock transformation protocol.
2.
Screen plates for
transformant colonies and place in 5mL of ZYM-5052 broth with appropriate
antibiotic concentration at 37oC with shaking until OD600¬ is 2.0
– 3.0.
3.
Take whole volume
and place in 45mL of ZYM-5052 broth in a sterile conical flask containing
appropriate antibiotic concentration. Incubate at 37oC with shaking.
4.
Cell pellets were
collected through centrifugation at 12000 rpm for 5 mins.
5.
Cell pellets were
resuspended in re-suspension buffer (Containing: 10% glycerol w/v, 50mM Tricene NaOH pH 8.0, 2mM MgCl2,
1mM DTT) and whole lysate was collected through a French Press for functional
assays of operon protein products in lysate.
MgPE enzymatic functional assay
1. Collect whole lysate, from induced operon 3 containing ChlM
expressed proteins, using ChlM pET
(L. Meinecke et al.2010)as a
negative control.
2.
Prepare a master
mix of recombinant ChlM, 25µl of S-Adenosyl-L- methionine and 65µl
of 2µM of Mg-protoporphyrin IX.
3.
Incubate at 30℃ with gentle
agitation for 8-10min
4.
100µL of acetone
was added to precipitate protein.
5.
The sample was
spun down, to separate the protein and supernatant.
6.
Supernatant was
then subjected to UHPLC to and observed peaks.
UHPLC protocol
The UHPLC protocol was performed
according to Sawicki and Willows (2007). Briefly, a
Shimadzu HPLC system was used at 2ml/min with an Alltech
C8 column. A 5 min linear gradient was used for separation of Mg-protoporphyrin IX and Magnesium-protoporphyrin
IX mono-methyl ester. Compounds were detected at an excitation wavelength of
excitation of 416nm and emission of 595nm.
Auto induction recipe
|
1.ZYM-5052 Medium ● ZY media 9.57ml ● 1m MgSO4 20ul ● 50x5052 200ul ● 50 x M 200ul |
2. 50 x M ● DI water 80ml ● Na2HPO4 17.75g ● KH2PO4 17g ● NH4Cl 13.4 ● Na2SO4 3.55g |
|
3. 50 x 5052 ● Glycerol 25g (weigh in Beaker) ● DI water 73ml ● Glucose 2.5g ● alpha lactose 10g |
4. 1M MgSO4 ● MgSO4.7H2O 24.65g ● DI water 87ml ● 5. ZY Media (400mL) ● Trypton 4g ● Yeast extract 2g |
References
● sawicki,A.,Willows,R.D.(2007).S-Adenosyl-L-methionine:magnesium-protoporphyrin
IX O-methyltransferase from Rhodebacter
capsulatus: mechanistic insights and stimulation with
phospholipids. Journal of Biochemistry, 406, 469-478. doi: 10.1042/BJ20070284
● William,F., Studier. (2014). Stable Expression
Clones and Auto-Induction for Protein Production in E.coli. Structural Genomics: General Applications, Methods in
Molecular Biology.1091, 17-32 DOI 10.1007/978-1-62703-691-7_2
● Meinecke, L., Alawady, A., Schroda, M., Willows, R., Kobayashi, M.C,.
Niyogi, K.K,. Grimm, B.,
Beck, C.F. (2010). Chlorophyll-deficient mutants of Chlamydomonas
reinhardtii that accumulate magnesium protoporphyrin IX. doi:
10.1007/s11103-010-9604-9