Team:Hong Kong-CUHK/Laboratory/protocol

Protocols Contents

1. Cloning

1.1 PCR Amplification of the gene

1.2 Double Digestion

1.3 PCR Purification

1.4 Agarose Gel electrophoresis

1.5 Ligation

1.6 Gel Extraction

1.7 Bacterial Transformation

1.8 Inoculation

1.9 Extraction of Plasmid DNA

2. Electrodesfunctional test

3. Bacteria light-couple electricity generation

4. Measuring voltage vs. sodium chloride concentrationrelation of mixing entropy electrode

5. Cell growth optimization

5.1 Preparation of growth medium

5.2 Inoculation

5.3 Salt concentration test

6. Measurement of chloride concentration in bacterial lysate using fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinoliniumbromide (MQAE)

7. Excitation of bacteria carrying halorhopsin

8. Measurment on controlled bacterial cell movement

9. Laser excitation protocol

9.1 General, wavelength

9.2 Laser power

9.3 excitation length

 

 

Cloning

PCR Amplification of the gene

 

- Reagents

1) PCR SuperMix High Fidelity

22 U/ml DNA polymerase mixture in:

- 66 mM Tris-SO₄ (pH 9.1 at 25°C)

- 19.8 mM (NH₄)₂SO₄

- 2.2 mM MgSO₄

- 220 μM dGTP

- 220 μM dATP

- 220 μM dTTP

- 220 μM dCTP

- stabilizers

2) Forward and Reverse Primer

3) DNA template

 

- Protocols

PCR Assembly from Reaction Components:

1) Set up reaction tubes on ice.

2) Add the following components in any order to each reaction tube:

a) 45 μl PCR SuperMix High Fidelity

b) Primer solution (200 nM final concentration of each is recommended)

c) Template DNA solution (Total volume of primer and template solutions added can be 0.5-5 μl)

3. Mix contents of tubes and cover with mineral or silicone oil. Depending on the model of thermal cycler used, mineral or silicone oil may notbe necessary.

4) Cap tubes and load thermal cycler at 94°C.

5) Start cycling program file

 

*Thefollowing general procedures are suggested as guidelines for use of PCRSuperMix High Fidelity. Assembly of the reactions on ice from pre-chilledcomponents is recommended. Final reaction volumes described below is areapproximately 50 μl. Reaction size may be altered to suit user preferences.

 

**Recommended Cycling Program

1 cycle of:  94°C for 30 seconds (Pre-amplification denaturation)

30–35 cycles of:

 94°C for 30 seconds (Denaturation)

 55–68°C for 30 seconds (Annealing)

 68–72°C for 60 seconds per kb target length(Extension)

 Use of 68°C during extension step of PCR is strongly recommended for fragments > 5 kb in length

 

Double Digestion

 

- Reaction mixtures

Total volume 50μl

37.6μl Insert/Vector + ddH₂O_*

5μl 10X NEB Buffer3

5μl 10X BSA

1.2μl Enzyme 1

1.2μl Enzyme 2

_*Water is addedfirst and the template the last

 At least 200ng DNA should be added

 

- Protocol

Reaction mixture at 37^oc for 2 hours

Buffer Chart

or http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp

 

PCR Purification

 

- *Protocol

1) Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not        necessary to remove mineral oil or kerosene.

2) If pH indicator I has been added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate (pH 5.0), and mix. The color of the mixture will turn to yellow.

3)Place a QIAquick spin column in a provided 2 ml collection tube.

4) To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60s.

5)Discard flow-through. Place the QIAquick column back into the same tube.

6) To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.

7)Discard flow-through and place the QIAquick column back in the same tube. Centrifugethe column for an additional 1 min. Protocol

8)Place QIAquick column in a clean 1.5 ml microcentrifuge tube.

9) To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl elution buffer to thecenter of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.

10) If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to

5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick

membrane for complete elution of bound DNA. The average eluate volume is 48 µl

from 50 µl elution buffer volume, and 28 µl from 30 µl elution buffer.

*Protocol adopted from QIAquick PCR purification kit protocol

 

 

 

Agarose Gel electrophoresis

 

- Set gel

1) Dissolve 0.55g Agarose into 55ml 0.5X TBE buffer.

2) Microwave (high power, 800W) for 1 minute.

3) Cool it down using running water for 1 minute.

4) Add 1μl GelRed

5)Pour the solution to tightened tank with gates and gel comb and allow it to solidify.

6)Transfer the gel to gel tank once it is set hard.

 

-Run gel

1)Orient the gel with wells facing the black negative electrode. Check if the gel is covered by TBE buffer in the tank. If not, add TBE buffer to cover it to about 1mm.

2) Mix loading dye and the insert/plasmid before adding to the wells. For example, if the DNA we have got is 45μl, and the loading dye we have got is 10X, then add 5μl of loading dye to the samples. Mixture should be in blue.

3)To run the gel, add all samples to the wells of gel. Then add 1kb DNA ladder to a separate well. 1μl should be enough for detection under UV.

4)Connect the electrodes to the power supply with correct colour. Set the power supply to 120V. Check if there are bubbles on the negative electrodes.

5) Allow it to run for about 40 - 60 mins. To avoid running the band off the gel, the yellow band (position of the smallest fragments) should stay on the gel.

 

*Gel Extraction

 

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.

2. Weigh the gel slice in a 2 ml centrifuge tube. Add 3 volumes of Buffer QG to

1 volume of gel (100 mg ~ 100 μl).

3. Incubate at 50°C until the gel slice has completely dissolved (around 10  min). Mix by vortexing the tube every 2 mins during the incubation.

4. After the gel slice has dissolved completely, check that the color of the  mixture is yellow. If the color of the mixture is orange or violet, add 5-10 μl of 3 M sodium acetate (pH 5.0), and mix until it turns to yellow.

5. Add 1 gel volume of isopropanol to the sample and mix (for DNA fragments  <500 bp and >4 kb).

6. Place a QIAquick spin column in a provided 2 ml collection tube.

7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 30 s. (The maximum volume of the column reservoir is 800 μl. For sample volumes of more than 800 μl, simply load and spin again).

8. Discard flow-through and place QIAquick column back in the same  collection tube. Collection tubes are re-used to reduce plastic waste.

9. Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 30 s toremove all traces of agarose.

10. To wash, add 0.75 ml of Buffer PE to QIAquick column, stand for 2 mins and centrifuge for 30 s.

11. Discard the flow-through and centrifuge the QIAquick column for anadditional 30 s and air-dry for 2 mins (This step can ensure all ethanol is removed and the column is NOT over-dry)!

12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

 

13. To elute DNA, add 35 μl of 50 oC ddH2O to the center of the QIAquick

membrane, wait 2 mins and centrifuge the column for 2 mins at maximum speed.

*extracted from CUHK iGEM2010 protocol

 

Ligation

 

- Reaction mixture

Total volume 20μl

2μl 10X ligation buffer

1μl T4 DNA ligase

14μl Vector

3μl insert

 

- Protocol

Allow the reaction to be taken place at 16^oC overnight, or 22^oC for 1 hour.

 

Bacterial Transformation

 

- Transformation of E. coli (DH5α or BL21(DE)) with plasmid DNA

1) Thaw competent cell on ice.

2) Add 50 ~ 100 ng DNA to competent cell culture.

3) Put in ice for 30 min.

4) Heat shock at 42^oC for 1 minute.

5) Put in ice for 2 min.

6) Add 1 ml LB broth medium.

7) Incubate at 37^oC for 90 min with shaking (~ 250 rpm).

8) Spread plate (with suitableantibiotics):

9) Spin down the remaining cells and discard large amount supernatant (1ml), then resuspend cells.

10) Incubate at 37^oC overnight until colonies are obtained.

 

Inoculation

 

- Protocol

1) Pick colonies & culture in 2~ 3 ml LB broth with antibiotics.

2) Incubate at 37^oC for 12-16 hours with shaking at 250rpm.

 

Extraction of Plasmid DNA (Miniprep)

 

- Protocol

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a micro-

centrifuge tube.

2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times and incubate 2 min at room temperature. Do not vortex!

3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube

4–6 times.

4. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. A compact white pellet will form.

5. Apply the supernatants from step 4 to the QIAprep spin column

6. Centrifuge for 30–60 s. Discard the flow-through.

7. Recommended: wash the QIAprep spin column by adding 0.5 ml Buffer PB and

centrifuging for 30–60 s. Discard the flow-through.

8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for

30–60 s.

9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual

wash buffer.

10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.

add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep

spin column, let stand for 1 min, and centrifuge for 1 min to elute the DNA.

Electrodes functional test

 

- Protocol

Put electrodes to 100 ml double distilled water, measure the voltage by multimeter between two electrodes until the reading becomes steady. Then, put electrodes to 100 ml, 0.4 M sodium chloride solution. Parallel connect a voltmeter and an ammeter (both are multimeter actually) between the two electrodes to measure voltage and current. Take the readings every minute.

When the voltage reading achieve steady, replace sodium chloride solution with double distilled water. While keep taking readings every minute. Put the electrodes in fresh water again. Keep taking the readings every minute until the voltage achieve steady.Plot voltage vs. time and current vs. time. Calculate the power for every time point. Estimate the total electric work.

 

Bacterialight-couple electricity generation

 

- Protocol

The experiment was conducted within a Class 2 biosafety cabinet and under sterile condition. Before each experiment the electrodes (4 ply carbon cloth, 3x4 cm each) were rinsed twice by immersing in tap water for 1-2 minutes, then wrapped with aluminium foil and autoclaved at 121⁰C for 25 minutes. Other instruments such as multimeter were wiped thoroughly with 70% ethanol before the experiment starts. The electrodes were fixed at the wall of the beaker with maximum possible separation (~3 cm) and connected to a RIGOL DM3051 digital multimeter with unshielded cable. The background open-circuit potential was about -0.1 to +0.2 mV.

25 mL of e. coli bacterial suspension (see below) were poured into the 100mL beaker. We define the moment where pouring was completed as t=0. Potential reading was then taken every minute afterwards using automatic data logging function of the multimeter. The experiment was done at room temperature.

To minimize the impact of external lighting on the experiments, most lights in the laboratory was turned off and the front glass of the biosafety cabinet was completely blocked by a polystyrene board after the bacterial suspension has been added. This is what we define as “dark condition” (<0.5 lux). The “light condition” was achieved by illuminating the beaker with a white LED (appox. 4160 lux at the beaker). The LED was a few centimeters above and aside from the beaker.

 

Measuring voltage vs. sodium chloride concentration relation of mixing entropy electrode

 

- Protocol

To start with, 0.6M stock sodium chloride solution was prepared by dissolving 5.310g of sodium chloride (Sigma Aldrich, Lot no: 090M01531V) in a small beaker and subsequently transferred into a 100 mL volumetric flask and made up to the graduation mark using deionized water. Other concentrations of sodium chloride solutiosn were prepared by mixing proper proportions of stock solution and deionized water in a 100 mL measuring cylinder.

The sodium manganese electrode and the inert electrode, with the dimension of 3x4 cm each, were immersed in a random sequence into different concentrations of salt water, which was contained within a 100 mL beaker. The solution used each time was approximately 25 mL. Less than 1 cm of each electrode was immersed in the solution.

Three potential readings were taken after the potential reading became steady with a RIGOL DM3051 digital multimeter. This procedure is duplicated with another batch of stock solution to give 6 replicates at each concentration. It could take 30 minutes to a few hours for the potential to become steady, depending on the previous salinity that the electrode was exposed to. Sometimes the tendency for electrodes to repel or attract to each other can be observed. Thereby the electrodes must be fixed carefully to avoid undesirable short-circuit.

Final concentration (M)	Total volume (ml)	Weight of total sodium chloride (g)	Weight of added sodium chloride (g)
0.10	100	0.58	0.29
0.15	100	0.88	0.29
0.20	100	1.17	0.29
0.25	100	1.46	0.29
0.30	100	1.75	0.29
0.35	100	2.05	0.29
0.40	100	2.34	0.29
0.45	100	2.63	0.29
0.50	100	2.92	0.29
0.55	100	3.21	0.29
0.60	100	3.51	0.29

 

4) Put the electrode in double distilled water until the reading become steady to wash away sodium chloride. Repeat thisstep for 2 times. Then take the reading for concentration 0 M.

5) Repeat the whole process for two moretimes.

 

Cell growth optimization

Preparation of growth medium (500ml LB (Luria Broth), 500ml 1M NaCl LB and 500ml 1M KCl LB)

 

- Protocol

1) Weigh 12.5g LB powder and add the powder into a 500ml bottle.

2) Add 500ml distilled water to the bottle.

3) Autoclave the solution.

4) 29.22g NaCl and 37.25g KCl is added respectively to prepare 500ml 1M NaCl LB and 500ml 1M KCl LB.

 

Inoculation

 

-Protocol

1) Add 5ml LB solution to a falcon.

2) Add 5ul antibiotic K.

3) Add 2.5ul IPTG.

4) Pick a single colony from plate spread with DH5α transformed with pET27b HR and add it into the falcon.

5) Put the start culture inside the incubator at 37℃ and shaking 200rpm.

6) Collect thestart culture the other day.

 

Salt concentration test

 

- Protocol

 

A. Preparation of 6 sets of solution:

0.1-1M NaCl with IPTG

0.1M-1M NaCl without IPTG

0.1M-1M KCl with IPTG

0.1M-1M KCl without IPTG

0M salt with IPTG

0M salt without IPTG

1) Prepare the medium as shown:

[Salt]/M	0	0.1	0.2	0.3	0.4	0.5	0.6	0.7	0.8	0.9	1.0
LB +1M salt/ml	0	0.4	0.8	1.2	1.6	2.0	2.4	2.8	3.2	3.6	4
LB/ml	4.0	3.6	3.2	2.8	2.4	2.0	1.6	1.2	0.8	0.4	0

 

2) Add 2ul IPTG (only applicable to those with IPTG, otherwise go to step 3).

3) Add 40ul of DH5α transformed with pET27b HR start culture into each tube.

4) Use a test tube rack to hold all the tubes and put the rack inside the incubator at 37℃. and shaking 200 rpm(?).

 

 

B.Measurement of absorbance

1) Set the wavelength of spectrophotometer to 600nm.

2) Set blank with 1ml LB solution.

3) Pipette up and down to mix the solution by P1000 pipette.

4) Transfer 1ml solution to a cuvette.

5) Record the OD600.

 

C. Measurement of bacterial growth curves

1) Inoculate the cells overnight.

2) Autoclave conical flasks with 100 ml LB solution.

3) Measure the OD600 and calculate the relative amount of bacteria needed and add into the conical flasks (usually 1% inoculums).

4) Measure the OD600 after adding the bacteria intothe conical flasks. This is time 0.

Measure the OD600 every half hour for eight hours.

 

Measurementof chloride concentration in bacterial lysate using fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinoliniumbromide (MQAE)

 

- Protocol

1) Culture the cells overnight.

2) Centrifuge for 1 min in maximum speed andcollect the cell pellet.

3) Lyse the cells by mixing well with 2% SDS and boil at 99℃ for 5 min.

4) Centrifuge for 1 min in maximum speed and obtain supernatant .

5) Dilute the supernatant to desired concentrations.

6) Add 95ul of each sample to 96well-microplate.

7) Add 5ul 0.5mM MQAE dissolved in DMSO afterwards.

8) Incubate for 1 hour at 37℃.

9)Measure the fluorescence by microplate reader using 350 nm absorption wavelength and 460 nm emission wavelength

Later found that the halorhodopsin’s colour affect the measurement ofthe MQAE fluorescence, need to change to centrifuge for 1 h in maximum speed at 4℃ and obtain supernatant after boiling with SDS to separate the halorhodopsin from the cell lysate.

 

Excitation of bacteria to trigger the halorhopsin

 

-Protocol

1) Use aluminum foil to wrap start cultures.

2) Excite the bacteria using either 405, 530, 670 nm lasers with the confocal microscope with live cell imaging for 2 min.

3) Adjust to the target laser power.

 

Controlled bacterial cell movement measurement

 

- Materials and Reagents

Protein A magnetic beads (New EnglandBioLabs)

Anti- E. coli antibody (Bioon)

Antibody coating buffer solution - Protein A magnetic bead binding buffer (New England BioLabs)

LB medium (USB)

Curvette (Sarstedt)

Spectrophotometer

40X bright-field microscope

 

- Protocol

1)Wash the Protein A magnetic bead with antibody coating buffer 3 times.

2)Resuspend the Protein A magnetic bead in antibody coating buffer.

3) Add anti- E. coli antibody and incubate at 4C overnight with gently shaking.

4)Wash away the excess antibody with with antibody coating buffer 3 times.

5)Addition of the E. coli cell to resuspend the magnetic bead and incubate for 2 hour room temperature with gently shaking.

6) The magnetic beads were transferred to a new tube and rinsed with fresh cell medium

7) The cells are attached with magnetic particles and subjected to movement by magnetic field.

8) The number of cells captured by fixed antibody-tagged magnetic particles are monitored by disrupting the antibody through heat or chemical and detected spectrophotometry or microscopy.

9) The bacterial cell tagged on magnetic particles, which can be observed with bare eyes with a dark brown colour, was attracted by a fixed magnetic field and the movement rate is calculated by distance over time until all brown colour particles bind on the wall

of the container.

 

Laser excitation protocol

General,wavelength

 

-Protocol

1)Grow transformed bacteria BL121(DE)in LB with 0.4 M of NaCl in the presence of antibiotic at 37°C.

2)Collect 500 μl of bacterial sample with OD600 reaches 0.4.

3)Transfer the bacterial sample to cell chamber.

4)Excite bacterial sample by laser confocal scanning microscope (FV1000D, Nikon).

5)Three wavelengths are chose: 405nm, 530 nm and 670 nm.

6)Length for excitation is 2 minutes.

7)Collect 200 μl of excited bacterial sample.

8)Spin sown bacterial sample.

9)Remove supernatant.

10)Lysis bacterial in 90 μl of 2% SDS.

11)Boil at 99°C for 10 minutes.

12)Determine the intracellular Cl^- concentration by MQAE.

 

Laser power

 

-Protocol

1) Grow transformed bacteria BL121(DE) in LB with 0.4 M of NaCl in the presence of antibiotic at 37°C.

2) Collect 500 μl of bacterial sample with OD600 reaches 0.4.

3) Transfer the bacterial sample to cell chamber.

4) Excite bacterial sample by laser confocal scanning microscope (FV1000D, Nikon).

5) Wavelength chose 530 nm.

6) Length for excitation is 2minutes.

7) Power of laser: 0-65%; 5% as interval.

8) Collect 200 μl of excited bacterial sample.

9) Spin sown bacterial sample.

10) Remove supernatant.

11) Lysis bacterial in 90 μl of 2%SDS.

12) Boil at 99°C for 10 minutes.

13)Determine the intracellular Cl^- concentration by MQAE.

 

Excitation length

 

- Protocol

1) Grow transformed bacteria BL121(DE) in LB with 0.4 M of NaCl inthe presence of antibiotic at 37°C.

2) Collect 500 μl of bacterial sample with OD600 reaches 0.4.

3) Transfer the bacterial sample tocell chamber.

4) Excite bacterial sample by laser confocal scanning microscope (FV1000D, Nikon).

5) Wavelength chose 530 nm.

6) Length for excitation: 0-600s; 30s as interval.

7) Power of laser: 25%.

8) Collect 200 μl of excited bacterial sample.

9) Spin sown bacterial sample.

10) Remove supernatant.

11) Lysis bacterial in 90 μl of 2%SDS.

12) Boil at 99°C for 10 minutes.

13) Determinethe intracellular Cl^- concentration by MQAE.