Construction of Bacteria

Bacterial Transformation

Performed and Documented by Chenni Wang

Materials

Plasmid vector DNA (pET23b)

Insert DNA

DNA ligase and buffer

Competent state E. coli (strain Bl21 (DE3))

LB media

LB plate with appropriate antibiotic for the LB agar

Equipment:

Eppendorf tube

Shaking incubator at 37 °C

Stationary incubator at 37 °C

Water bath at 42 °C

Bucket of ice

Microcentrifuge tubes

Sterile spreading device (i.e. pipette tip)

Procedures:

1. Combine the following in a PCR or Eppendorf tube:

   • Plasmid Vector DNA

   • Insert DNA

   • Ligase Buffer (1μL/10μL reaction for 10X buffer)

   • 1μL DNA Ligase

   • H2O to a total of 10μL

   • For a control group, add H2O instead of insert DNA.

2. Incubate at room temperature (approximately 25°C) for 2 hours before proceeding to bacterial transformation.

3. Incubate at room temperature (approximately 25°C) for 2 hours before proceeding to bacterial transformation.

4. Retrieve competent cells from -80°C refrigerator, and defrost on ice for approximately 30 minutes.

5. Retrieve agar plates containing the appropriate antibiotic from storage at 4°C, and let it warm up to room temperature. Leave in 37°C incubator for later use.

6. Mix 5μL of plasmid DNA into 50 μL of competent cells in a microcentrifuge tube. Gently mix the components. For a control group, add 1μL of water to verify that the LB agar’s antibiotic functions properly.

7. Incubate the competent cell/DNA mixture on ice for approximately 30 minutes.

8. Heat shock each transformation tube by placing the bottom half of the tube into a 42°C water bath, leaving it for approximately 45 seconds.

9. Put the tubes back on ice for 2 minutes.

10. Add 250 μL LB media (without antibiotic) to the bacteria, and grow in 37°C shaking incubator for 45 min.

11. Plate an appropriate amount of the transformation onto the 10 cm LB agar plate containing the corresponding antibiotic.

12. Incubate plates at 37°C overnight.

Liquid Culture & Glycerol Stocks

Materials:

LB media

Appropriate antibiotic for LB agar

Previously cultured bacteria

50% filter sterilized glycerol

Equipment:

Sterile 1.5 mL Eppendorf Tubes

Sterile loop for bacteria retrieval

Procedures:

1. Add liquid LB to a tube or flask and add the appropriate concentration of antibiotic.
2. Using a sterile loop, select a single colony from the LB agar plate.
3. Drop the loop into the liquid LB mixture and swirl.
4. Loosely cover the culture with a sterile cap that is not air tight. Incubate bacterial culture at 37°C for 12-18 hours in a shaking incubator.
5. After observed bacterial growth, add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube, and gently mix.
6. Freeze the glycerol stock tube at -80°C.

Restrictive Endonuclease Digestion

Documented by Yunxian Ding and Yunzhu Chen

Recovering Plasmid DNA:

Materials:

Overnight culture of bacteria transformed with plasmid

PBS buffer

Denaturing solution

Renaturing solution

100% and 70% ethanol

Equipment:

Desktop microcentrifuge

Desktop vortexer

Procedures:

1. Grow an overnight culture of bacteria.

2. Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation.

3. Remove the supernatant and resuspend the bacteria in buffer.

4. Add a denaturing solution from the DNA extraction kit to the resuspended bacteria.

5. Add a renaturing solution from the DNA extraction kit to the denatured bacteria.

6. Pellet the proteins and genomic DNA by centrifugation, and remove the plasmid-containing supernatant.

7. Add ethanol (or isopropanol) to precipitate the plasmid DNA.

8. Centrifuge at 12000 rpm for 10 minutes to pellet the DNA.

9. Wash the pellet with 70% ethanol to remove excess salt.

10. Resuspend the DNA pellet with PBS buffer.

Diagnostic Endonuclease Digest & PCR

Materials*:

Plasmid DNA

Restriction enzyme

Restriction digest buffer

Forward Primer

Reverse Primer

5 μL 10X Taq buffer with MgCl2

1 μL dNTP mix

Taq DNA Polymerase

*A TaKaRa PCR amplification kit was used in our experiments. Its details can be found here: https://www.takara.co.kr/file/manual/pdf/R011_e.v1906Da.pdf

Equipment:

Thin-walled PCR tube

Ice Bucket

PCR Machine

Procedures:

1. In a 1.5mL tube combine the following:

   • 1 μg of plasmid DNA sample

   • 1 μL of each restriction enzyme

   • 3 μL restriction digest buffer

   • H2O up to total volume of 30 μL

   Mix gently by pipetting.

2. Incubate tube at 37 °C for 1 hour.

3. Place thin-walled PCR tubes on ice. Set up a 50 μL reaction:

   • 2 μL template DNA

   • 5 μL 10X Taq buffer with MgCl2

   • 1 μL dNTP mix

   • 2.5 μL of 10 μM Forward Primer

   • 2.5 μL of 10 μM Reverse Primer

   • 0.2 μL 5 units/μL Taq DNA Polymerase

   • H2O up to total volume of 50 μL

4. Place reaction tubes in PCR machine. Set the standard time and temperature for each step in a PCR cycle:

   Initial Denaturation for 2 minutes at 94°C.

   Denature for 30 seconds at 94°C.

   Anneal primers for 30 seconds at 5°C below Tm.*

   Extend DNA for 2 minutes at 72°C.

5. Repeat for 20-30 cycles. Final Extension for 5 minutes at 72°C.

*We amplified 3 different sequences with different primer Tm, so they were not performed simultaneously.

Agarose Gel Electrophoresis:

Materials:

DNA samples

Gel loading dye

Electrophoresis buffer

TAE

Agarose

Gelred

Equipment:

Well combs

Gel box

Microwave

Electrophoresis chamber

Voltage source

UV light source

Casting tray

Procedures:

• Mix 1g of agarose powder and 8μL of GelRed with 100 mL 1xTAE in a microwavable flask.
• Microwave for 1-3 min until the agarose is completely dissolved.
• Let agarose solution cool down to about 50 °C in about 5 mins.
• Pour the agarose into a gel tray with the well comb in place.
• Place the gel at room temperature for 20-30 mins, until it has completely solidified.
• Place the gel into the gel box of electrophoresis unit. Fill gel box with 1xTAE until the gel is covered.
• Carefully load a molecular weight ladder.
• Add loading buffer to each DNA sample. Carefully load samples into the additional wells of the gel.
• Run the gel at 120 V for approximately 90 minutes.
• Turn off power, disconnect the electrodes, and remove the gel from the gel box.
• Visualize DNA fragments using UV light source.

Protein Expression Analysis

Performed and Documented by Qinyi Wang and Zhiwen Ji

Cell Fractionation

Materials:

Bacteria Samples

0.1mM IPTG inductor

PBS buffer

0.1mM EDTA

10 μg/mL lysozyme

0.01mM MgCl2

2% Triton X-100

Equipment:

Microcentrifuge tubes

Ultracentrifuge tubes

Desktop centrifuge

Ultracentrifuge

Ultrasound sonicator

Ice bucket and beaker

Procedures:

• The bacteria were grown at 37°C for 12h after 0.1mM IPTG induction.

• Cells were collected by centrifuge of 6000 rpm for 10 min.

• After 2 washes with PBS buffer, cells were resuspended in PBS containing 0.1mM EDTA and 10 μg/mL lysozyme.

• After incubation of 2 hours on ice, the cell suspensions were treated with ultrasound sonication on ice (5 minutes, 1 cycle).

• Intact cells were separated from the lysates with a slow centrifuge of 6000 rpm for 10 min.

• At this point, the supernatant contained the whole cell lysates. We retrieved it with ultracentrifugation at 50,000 rpm for 1h to pellet the total membrane fraction.

• The supernatant containing soluble cytoplasmic and periplasm fractions was collected and stored at -4°C for later assays.

• The pellet of total membrane fraction was resuspended into PBS buffer containing 0.01 mM MgCl2 and 2% Triton X-100 for solubilizing the inner membrane, and incubated at room temperature for 30 minutes.

• The suspension was ultracentrifuged again at 50,000 rpm for 1h. The supernatant contained the inner membrane proteins, and the pellet contained the outer membrane proteins.

• All samples were collected and stored at -4°C for later assays.

SDS-PAGE & Coomassie

Materials:

1. 1X PBS

2. Protein loading buffer

3. Precast SDS-PAGE gel

4. SDS-PAGE running buffer

5. Prestained protein ladder

6. Distilled deionized (DDI) water

7. Coomassie Stain Solution:

   • Ethanol 75 mL

   • Glacial Acetic Acid 25 mL

   • DDI H2O 150 mL

   • Coomassie Brilliant Blue R-250 0.5 g

8. Destain Solution:

   • Ethanol 60 mL

   • Glacial Acetic Acid 20 mL

   • DDI H2O 120 mL

Equipment:

Heat block

SDS-PAGE gel tank & chamber

Power supply

Spatula

Platform shaker

Gel dryer

Procedures:

• Prepare the precast gel as follows:

  a)Remove the gel from the plastic packaging.

  b)Remove tape and plastic comb.

  c)Rinse the wells with DDI water 3 times, Shake the gel gently between washes to remove residual water.

  d)Load the gel into the chamber of the SDS-PAGE gel tank, fill the chamber with the 1X running buffer.

• Add 16 µL of loading buffer to 20 µL of each sample. The mixture was heated at 100°C for 5 minutes.

• Load 20 µL of each samples into the gel.

• Load 16 µL of the prestained protein ladder.

• Place the lid on the tank and plug the electrode cords into the power supply.

• Run the gel at 100 V for approximately 15 minutes, until the dye has migrated into the running gel.

• Increase the voltage to 150 V and continue for approximately 40 minutes, until the dye front has reached the bottom of the gel.

• Remove the run gel from the apparatus and remove the spacers and glass plates. Place the gel into a small tray.

• Rinse the gel 3 times for 5 minutes with 100 ml deionized water to remove SDS and buffer salts. Discard each rinse.

• Add 50 mL Coomassie Bright Blue R-250 staining solution and stain for 30 minutes with gentle shaking at 100 rpm. Discard the stain.

• Add 30 ml destain solution and destain for 1 minute with gentle shaking at 100 rpm.

• Pour off and discard the destain solution. Add 50 mL of destain solution.

• Destain with gentle shaking until the gel is visibly destained, for approximately 2.5 hours. Discard the destain solution.

• Add 50 ml DDI H2O and rinse for 5 minutes with gentle shaking at 100 rpm.

• Dry the gel on the gel dryer at 60°C for 1 hour with a sheet of filter paper below the gel.

Degradation Assay

Performed and Documented by Qinyi Wang and Zhiwen Ji

Materials:

MC-LR (This study used MC-LR synthesized by Taiwan Algal Science Inc.)

Bacteria from glycerol stock

Methanol and trifluoroacetic acid as the mobile phase of HPLC

PBS buffer

LB agar plate and media

Equipment:

Desktop microcentrifuge

Microcentrifuge tubes

Shaking incubator at 37 °C

HPLC vials

High-performance liquid chromatography machine

Mass spectrograph machine

Procedures:

• 1.Samples with MC-LR at concentrations 0.1 mg/L, 0.25 mg/L, 0.5 mg/L, 1 mg/L, 2.5 mg/L, 5 mg/L, 10 mg/L in PBS buffer were analyzed using HPLC to derive the standard curve.

  

• Recombinant strain of pET23b-inaK+mlrA was incubated overnight on a 120 rpm shaker at 37 °C.

• After cell growth, retrieved cell colony were centrifuged at 8000 rpm for 5 min and wash with PBS buffer twice. The cells were resuspended in PBS reaction system of 20mL containing MC-LR and cultivated on a 120 rpm shaker at 37 °C for 10 hours. The same culture medium without any bacteria was used as a control. Both the treatment group and the control group were triplicated.

• Samples were collected from the cultures every hour, and centrifuged at 10,000 rpm for 1 min to separate the bacteria from the medium.

• Supernatant from the sample were placed into HPLC vials for concentration assay. The concentration of cyclic MC-LR was measured by the area in HPLC graph and compared to the standard curve.

  

  FIG.5 Degradation Assay Results (error = standard deviation)

• Samples from the 10th hour were analyzed with mass spectrograph to determine the final product of linearized microcystin.

  

  FIG.6 Mass Spectrometry of Degradation Product

Co-Culture with microcystis aeruginosa

Performed and Documented by Qinyi Wang, Yunxian Ding, and Chenni Wang

Culture Setup

Materials:

Microcystis aeruginosa

Engineered E. coli

PBS buffer

Equipment:

Spectrophotometer

250 mL Erlenmeyer flask

Procedures:

• 1.Grow an overnight culture of bacteria and microcystis aeruginosa.

• 2.Add the two types of cells into an 250 mL Erlenmeyer flask. Adjust the culture so that all experimental groups had cyanobacteria density of 8.96±0.32×106 CFU/mL and E. coli density of 1.52±0.24×109 CFU/mL.

• Control group was cultured with only microcystis aeruginosa. Both the treatment group and the control group were triplicated.

• 3.The flasks were kept at 25 °C for 7 consecutive days.

Population of microcystis aeruginosa

Materials:

Co-culture of microcystis aeruginosa and engineered E. coli

Distilled water

Equipment:

Hemocytometer

Microscope

Procedures:

1. Gently swirl the flask to ensure the cells are evenly distributed.Before the cells have a chance to settle, take out 0.5 mL of cell suspension using a sterile pipette and place in an Eppendorf tube. Add distilled water to total volume 2.5 mL.

2. Pipette 100 µL of diluted cell suspension and apply to the hemocytometer.

3. Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective. Count the live cells in one set of 16 squares. If necessary, dilute again until all cells are distinct from each other.

   Note: although the E. coli and cyanobacteria were not separated, only cyanobacteria are identifiable under optical microscope precision. Therefore, the counting is not interfered.

   

   FIG.7 microcystis aeruginosa Population Density (error = standard deviation)

RNA extraction and reverse transcription

Materials:

Co-culture of microcystis aeruginosa and engineered E. coli

PBS buffer

TRIzol

Chloroform

Isopropanol

Ethanol

RNAse-free water

Reverse Transcription Buffer

MgCl2

dNTP mix

DTT

RNase Inhibitor

MultiScribe Reverse Transcriptase

DEPT-treated water

RNA-specific primer

Equipment:

Desktop microcentrifuge

Desktop vortexer

Ice bucket

RNAse-free tube

Thermal cycler

Procedures:

1. 1 mL of cell culture was retrieved and centrifuged at 8000 rpm for 10 minutes.

2. The pellet was resuspended in PBS and TRIzol to be lysed, and sat at room temperature for 5 minutes.

3. Extract RNA from the samples: add 0.2 mL of Chloroform per 1 mL of TRIzol used. Shake by hand for 10 seconds.

4. Incubate the samples for 3 minutes on ice and centrifuge for 15 minutes at 10,000 rpm at 4°C to separate RNA from the rest of the cell lysate.

5. Transfer the aqueous layer to a new RNAse-free tube. Add 1 volume of Isopropanol to the extracted aqueous layer. Incubate at -20°C for 2 hours.

6. Centrifuge for 20 minutes at 10,000 rpm at 4°C and discard the supernatant.

7. Wash the pelleted RNA by resuspending the pellet in 0.5 ml of 75% ethanol and vortex for a few seconds. Centrifuge for 5 minutes at 10,000 rpm at 4 °C.

8. Remove the ethanol wash and air-dry the pellet for 5 minutes.

9. Resuspend RNA pellet in RNase-free water.

10. Prepare reverse transcription master mix: (listed are final concentrations)

    • 1X Reverse Transcription Buffer

    • 1.75 mM MgCl2

    • 0.5mM (each) dNTP mix

    • 5.0 mM DTT

    • 1.0 U/µL RNase Inhibitor

    • MultiScribe Reverse Transcriptase 2.5 U/µL

    • DEPT-treated water to total volume

11. 11.Add 1 µL of template RNA and 1 µL of 10 µM primer into 20 µL reaction system containing the above master mix.

12. 12.Incubate at:

    • 65 °C for 5 minutes

    • 4 °C for 2 minutes

    • 37 °C for 30 minutes

    • 95 °C for 5 minutes

    • 4 °C until retrieved.

Quantitative PCR

Materials:

Reverse transcribed cDNA

SyBr Green PCR Mix

qPCR primers

Distilled water

Equipment:

Desktop microcentrifuge

Desktop vortexer

qPCR machine

96 well plate

Procedures:

1. Prepare reaction mix for each well as follows:

   • 2 µL 1:10 diluted cDNA (or water, for control groups)

   • 5 µL 2x SyBr Green PCR Mix

   • 0.05 µL 10uM forward primer

   • 0.05 µL 10uM reverse primer

   • 2.9 µL water

2. Run qPCR at the following temperatures:

   • 50°C,2 minutes

   • 95 °C,10 minutes

   • 95°C, 15 seconds; 60°C,1 minute for 40 cycles.

Note: although the E. coli and cyanobacteria were not separated, only genes unique to microcystis aeruginosa were examined in qPCR. Moreover, the 16S gene not present in E. coli was used as reference gene, so results were not interfered.