Team:Macquarie Australia/Experiments

Experiments and Protocol
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Figure 1. An overview of the methodology. A modified BioBrick Standard Assembly was used. Alkaline phosphatase (referenced below) on the target vector backbone for the genes; ChlM and those from Photosystem II. The Gibson Assembly method was used in order to insert composite part ChlH into the BioBrick vector plasmid (referenced below).

Experiments and Protocol

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.
  11. Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice.
  12. Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen.
  13. 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.
SOB Media (for competent cells)
Ingredients: 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl NaCl, 417µL KCl, 1.205g Mg2SO4 & 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).

AGAROSE GEL ELECTROPHORESIS
  1. Mix 1ul of 1kbp DNA ladder with 6ul of loading dye (bromophenol blue) and 4ul of 1x TAE buffer and load into first well
  2. Mix 5ul of plasmid DNA with 1ul of loading dye and load into wells.
  3. Run gel at 90V for 45minutes approximately
  4. Gel image is taken using Biorad GelDoc
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.
50 X 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: Components were combined then make up to 500ml with distilled water.
Making Agarose Gel
Ingredients: 1g agarose powder, 100ml of 1 x TAE
Methods: Dissolve agarose in TAE by microwaving for 1 minute, wait till cool to touch, then add 2ul of GelRed and pour into the casette.

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.


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 by adding 30µl of water, stand for 1min, centrifuge for 1min
  15. Plasmid concentration is measured using NanoDrop 2000 spectrophotometer


GENERAL PCR RECIPE
  • 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.


TRANSFORMATION BY 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 30 min.
  4. Heat shock in 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 1h
  6. For each tube of cells, spread 50µl onto one LB plate with appropriate antibiotic, and 100µl onto a second plate, using aseptic technique. Place your plate upside-down in the 37oC incubator.
LB Agar Plate Preparation
Ingredients: 15g Bacto agar, 1000mL LB media
Methods: Dissolve agar in LB media, autoclave for 15 mins at 121oC; wait until cool to touch, then add appropriate antibiotics before pouring into petri dishes.
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
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).

PROTEIN EXPRESSION
  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: 50mM Tricine 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.
Auto induction Medium (ZYM-5052)
  • 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


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

The UHPLC 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.



SDS-PAGE ELECTROPHORESIS
  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.


MANUAL TRYPSIN IN-GEL DIGESTION PROTOCOL FOR COOMASSIE STAINED GELS
  1. Wash 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 SPECTROMETRY
  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.


Gun4 PPIX binding by fluorescence spectrophotometry
Preparation of PPIX
  1. Approximately 1mg of dried PPIX was dissolved in 5ul of 1M NaOH, and diluted with 200ul of deionized water.
  2. Pellet any undissolved particulate by centrifugation, and dilute the supernatant 100-fold with 5% HCl.
  3. The concentration of the PPIX solution was calculated by measuring the absorbance of the diluted solution at 404nm, using the Beer-Lambert equation with molar extinction coefficient of 278000.
  4. A 2uM concentration solution of PPIX was used for the binding assay.
Gun4 PPIX binding
  1. Over-expressed Gun4 containing cell lysates was prepared as described above.
  2. The protein concentration was estimated using Bradford reagent, and the lysate diluted to approximately 0.2mg/ml.
  3. 100ul of the 2uM PPIX solution was added with varying amounts of protein lysate to a total volume of 200ul, and transferred to a clean quartz cuvette for fluorescence measurements in a Cory Eclipse fluorescence spectrophotometer (Agilent)
  4. The excitation wavelength was set at 280nm and the emission at measure from 580nm to 800nm. The emission maxima for protein bound PPIX is at 635nm.
References
  1. 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
  2. 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
  3. 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