Revision as of 14:02, 30 October 2017

Protocol

I. PCR amplification of DNA fragment

Pipette the 50 uL reaction mixture into a PCR tube

25 uL Tiangen 2×Taq PCR MasterMix (with loading dye)（Tiangen Catalog # KT201-03）

1 uL plasmid template

2.5 uL forward primer（10uM）

2.5 uL reverse primer（10uM）

19 uL ddH2O

PCR cycle

94℃ 3 min

30 cycles

94℃ 30 sec

55℃ 30 sec

72℃ 1 min

72℃ 5 min

Products are purified using 1% agarose gel and TIANgel Mini DNA Purification Kit（DP208-02）

II. DNA digestion

1. Pipette the following volume into a PCR tube

For PCR product digestion (20uL)

16 uL DNA (PCR product)

2 uL 10×H buffer

1 uL Takara EcoRI

1 uL Takara XbaI

For plasmid digestion (20uL)

16 uL DNA (part plasmid)

2 uL 10×M buffer

1 uL Takara EcoRI

1 uL Takara XbaI

2. Incubation

Incubate the tubes at 16℃ overnight (≥12h)

3. Purification

Fragment is purified using TIAN quick Midi Purification Kit (DP204-02), and the vector is purified using 1% agarose gel and TIAN gel Mini DNA Purification Kit.

Concentrations of digestion final products were measured by A260 with a Nanodrop machine.

III. DNA Ligation

(a) T4 ligase based ligation

1. Pipet the following system into a PCR tube

10 uL reaction system (for DNA concentration >10 ng/uL)

8 uL total DNA (vector and fragment with molar ratio around 1:5)

1 uL 10×ligation buffer

1 uL Takara T4 DNA Ligase

50 uL reaction system (for DNA concentration <10ng/uL)

44 uL total DNA (molar ratio = 1:5)

5 uL 10×ligation buffer

1uL Takara T4 DNA Ligase

2. Incubation

Incubate the tubes at 16℃ overnight (≥12h)

3. Transformation

Add 1uL of the mixture directly to 20ul competent DH5α cell for transformation. Standard transformation protocol (please give a paper reference here) was used.

(b) Molecular cloning with Gibson kit

1. Design primers to amplify fragments (and/or vector) with appropriate overlaps.

2. PCR amplify fragments using a high-fidelity DNA polymerase.

3. Prepare linearized vector by PCR amplification using a high-fidelity DNA polymerase or by restriction digestion.

4. Confirm and determine concentration of fragments using agarose gel electrophoresis, a Nanodrop™ instrMent or other method.

5. Add DNAs to Gibson Assembly Master Mix and incubate at 50°C for 15 minutes to 1 hour, depending on number of fragments being assembled.

6. Transform into E. coli or use directly in other applications.

(c) Molecular cloning by Infusion homologous recombination

1. Construction of linearized vector:

linearize 1 uL vector by PCR. Digest the PCR product with DpnI at 37 ° C for 5 hours to digest the template plasmid.

2. Insertion of the fragment of the linear:

Take 1 uL plasmid with the desired fragment，and linearize the fragment of interest by PCR.

3. Carrier and insert fragment purification:

Amplify the linearized fragments and vectors after PCR system by agarose gel electrophoresis, and the electrophoresis system was 5uL. If the bands are clear and bright, purify the product using DNA extraction purify kit. If not, purify the remaining PCR products from agarose gel.

4. Infusion homology reorganization:

Take 0.03 pMol vector and insert fragments , (simple calculation method: base number x0.02 (unit: ng);

If the vector or insert is not purified, the total volume of the carrier and the fragment is not more than 4uL; if the calculated volume is less than 0.5uL, 0.5uL is taken;

Add 4uL "4 x Infusion Mix", 2uL Infusion recombinase, make the total volume to 20uL by adding 18MΩ double distilled water;

Put the system into the PCR instrument, react at 37 degrees for 30min. Place it on ice immediately for 5min, to prevent the subsequent transformation efficiency.

5. Take 1uL Infusion product for transformation in 50uL system directly.

(d) 3A assembly

Linearize two target PCR fragments and vectors by PCR.
Digest two parts and construction plasmid backbonedestination vector with the following enzymes

§ Left part with EcoRI and SpeI

§ Right part with XbaI and PstI

§ Construction plasmid backbone with EcoRI and PstI. Also digest the construction plasmid backbone with DpnI if possible to eliminate any plasmid remaining from the PCR.

3. Combine 1 ul of each restriction digest reaction with 1 ul of ligase in a 25 ul reaction.

4. Transform the ligation product.

IV. Point mutagenesis

Introduce a mutation point in the PCR primers, and proceed according to the PCR protocol above

V. HPLC sample preparation

To test the function of generator

Samples were prepared as the following: Inoculate bacteria transformed with QS generator plasmid into liquid LB at a ratio of 1: 100. Put the bacteria into the rocking device with 220rpm and 37℃ (t=0h). After 2 hours, 4 hours, 6 hours and 8 hours, take 1ml of bacteria in a small tube, centrifuge at 7000rpm for 5min, filter the supernate with 0.22mm bacteria filter membrane, put the filtered liquid in a tube at 4℃. Add another 1ml liquid LB to a tube, and add 1ul small molecules of the concentration of 10^-2M, as a positive control. For negative control, 1ml LB with chlorampenicol without small molecules.

To Test the attenuation of signal molecules

Take overnight grown lasR-GFP bacteria, inoculated to 20ml LB with the ratio of 1: 100. Culture at 37 ℃ in shaker of 220rpm to OD₆₀₀= 0.1. Aliquot the bacteria to 8 tubes, marked as 1-7h and blank, each tube 2ml. Add 2ul 0.01M small molecules to the first 7 tubes, and put them back to the shaker. Then according to the time on the tube, remove a tube to collect 1ml supernatant every 1h. Collect the blank supernatant at t= 7 hours. Preparation of standard solution: add 1ul 0.1M small molecules to 1ml chloramphenicol LB.

VI. Measure the relationship of bacteria concentration and GFP expression

1. Take 1200ul bacteria solution, centrifuge at 5000rpm for five minutes, discard the supernatant, and then add 1000ul H20 and 200ul 60% glycerol. Mix completely.

2. Gradient preparation: mark the mixed bacteria from the previous step as 1, take 600ul 1 and add 600ul 10% glycerol, mix thoroughly and mark it as 1/2. Repeat this procedure until we got the marks of 1, 1/2, 1/4, 1/8, 1/16, and the 10% glycerol solution was labeled as zero. Add 150ul into the 96-well plate and read the data in the Multimode Reader. Save the data and the original result.

VII. Fluorescence measurement induced by gradient concentration of Signal Molecules

Bacteria cells were prepared for fluorescence plate reader experiments as follows: Strains of bacteria were grown overnight and then reseeded in a 1:100 dilution into fresh media containing corresponding antibiotics. The dilution was allowed to grow for about 2 h until it reached OD ₆₀₀ ≈ 0.1. Then, the cell culture were evenly distributed into the number of tubes and induced with a range of AHL (the signal molecules) concentrations (1:10 serial dilutions from 1 × 10^–4M to 1 × 10^–10 M or 1 × 10^–14 M). Choose different types of AHLs with different concentrations as shown in figures. AHLs stocks were dissolved in proper solvents. After induction, the cells were allowed to grow for about 7 h. We took out 5 ul samples to check the green or red fluorescence under a microscope. If the fluorescence can be seen, we centrifuged the remaining samples with 7000rpm for 5min and discarded the supernatant. We used 10% glycerol to resuspend the cells, and then add 150ul into the 96-well plate to measure for OD₆₀₀and GFP fluorescence with a Tecan infinite M200Pro. We used the following fixed settings: no top, fixed gain of 61, excitation of 485 nm, emission of 520 nm, and Z-position of 19500. All GFP measurements were normalized by dividing their raw values by the OD₆₀₀ of that well to give a “per-cell” measurement.

VIII. GFP induction by signal molecules produced by QS generator bacteria

GFP induction directly by QS generator bacteria

The overnight bacteria 1 and the overnight bacteria 2 were inoculated into 15 ml of liquid LB (with proper antibiotics). Put the bacteria into the rocking device with 220rpm and 37℃ for 2 hours and then measure OD₆₀₀. Take 6 tubes of 1ml bacteria 1, centrifuge at 7000rpm for 5min, discard the supernatant, add 1ml fresh LB to resuspend. Similarly, take 5ml, 2ml, 1ml, 0.5ml, 0.2ml bacteria 2, centrifuge at 7000rpm for 5min, discard the supernatant, add 1ml fresh LB to suspend. These bacteria are 5 *, 2 *, 1 *, 0.5 * and 0.2 *. Mix the bacteria 1 and bacteria 2 according to the following table:

Number	1	2	3	4	5	6	7
bacteria1(ml)	0	1	1	1	1	1	1
bateria2 (ml)	1ml1*	1ml5*	1ml2*	1ml1*	1ml0.5*	1ml0.2*	0
Concentration (bateria1：bacteria2)	-	1:5	1:2	1:1	2:	5:1	-

Put the mixed bacteria into the rocking device with 220rpm and 37℃. After observing a fluorescence under the microscope, the mixture was taken 1ml into a tube, centrifuged at 7000rpm for 5min. Take the supernatant and the filter it using membrane. The precipitated bacteria were resuspended with 400 ul of glycerol, and added to 96 empty plates for detection.

GFP induction with generator bacterial supernatant

Pick a single clone from the pcon-lasI transformation bacteria plate and inoculate into 3 ml medium. Culture overnight.

25 hours before induction, inoculate the pcons-lasI bacteria to 3 tubes of 3ml fresh medium at the ratio of 1:100, put the original bacteria back to the rocking device (labeled lasI-25H).

20 hours before induction, inoculate the pcons-lasI bacteria to 3 tubes of 3ml fresh medium at the ratio of 1:100, put the original bacteria back to the rocking device (labeled lasI-20H）。

Pick a single clone from the LasR-GFP transformation bacteria plate and inoculate into 3 ml medium. Culture overnight.

15 hours before induction, inoculate the pcons-lasI bacteria to 3 tubes of 3ml fresh medium at the ratio of 1:100, put the original bacteria back to the rocking device (labeled lasI-15H）。

10 hours before induction, inoculate the pcons-lasI bacteria to 3 tubes of 3ml fresh medium at the ratio of 1:100, put the original bacteria back to the rocking device (labeled lasI-10H）.

5 hours before induction, inoculate the pcons-lasI bacteria to 3 tubes of 3ml fresh medium at the ratio of 1:100, put the original bacteria back to the rocking device (labeled lasI-5H）.

……（Inoculate the pcons-lasI bacteria to 3 tubes of 3ml fresh medium at the ratio of 1:100 at any time required.）

2 hours before induction, inoculate the LasR-GFP bacteria to 30ml fresh medium at the ratio of 1:100. Culture the bacteria until OD₆₀₀>=0.1

Dispense the lasR-GFP bacteria into the number of tubes required and 1 ml per tube, centrifuge them at 7000rpm for 5min. Add1 ml of the supernatant with corresponding marker (lasR-lasI - the number of hours - parallel markers) of the LasR-GFP bacteria (to ensure that the bacteria in each tube is uniform, adjust the amount of bacteria according to the situation). The small molecule’s final concentration is 10 ^-10, 10^-9, 10^-8, 10 ^-7 , and to keep three tubes in parallel (labeled lasR-P-induced concentration)

After seven hours inducing: observe the bacteria under fluorescence microscopic. Add 150ul samples into the 96-well plate and read the data in the Multimode Reader. Save the data and the original result.

IX. Test the function of convertor

Day1

Transform the convertor plasmid.

Day2

Pick rpaR+lasI, lasR +GFP each for two from plate, to 6 tubes for 5ml liquid LB with chloramphenicol of each.

Day3