Experiments

Describe the research, experiments, and protocols you used in your iGEM project.

Protocols

Preparation of 0.8% Agarose Gel

  1. Mix 0.8 g of agarose and 100 ml of 1x TAE;
  2. Heat up for 2 min;
  3. Leave to cool down;
  4. Add 5 µl of Ethidium Bromide (10mg/ml);
  5. Put on gel caster;
  6. Let it cool down and gelify;
  7. If not to use right away, store in fridge.

Running DNA on Gel

  1. Put gel on running apparatus;
  2. Load 10 µl of ladders
  3. Load samples ( x amount sample + 6x loading buffer + H2O)
  4. Run 90 mV for 45 min.

Loading buffer

  • 333 µl of 60% glycerol
  • 10µl of 0.5M EDTA
  • 20-100µl 6xDye
  • Complete till 1ml with H2O

Competent cell preparation

  1. 1 ml of an overnight E. coli culture was transferred to 100 ml of LB media and incubated at 37 °C and 200 rpm until OD = 0.4 - 0.6.
  2. The culture was poured into 50 ml falcon tubes, cooled on ice for 30 minutes and centrifuged at 4 °C and 4000 rpm for 5 minutes.
  3. The media was discarded, and the pellet was resuspended in 25 ml 0.1 M of ice cold MgCl2.
  4. The cells were cooled on ice for 30 minutes and spun down at 4 °C 4000 rpm for 5 minutes.
  5. The supernatant was discarded, the pellet was resuspended in 25 ml 0.1 M of ice cold CaCl2.
  6. The cells were cooled on ice for 30 minutes and spun down at 4 °C at 4000 rpm for 5 minutes.
  7. The supernatant was discarded, and the pellet was resuspended in 1.25 ml ice cold CaCl2/glycerol solution (1.7 mL 0.1 M CaCl2, 0.3 ml 100 % glycerol).
  8. The suspension was divided into 100 µl aliquots that were flash frozen in liquid nitrogen and stored at -80 °C.

Minipreps

  1. An overnight culture was grown and centrifuged at 4000 rpm for 5 minutes.
  2. The media was removed, and the pellet resuspended in 250 µl P1 buffer.
  3. 250 µl of P2 buffer were added, and the mixture was mixed well.
  4. 350 µl of N3 buffer were added, and the mixture was mixed well.
  5. The tube was centrifuged at 13000 rpm for 10 minutes, 800 µl of the supernatant were added to the QIAprep spin column.
  6. The column was centrifuged at 13000 rpm for 1 minute.
  7. 0.75 ml PE buffer were added to the column.
  8. The tube was centrifuged at 13000 rpm for 1 minute.
  9. The column was transferred to a new Eppendorf tube, 50 µl of EB buffer were added to the column.
  10. DNA was eluted after centrifuging 13000 rpm for 1 minute.

PCR Cleanup (Qiagen)

  1. 6X PB buffer was added to the PCR mixture and moved to the QIAquick spin column.
  2. The column was centrifuged at 13000 rpm for 1 minute, the flow through was discarded.
  3. 0.75 ml PE buffer were added to the column.
  4. The column was centrifuged again at 13000 rpm for 1 minute, the flow through was discarded.
  5. The column was centrifuged at 13000 rpm for 1 minute to remove any residual buffer.
  6. The column was transferred to a new Eppendorf tube and 30 µl of EB buffer was added.
  7. Purified DNA was eluted by centrifuging the column at 13000 rpm for 1 minute.

PCR Cleanup (Monarch, 5 μg)

All centrifugation steps should be carried out at 16,000 x g (~13K RPM in a typical microcentrifuge).

  1. Dilute sample with DNA Cleanup Binding Buffer (ensure that isopropanol has been added, as indicated on the bottle label)* according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex. A starting sample volume of 20–100 μl is recommended. For smaller samples, TE can be used to adjust the volume. For diluted samples larger than 800 μl, load a portion of the sample, proceed with Step 2, and then repeat as necessary.

    *Beginning in April 2021, the DNA Cleanup Binding Buffer will be changed to a concentrated format which requires the addition of isopropanol by the user. Please refer to the instructions inside of the product that you receive.

    SAMPLE TYPE RATIO OF BINDING BUFFER: SAMPLE EXAMPLE
    dsDNA > 2 kb (plasmids, gDNA) 2:1 200 μl:100 μl
    dsDNA < 2 kb (some amplicons, fragments) 5:1 500 μl:100 μl
    ssDNA > 200 nt** 7:1 700 μl:100 μl

    ** Please note that recovery of ssDNA < 200 nts can be increased by using the Oligonucleotide Cleanup Protocol, but doing so will shift the cutoff size for DNA binding to 18 nt (versus 50 nt).

  2. Insert column into collection tube and load sample onto column and close the cap. Spin for 1 minute, then discard flow-through. To save time, spin for 30 seconds, instead of 1 minute.
  3. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
  4. Repeat wash (Step 3).
  5. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
  6. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

    Note: Typical elution volumes are 6–20 μl. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield. Care should be used to ensure the elution buffer is delivered onto the matrix and not the wall of the column to maximize elution efficiency. To save time, spin for 30 seconds, instead of 1 minute.

Transformations

  1. 10 µl of plasmid DNA or reaction mixture were added to 100 µl of competent E. coli cells and left on ice for 30 minutes
  2. The cells were heat shocked at 42 °C for 30 seconds, then returned to ice for 30 seconds.
  3. 1 ml of SOC media was added.
  4. The cells were incubated at 37 °C and 200 rpm for 45 minutes.
  5. The tube was spun down at 13000 rpm for 1 minute, and the media was removed.
  6. Bacterial pellet was resuspended in 100 µl of SOC, then plated.

High Efficiency Transformation Protocol (Genomic DNA Production)

This protocol was performed to produce genomic DNA from which genes could be extracted.

Use this procedure to transform MG1655 chemically competent cells. We recommend verifying the transformation efficiency of the cells using the pUC19 control DNA supplied with the kit. Do not use these cells for electroporation.

  1. Remove competent cells from the -80 °C freezer and thaw completely on wet ice (10-15 minutes).
  2. Aliquot 1-5 µl (1 pg-100 ng) of DNA to the chilled microcentrifuge tubes on ice.
  3. When the cells are thawed, add 50 μl of cells to each DNA tube on ice and mix gently by tapping 4-5 times. For the pUC19 control, add 1 µl of (10 pg/µl) DNA to a chilled microcentrifuge tube, prior to adding 50 µl of cells. Mix well by tapping. Do not pipette up and down or vortex to mix, as this can harm cells and decrease transformation efficiency.
  4. Incubate the cells with DNA on ice for 30 minutes.
  5. After the 30-minute ice incubation, heat shock the cells at 42 °C for 45 seconds.
  6. Transfer the tubes to ice for 2 minutes.
  7. Add 950 µl of Recovery Medium or any other medium of choice to each tube.
  8. Incubate tubes at 37 °C for 1 hour at 210 rpm.
  9. Spread 50 μl to 200 μl from each transformation on pre-warmed selection plates. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies. For the pUC19 control, plate 50 μl on an LB plate containing 100 μg/ml ampicillin. Use a sterilized spreader or autoclaved ColiRoller™ plating beads to spread evenly.
  10. Incubate the plates overnight at 37 °C.

Restriction Digests

  1. For a typical 20 µl reaction using Restriction enzyme, the reaction was set up as follows:

    2. Reagent Volume or Mass Added
    DNA 1 µg
    10X reaction buffer 2 µl
    Restriction Enzyme 1 µl
    H2O Up to 20 µl

  2. The mixture was incubated at 37 °C for 1 hour.
  3. The completed reaction was run on an agarose gel to visualize the products.

PCR (Extraction, Domestication and Troubleshooting)


For Q5® High-Fidelity DNA Polymerase (M0491)

Annealing temperatures were calculated on the NEB Tm calculator (NEBTm Calculator)


Reaction Setup:
Component 25 µl Reaction 50 µl Reaction Final Concentration
5X Q5 Reaction Buffer 5 µl 10 µl 1X
10 mM dNTPs 0.5 µl 1 µl 200 µM
10 µM Forward Primer 1.25 µl 2.5 µl 0.5 µM
10 µM Reverse Primer 1.25 µl 2.5 µl 0.5 µM
Template DNA variable variable < 1,000 ng
Q5 High-Fidelity DNA Polymerase 0.25 µl 0.5 µl 0.02 U/µl
5X Q5 High GC Enhancer (optional) (5 µl) (10 µl) (1X)
Nuclease-Free Water to 25 µl to 50 µl

Thermocycling Conditions for a Routine PCR:
Step Temp Time
Initial Denaturation 98°C 30 seconds
25–35 Cycles 98°C
50–72°C
72°C		 5–10 seconds
10–30 seconds
20–30 seconds/kb
Final Extension 72°C 2 minutes
Hold 4–10°C

OneTaq® DNA Polymerase (M0480)

Annealing temperatures were calculated on the NEB Tm calculator (NEBTm Calculator)


Reaction Setup:
Component 25 μl reaction 50 μl reaction Final Concentration
5X OneTaq Standard Reaction Buffer* 5 µl 10 μl 1X
10 mM dNTPs (#N0447) 0.5 µl 1 μl 200 µM
10 µM Forward Primer 0.5 µl 1 μl 0.2 µM
10 µM Reverse Primer 0.5 µl 1 μl 0.2 µM
OneTaq DNA Polymerase 0.125 µl 0.25 µl 1.25 units/50 µl
Template DNA variable variable < 1,000 ng
Nuclease-free water to 25 µl to 50 µl

Thermocycling Conditions for a Routine PCR:
Step Temp Time
Initial Denaturation 94°C 30 seconds
30 Cycles 94°C
45-68°C
68°C		 15-30 seconds
15-60 seconds
1 minute per kb
Final Extension 68°C 5 minutes
Hold 4-10°C

SuperFi DNA Polymerase (M0480)

Invitrogen info sheet found here.


Reaction Setup:
Component Final concentration 20-µL Reaction 50-µL Reaction
5X SuperFit II Buffer 1X 4 µL 10 µL
Forward primer 0.5 µM x µL x µL
Reverse primer 0.5 µM x µL x µL
10 mM dNTPs 200 µM each 0.4 µL 1 µL
Template DNA 0.1-10 ng plasmid DNA (5-100 ng genomic DNA) x µL x µL
PlatinumTM SuperFiTY 11 DNA Polymerase 1X 0.4 µL 1 µL
Water, nuclease-free to 20 µL to 50 µL

Thermocycling Conditions for a Routine PCR:
Cycle step Temperature Time Cycles
Initial denaturation 98°C 30 seconds 1
Denaturation
Annealing
Extension		 98°C
60°C
72°C		 5-10 seconds
10 seconds
15-30 seconds per 1 kb		 25-35
Final extension 72°C 5 minutes 1
Hold 4°C

Colony PCR

  1. Put 50 μl sterile water into PCR tubes. One for each of your chosen colonies.
  2. Put 1 ml of LB media into a sterile Eppendorf tube. One for each of your chosen colonies.
  3. Using your pipette set to 40 μl, pick up the colony of interest.
  4. Resuspend the colony in 50 μl of water in a PCR tube.
  5. Once the colony is resuspended, take 40 μl and inoculate the 1 ml of LB media.
  6. You will be left with 10 μl of resuspended colony in your PCR tube. This is your DNA for your PCR.
  7. Once you have done all your samples, freeze them at -20 °C for 10 minutes.
  8. During this 10 minutes, make your master mix.

GoTaq® DNA Polymerase

Promega info sheet found here.


Reaction Setup:
Component Final Concentration Volume
5x Green GoTaq reaction buffer 1X (1.5 mM MgCl2)2 5 µL
dNTP mix (10 mM) 0.2 mM 0.5 µL
Forwards primer (cPCRFor) 1:00 PM 2.5 µL
Reverse primer (cPCRRev) 1 µL 2.5 µL
GOTaq G2 DNA polymerase (Demonstrator has this) 1.25 u 0.125 µL
Template DNA 5 µL*
Water 9.375 µL
Total 25 µL

Reaction Setup:
Step Temperature Time Number of Cycles
Initial Denaturation 95°C 2 minutes 1 cycle
Denaturation 95°C 0.5-1 minute
Annealing 42-65°C* 0.5-1 minute 25-35 cycles
Extension 72°C 1 minute per kb
Final Extension 72°C 5 minutes 1 cycle
Soak 40°C Indefinite

* Annealing temperature should be optimized for each primer set based on the primer Tm.

MAD Medium (1 L)

Component Quantity
NaCl 18 g
MgSO4·7H2O 5 g
A+ modified 100X 10 mL
Tris (125 g/L, pH 8.2) 8.3 mL
Modified D7 Metal 1000X 1 mL
NaNO3 7 g
KH2PO4(1 M) 0.83 mL

Autoclave OR sterilise using 0.22 µm filter and cool down to room temperature before adding the following:

Component Quantity
FeCl3·6H2O (480 mM) 0.5 mL
Vitamin B12 - Cobalamin 1 mL

MAD Medium Stock Solutions

Modified D7 Metal 1000X (1 L)
Component Quantity
H3BO3 2.86 g
MnCl2·4H2O 1.81 g
ZnSO4·7H2O 222 mg
Na2MoO4·2H2O 1.26 g
CuSO4·5H2O 79 mg
CoCl2·6H2O 40.3 mg

A+ Modified 100X (1 L)
Component Quantity
KCl 60 g
NaNO3 100 g
CaCl2·2H2O 37 g
Na2EDTA 3 g
KH2PO4 5 g

BG11 100X Medium (1L)

All components, with exception of buffers, must be autoclaved


Main Components
Component Quantity
NaNO3 149.6 g
MgSO4.7H2O 7.49 g
CaCl2 3.60 g
Citric acid 0.60 g
Na2EDTA 1.12 mL of 0.25M solution, pH 8.0

Trace Elements
Component Quantity
H3BO3 2.860 g
MnCl2·4H2O 1.810 g
ZnSO4·7H2O 0.220 g
Na2MoO4·2H2O 0.390 g
CuSO4·5H2O 0.080 g
Co(NO3)2·6H2O 0.050 g

Stock Solutions (g/100ml)


Iron Stock
Component Quantity
Ferric citrate 0.6
(ALTERNATIVELY) Ferric ammonium citrate 1.11

Phosphate Stock
Component Quantity
K2HPO4 3.05

Na2CO3 Stock
Component Quantity
Na2CO3 2.0

Glucose Stock
Component Quantity
Glucose 9.0 g (= 0.5 M)

store in the fridge


Buffers (g/100ml)


TES buffer
Component Quantity
TES 22.9

Adjust with 1 M NaOH to pH 8.2


HEPES buffer
Component Quantity
HEPES 23.8

Adjust with 1 M NaOH to pH 7.8


Other (g/100ml)


Component Quantity
NaHCO3 8.4

Cyanogate Assembly Protocol


Reaction Setup:
Bpil Assembly Components (Level 0, T) Bsal Assembly Components (Level 1)
50-100 ng of acceptor vector 50-100 ng of acceptor vector
For each vector/part to insert, use a 2:1 ratio of insert: acceptor vector. For each vector/part to insert, use a 2:1 ratio of insert: acceptor vector.
2 µL 10 mM ATP (Table of Materials) 2 µL 10 mM ATP (Table of Materials)
2 µL buffer G (buffer for Bpil/Bsal) 2 µL buffer G (buffer for Bpil/Bsal)
2 µL BSA (10x) (Table of Materials) 2 µL BSA (lox) (Table of Materials)
10 units Bpil (1 µL 10 U/µL Bpil, Table of Materials) 10 units Bsal (1 µL 10 U/µL Bsal, Table of Materials)
Bring to 20 µL with dH2O. Bring to 20 µL with dH2O.
200 units T4 DNA ligase (1 µL 200 U/µL, Table of Materials) 200 units T4 DNA ligase (1 µL 200 U/µL, Table of Materials)

*Experimentation showed that ATP is optional.


Thermocycler Protocol:
Level 0, T Level 1
37 °C for 10 min
16 °C for 10 min		 (cycle x 5) 37 °C for 10 min
16 °C for 10 min		 (cycle x 5)
37 °C for 20 min 37 °C for 20 min
65 °C for 10 min 65 °C for 10 min
16 °C (hold) 16 °C (hold)

JUMP Assembly (Level 1)

  1. Create a reaction mix in a PCR tube with the following components:
    2. Reaction Component Amount (μl)
    ggpS epPCR digest (20fmol/μl) 1
    pJUMP29-1A acceptor vector (20fmol/μl) 1
    PJ23100 Promoter in level 0 (20fmol/μl) 1
    L3SAP51 terminator in level 0 (20fmol/μl) 1
    pET Ribosome Binding Site in level 0 (20fmol/μl) 1
    T4 DNA ligase - HF 0.25
    BsaI-HF 1
    T4 Ligase Buffer 2
    ddH2O Fill to 20
  2. Set the Thermocycler to run in the following cycles:
    • 1 cycle at 37°C for 15 minutes
    • 25 cycles at 37°C for 3 minutes and 16°C for 3 minutes
    • 1 cycle of 37°C for 15 minutes, 50°C for 5 minutes, and 80°C for 5 minutes
    • 25 cycles of 37°C for 3 minutes and 16°C for 3 minutes
    • 1 cycle of 37°C for 15 minutes, 50°C for 5 minutes, and 80°C for 5 minutes
    • Finally, hold the reaction at 4°C

Preparation of NaCl Lysogeny Broth and BL21 Growth Curve

  1. Prepare Sterile 34% (w/v) Saline stock solution.
  2. Add Saline solution in increasing amounts to LB in a falcon tube to create a medium of varying salt concentrations and then inoculate with 1ml of overnight culture BL21 using the table below:
    3. Concentration of NaCl in LB media (w/v) Volume of 34% Saline Solution (mL) Volume of LB media (mL)
    0.5% 0 29
    1% 0.441 28.559
    1.5% 0.882 28.118
    2% 1.323 27.677
    2.5% 1.764 27.236
    3% 2.205 26.795
    3.5% 2.646 26.354
    4% 3.087 25.913
    4.5% 3.528 25.472
    5% 3.969 25.031
    5.5% 4.380 24.590
    6% 4.821 24.149
  3. Take an initial OD600 from each of the cultures.
  4. Incubate the cultures at 37°C, 250rpm.
  5. Take a 1ml sample every hour and record the OD600 of each culture. If the OD600 of a culture is significantly more than 1, dilute the sample and measure the OD600.

Error Prone PCR

  1. Create a reaction mix in a PCR tube with the following components:
    2. Reaction Component Amount
    10x NEB Taq DNA polymerase Thermopol buffer 5μl
    Genomic Forward Primer 1μl
    Genomic Reverse Primer 1μl
    Taq polymerase 0.5μl
    ggpS Level 0 Template DNA 50ng
    MgCl2 2.95mM
    MnCl2 0.1mM
    ddH2O Fill to 50μl
  2. Set the thermocycler to have an initial denaturation at 95°C for 1 minute, 25 cycles of 30 seconds at 95°C, 45 seconds at 53°C, and 90 seconds at 68°C, a final elongation at 68°C for 5 minutes, and hold at 4°C.
  3. Purify the product with the Monarch DNA Gel Extraction kit (New England Biolabs) and store at -20°C.

Special Protocols


Membrane fractionation by cold osmotic shock

The membrane fractionation protocol allows to separate the contents of E. coli to the cytoplasmic,periplasmic and membrane components. Doing this in Escherichia coli (E. coli) would allow for the identification of wanted membrane,periplasmic proteins (mtrB,mtrA respectively) in our engineered cells. :

Materials
  • Overnight culture of E. coli
  • Microcentrifuge 
  • Icebath
  • 0.2 µm filter & filtration syringe 
  • Cell fractionation buffer (CFB) 1 (pH 8):
    • 0.2M Tris
    • 200 g/L sucrose
    • 0.1M EDTA
  • Cell fractionation buffer (CFB)  2.1 (pH 8) :
    • 0.01M Tris
    • 0.005M MgSO4
  • Cell fractionation buffer (CFB) 2.2 (pH 8) :
    • 0.01M Tris
    • 0.005M MgSO4
    • 1% Triton
    • 2% SDS
  • Cell fractionation buffer (CFB) 2.3 (pH 8) :
    • 0.01M Tris
    • 0.005M MgSO4
    • 1% Triton
    • 0.2% SDS
  • Cell fractionation buffer (CFB) 2.4 (pH 8) :
    • 0.01M Tris
    • 0.005M MgSO4
    • 1% Triton
    • 1% SDS
  1. Centrifuge E. coli cell suspension for 5 min at 14,000 g (4 °C) to collect the cells
  2. Discard the entire supernatant
  3. Resuspend the cells in ice-cold CFB1. The resulting volume should be 1/4 of the former suspension volume
  4. Incubate for 20 min on ice. Invert the suspension at regular intervals to counteract sedimentation
  5. Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
  6. Discard the entire supernatant
  7. Resuspend the cells in ice-cold CFB2. The resulting volume should be 1/4 of the former suspension volume
  8. Incubate for 10 up to 20 min on ice under regular invertion
  9. Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
  10. Save the supernatant, which contains the periplasmatic proteins and membrane proteins when using 2.2 and 2.3
  11. If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter

Ferricyanide Assay Protocol

Materials:

  • Potassium hexa-cyanoferrate (III) (CAS Number: 13746-66-2)
  • 20 mL cyanobacterial cultures
  • 0.22 µm syringe filter
  • Spectrophotometer
  • Aluminium foil
  • Shaking incubator
  • 15 mL Synechocystis sp. pcc 6803 cultures at exponential growth phase (OD~1 at 630 nm), doublets for each experimental condition

Protocol:

  1. Cultures were removed from the incubator and placed under dark conditions for 4 hours prior to the start of the experiment.
  2. Ferricyanide stock solution was made using water to 100 mM.
  3. Cultures in light-absent conditions were covered in aluminium foil.
  4. Ferricyanide was added to the culture, reaching a final concentration of either 1 or 5 mM.
  5. 1 mL volumes were removed and passed through a 0.22 µm syringe filter. The filtrate was used to blank the spectrophotometer at 390 nm.
  6. Cultures were placed in a shaking incubator.
  7. At specified time intervals, cultures were removed from the incubator, and a 1 mL volume was removed and passed through a 0.22 µm syringe filter. Filtrate absorbance was measured at 390 nm and compared against the relevant blank (1 or 5 mM ferrocyanide).
  8. Recorded data were used to plot a linear regression graph for analysis.

SDS-PAGE Protocol

Procedure:

1. Setting up the SDS-PAGE Tank

  • The tank for SDS-PAGE is set up with the inner chamber and half of the outer chamber filled with 1X running buffer.

2. Sample Preparation

  • Mix 8 μl of sample lysate with 10 μl of 2X Laemmli sample buffer and 2 μl of β-mercaptoethanol under a fume hood.

3. Boiling the Sample

  • Boil the sample at 98°C for 5 minutes.
  • Spin down the sample.
  • You can either freeze the sample at -20°C or proceed to load it onto the gel.

4. Gel Loading

  • Load the prepared sample onto a precast 4-15% Mini-PROTEAN TGX SDS-PAGE gel along with a protein standard.

5. Electrophoresis

  • Connect the power supply unit to the electrodes.
  • Run the electrophoresis at 180V until the desired separation of protein bands has been achieved.

6. Gel Staining

  • Remove the gel from the tank.
  • Carefully break the gel out of the plastic mold.
  • Wash the gel with water to remove excess buffer and impurities.
  • Pour off the water.
  • Add enough Coomassie Blue to cover the entirety of the gel.
  • Incubate the gel while shaking at an orbital shaker at approximately 35 rpm until protein bands become clearly visible.

Heme Stain Protocol

Materials

  • TMB (N, N, N', N'-tetramethylbenzidine)
  • MeOH
  • 0.5 M Na Acetate, pH 5.0
  • H2O2

Method

  1. Dissolve 30 mg TMB (N, N, N', N'-tetramethylbenzidine) in 15 ml MeOH. (Make sure it dissolves well before the next step.)
  2. Add 35 ml of 0.5 M Na Acetate (pH 5.0).
  3. Carefully place the gel in the stain.
  4. Wait for 3-5 minutes.
  5. Add 300 µl of H2O2 and mix.
  6. Wait for 5 minutes.
  7. Check for blue bands.

Notes

  • Load 50 µg of total protein per well.
  • Keep the reaction in the dark at all times. TMB is light-sensitive.

Optimal Transformation Protocol for Synechocystis sp. pcc6803

Before Starting:

  • Make sure you have enough BG11, 2x BG11, and 2x BactoAgar for the protocol.
  • When making plates for plating cells after transformation, use a defined volume of BG11+Agar per plate:
    • Use 30 ml BG11-Agar media. This amount makes a thick gel which prevents gel breaking in the next step. As in the next step, we add antibiotics (here CAM and Spect in 500 μl BG11) underneath the agar.
  • Set up a fresh liquid Synechocystis culture in advance for the protocol.

Making the Preculture:

  • Set up a preculture of Synechocystis in BG11 media (e.g., 25 ml BG11 in 250 ml flasks) by taking a loopful of wild-type Synechocystis pcc6803 culture grown on BG11 plates.
  • Let the culture grow for 2-4 days (to reach OD750=1-5).
  • Use a diluted sample (1:10; 100 μl culture + 900 μl BG11 or H2O) to measure OD750.

Making the Main Culture:

  • Make the main culture with a starting OD750 of 0.1 or 0.2 (etc.) by inoculating BG11 with the preculture.
  • Let the culture reach mid-logarithmic growth phase (OD750=0.8). If high transformation efficiency is required, grow the cells (OD750=0.8) in BG11 with 2 mM EDTA for 2 days before the transformation.
  • Transformation in late latent growth phase (OD750=0.3-0.7) or plateau growth phase (OD750= >1) will lower the transformation efficiency severely.

Transformation:

  • Two days after the addition of EDTA to the culture with OD750=0.8:
    • Pipet 5 ml of culture into a sterile 12 ml falcon tube.
    • Pellet the cells with 3000 g, 5 min, RT centrifugation.
    • Resuspend the cell pellet in 1 ml of fresh BG11.
    • Add DNA 15-20 μg/ml (usually 3-20 μg/ml) to each sample.
    • Incubate the cells for 5 h in light conditions on a shaker (150 rpm, 30°C, 50 μE, 1% CO2).
    • Centrifuge at 3000 g, 5 min, RT, and discard the supernatant.
    • Resuspend the pellet in the remaining liquid.
    • Plate them in BG11 (plate contains 30 ml BG11) with NO antibiotics.
    • Keep at 30°C and 50 μE until a slight green film arises (ca. 2-4 days). Usually two days.
  • Selection: Addition of antibiotics
    • Take a sterile spatula and lift the agar at one side.
    • Fill 500 μl BG11 containing antibiotics (in our project CAM and Spect) underneath the agar.
    • The amount of antibiotics is calculated for the final BG11+Agar volume, here 30 ml.
    • We start with 3 μg/ml CAM (≃ 2.6 μl of 34 mg/ml CAM stock, 8.8 % of the final concentration; 34 μg/ml) and 6 μg/ml Spectinomycin (≃ 3.6 μl of 50 mg/ml CAM stock; 12 % of the final concentration; 50 μg/ml).
  • Cover the plates with paper sheets for 1-2 days to reduce light intensity (usually two days!).
  • Colonies should appear in 10-30 days (usually transformants appear in three weeks, but it may take longer time).

Increasing Selection Pressure:

  • Pick the colonies from the previous step (grown in BG11 plate + 3 μg/ml CAM + 6 μg/ml Spectinomycin) by loop and streak them to BG11 plates containing a higher amount of antibiotics:
    • 6 μg/ml CAM and 12 μg/ml Spectinomycin. Continue cultivation until colonies appear (1-3 weeks).
  • Pick the colonies from the previous step (grown in BG11 plate + 6 μg/ml CAM + 12 μg/ml Spectinomycin) by loop and streak them to BG11 plates with the final antibiotic pressure: 34 μg/ml CAM + 50 μg/ml Spectinomycin.
  • Finally, axenic cultures/transformants will be tested by culturing them on antibiotic-free LBA. Keep the plate at 37°C for a couple of days as some bacteria need longer time to grow!

Notes:

  • I did not transfer the colonies to 12 μg/ml CAM and 24 μg/ml Spectinomycin and directly streak them from 6 μg/ml CAM + 12 μg/ml Spectinomycin to the final concentrations.
  • All the colonies survived in plates containing 34 μg/ml CAM + 50 μg/ml Spectinomycin.

Ninhydrin Assay

The ninhydrin test is a widely used method to detect the presence of amino acids in a sample. It is often used to determine amino acid expression or the presence of free amino acids. Here's a protocol for performing the ninhydrin test on a live overnight culture of Escherichia coli (E. coli) to determine amino acid expression:


Materials
  • Overnight culture of E. coli
  • Ninhydrin solution (0.2% in acetone or ethanol)
  • 2M Sodium acetate buffer (pH 6.0-6.5)
  • Test tubes
  • Water bath or heat block
  • Pipettes
  • Microcentrifuge (optional)
  • Spectrophotometer (optional)

Protocol
  1. Prepare the Ninhydrin Solution:
    • Prepare a fresh ninhydrin solution by dissolving 0.2 g of ninhydrin in 100 ml of acetone or ethanol. Mix well until the ninhydrin is completely dissolved.
  2. Prepare Sodium Acetate Buffer:
    • Prepare a 2M sodium acetate buffer solution at the desired pH (usually around 6.0-6.5). Adjust the pH using acetic acid or sodium hydroxide as needed.
  3. Harvest Cells (Optional):
    • If necessary, harvest the E. coli cells from the overnight culture by centrifugation. Wash the cells with a suitable buffer (e.g., phosphate-buffered saline) and resuspend them in a small volume of the sodium acetate buffer.
  4. Reaction Setup:
    • Label test tubes for each sample or standard you will be testing.
    • To each test tube, add:
      • 1-2 ml of the sodium acetate buffer (pH 6.0-6.5)
      • 100-200 µl of the E. coli culture (or equivalent volume of harvested cells)
      • If using standards, prepare a set of standards containing known concentrations of amino acids in the same buffer.
  5. Add Ninhydrin Solution:
    • Add 1-2 ml of the prepared ninhydrin solution to each test tube containing the samples and standards.
  6. Heating:
    • Place the test tubes in a water bath or heat block preheated to around 80-100°C.
    • Incubate the tubes for about 10-20 minutes. The exact incubation time may vary based on the temperature and the specific amino acids being tested.
  7. Color Development:
    • After incubation, remove the tubes from the water bath and allow them to cool to room temperature.
  8. Measurement:
    • Measure the color development in each tube using a spectrophotometer at an appropriate wavelength (usually around 570 nm). Use the buffer with ninhydrin as a blank.
  9. Data Analysis:
    • Compare the absorbance readings of the samples with the standard curve generated using known concentrations of amino acids. The intensity of color development indicates the presence and quantity of amino acids in the samples.
  10. Interpretation:
    • Calculate the concentration of amino acids in the E. coli culture based on the standard curve and the absorbance readings.

Protocol for Preparing E. coli Samples for Mass Spectrometry

Materials and Supplies:

  • OP50 media - ~1200 ml (1L solution - 5g Tryptone, 2.5g Yeast Extract)
  • LB plate (1)
  • Autoclaved Test Tubes (2-4)
  • OD600nm Spectrometer
  • Autoclaved 250ml Beckmann centrifuge bottles (6)
  • Centrifuge with rotor that holds 250ml bottles
  • 37°C incubator
  • Roller or shaker that holds test tubes
  • Shaker for 1L Erlenmeyer flasks
  • Ice buckets w/ ice (2)
  • French Press with refrigerated cell (cleaned before use)
  • Autoclaved 1.5ml Eppies
  • Sterile 15ml Falcon conical tubes
  • Microcentrifuge
  • Disposable 25ml pipets
  • Bio-Rad RC DC Protein Assay Kit (Cat# 500-0122)
  • 0.2 % RapiGest SF Powder in 50mM Ammonium Bicarbonate pH 7.8 (Waters Corporation, 5 Pack of 1 ml Vials, 186001860)
  • 50mM Ammonium Bicarbonate buffer pH 7.8
  • 500mM DTT
  • 500mM IAA (Iodoacetamide - light-sensitive)
  • 500mM HCl
  • 250 ng/μl Trypsin, modified, sequencing grade in 0.01% Acetic Acid (Roche, 4 x 25 μg, Cat # 1418033)
  • 100 mM CaCl2

Sample Preparation

DAY 1 - Plate Growth

  1. Streak a plate of TJ2 strain of OP50 E. coli located in the freezer in J256.
  2. Use an LB Agar Plate found in J180C 4°C room.
  3. Grow up colonies overnight at 37°C (K231).

DAY 2 - Culture Growth part 1

  1. Inoculate 1-3 test tubes with 5 ml OP50 media with single colonies from the plate. Grow up several cultures in case some don't grow.
  2. Grow overnight (8-12 hrs) at 37°C on a roller or shaker (K231 or K310 hallway).

DAY 3 - Culture Growth part 2

  1. Remove 10 ml from 1L of OP50 media to use as a zero for spectrophotometer and add 10 ml overnight cultures to the liter of media for a 1:100 dilution.
  2. Grow bacteria at 37°C on a shaker for ~5 hours or until OD600nm reading on the spectrophotometer (in J180) is 0.5.
  3. Pour 200 ml of bacteria into 5 x 250 ml Beckmann centrifuge bottles and use the extra bottle as a balance with 200 ml of water.
  4. Chill on ice for 5 minutes (I usually chill the centrifuge bottles ahead of time as well).
  5. Spin down samples at 3000 rpm for 5 minutes at 4°C using a Beckman centrifuge in either J180 or J160.
  6. Remove supernatant from pellets.
  7. Add 2 ml of cold 50mM Ammonium Bicarbonate pH 7.8 to each pellet, making sure to fully resuspend the pellets in the buffer.
  8. Combine all resuspended pellets into a sterile 15ml Falcon conical tube.
  9. Freeze the sample at -20°C until ready to lyse the sample.

DAY 4 - Lysis

  1. Lyse a 10 ml sample with a French Press (J432) using Klevit lab cell in K464 (see separate protocol for French Press lysis technique).
  2. Spin the sample at 4000 rpm for 30 minutes at 4°C using Waterston lab centrifuge in K313 hall.
  3. Remove supernatant from pelleted debris.
  4. Spin the sample at 14K for 10 minutes to separate insoluble fraction from soluble.
  5. Use Bio-Rad Protein Assay Kit to quantitate protein (see separate protocol). The spectrophotometer is in Fangman/Brewer lab J135.

Sample Digestion

  1. Make 0.2% RapiGest diluted in 50 mM Ammonium Bicarbonate pH 7.8 (1 mg RapiGest per 500 μl 50 mM Ammonium Bicarbonate pH 7.8).
  2. Using a locking lid microcentrifuge tube, add 100 μl of 0.2% RapiGest per 100 μl protein mixture (1:1) - [final concentration of RapiGest should be 0.1% (w/v)]. If protein is in pellet form, add 25-50 μl of 0.1% RapiGest. Vortex the sample.
  3. Boil the sample at 99°C for ~2 minutes, then let it cool for a couple of minutes.
  4. Quick spin the sample and add DTT to a final concentration of 5 mM.
  5. Incubate the sample at 60°C for 30 minutes.
  6. Cool the sample to room temperature.