Team:Shanghaitech/Protocol

Protocols

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 primer10uM

       2.5 uL reverse primer10uM

       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 KitDP208-02

 

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 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 fragmentand 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

  1. Linearize two target PCR fragments and vectors by PCR.
  2. 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 OD 600= 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. Verify the relationship with expression of GFP and OD600

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 × 10–4 M 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 OD600 and 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 (bateria1bacteria2)

­-­

1:5

1:2

1:1

2:1

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

8:00

1 to 100 inoculation converter to 36ml liquid LB,

10:00

Split converter into 7 tubes of 4ml and 4ul of 3 gradients (10-1 to 10-6M) of signal molecular and one blank were added to induce converter.

14:00

Inoculated lasR+GFP to 36ml liquid LB at 1 to 100

17:00

Take 1 ml bacteria of rpaR+lasI, centrifuge at 7000 rpm for 5 minutes, let the supernatant go through the 0.2 filter membrane. The left liquid of bacteria was put back to be cultured.

Split lasR+gfp to 15 tubes with 2 ml of each one. Take 7 of them and add 1ml supernatant, the other 6 tubes of bacteria were added corresponding signal molecules. Add 2ul 10-7M las-AHL to the 14th tube and leave the last one as a blank sample.

24:00

Go on the microscope, using Microplate Reader to measure the related fluorescent.

Day4

8:00

Take the overnight bacteria of lasR+GFP and inoculate lasR+GFP to 36ml liquid LB at 1 to 100

10:00

Split lasR+GFP to 15 tubes with 2 ml of each one. Take 7 of them and add 1ml supernatant, the other 6 tubes of bacteria were added corresponding signal molecules. Add 2ul 10-7M las-AHL to the 14th tube and leave the last one as a blank sample.

Take 1 ml bacteria containging rpaR+lasI, centrifuge at 7000 rpm for 5 minutes, let the supernatant go through the 0.2 filter membrane. Add the supernatant to the bacteria containing lasr+GFP.

Take 1 ml bacteria containing rpaR+lasI, centrifuge at 7000 rpm for 5 minutes, let the supernatant go through the 0.2 filter membrane. Deliver the sample for HPLC-MS.

15:00

Go on the microscope, using Microplate Reader to measure the related fluorescent.

According to the requirements, the time gradient and shaking hours can be replaced.

 

X. Test the special tube designed for the liquid handling robot

Tube construction:

Heat the 15ml centrifuge tube with outer flame for 5s, then cut the tube at 0.6ml. The first layer of glue was applied to the cut flat cross section. Coat the first circle of glue in the cut section, so that the tube close contact with the membrane, so that the glue filled the gap.

The glue gun is aimed at the angle between the tube and the filter paper so that the glue fills the gap between the gun and the corner, smearing a circle of glue.

Put the filter paper up, in the edge of the filter paper, smear a circle of glue to ensure that the glue wrapped around the side of the filter paper, wrapped part of the overall integration with the previous one.

       Unscrew the lid of the 15 ml centrifuge tube and fill it with glue at the gap of the tube and the lid of the 50 ml centrifuge tube, taking care not to be higher than the thread.

 A set of special hardware designed by ourselves was constructed for conducting the experiments automatically. The method of constructing the hardware is shown in the following link:http://2017.igem.org/Team:Shanghaitech/Hardware

To test the tube:

1. Add 5ml LB and 5ul bacteria of lasi to Ai inner layer, and add 5ml LB to outer layer. Add 5ml LB to Bi inner layer, and add LB to 30ml scale line and 5ul mixed bacteria lasi to outer layer. And incubated overnight at 220 rpm in a shaker at 37 ° C

2. Take all liquid in Ai outer layer, and add it to AR outer layer. Add 5mlLB and 5ul mixed bacteria of lasR to inner layer.

     3. Take all liquid of Bi inner layer, and add it to BR inner layer. Add LB to scale 8 and 5ul mixed bacteria of lasR to BR outer layer

 

 

XI. Fluorescence measurement over time induced by Signal Molecules

Day 0

1. Preparation of autoinducer working solution with different concentration gradient

Dilute the autoinducers to 8-well PCR tubes such that they will have the desired concentration when added to the well plate.

Final Concentration for every column of wells:

10-7  10-8  10-9  10-10  10-11  10-12  0(NC)  0(M9 Medium)

Since that every well is set to contain 200μL samples, working solution concentration is:

2*10-5   2*10-6  2*10-7  2*10-8  2*10-9  2*10-10   0(NC)  0(M9 Medium)

Note:

• For every Homoserine Lactate autoinducer, it’s recommended to prepare concentration gradient

Working solution needed for all further experiments at the same time to ensure the repeatability.

• The gradient working solution could be stored at 4°C

• Most Homoserine Lactate autoinducers have a non-polar tail, we choose DMSO as solvent.

• DO NOT use solvent with high volatility such as Chloroform. Using such solvents may cause a greater variation in concentration.

Day 1

2. The night before testing, grow bacteria and blank controls overnight for all samples in M9 Media.

The above step takes about 15 minutes.

Day 2

3. Dilute all the cultures to an optical density of 0.05 (OD600).

4. Grow for approximately 3-4 hours. We monitored the Optical Density to make sure it did not grow above 0.5.

Steps above take about 15 minutes plus a ~3 hours waiting time.

5. When the OD of the samples reached 0.5, distribute samples into a 96 well plate, 200μL per well. Every concentration level occupied a row which gave us 12 parallel repeats.

6. Add pre-diluted Autoinducer concentration gradient working solution to all 12 columns.

7. Put the plate in a Synergy fluorescence plate reader and take regular measurements of OD and fluorescence.

For GFP use excitation frequency of 485nm and an emission frequency of 528nm

Steps above take about 15 minutes plus a ~10 hours waiting time.

8. Collect data from Synergy fluorescence plate reader. If needed, recycle the 96 well plate and get it ready for next measurement.

 

XII. Fluorescence measurement with Microplate Reader

    Please pay attention that each time the sample volume should be the same (150μl per hole recommended)

Program settings:

1. Boot, select the microplate reader control software.

2. Click "Plate" and select setting

3. Select following the order listed below:

        1. Monochromator, spectrometer mode.

        2. FL, Fluorescent mode

        3. Endpoint / Kinetic (depending on your needs)

1.     Endpoint mode:

Choose the proper wavelength.

The type of the boards, the default is that we use the opaque, select lidded depending on whether you are cap the boards.

Read the area, select the areas you want to read.

PMT & Optics don’t need any adjustment.

You’d better use the linear mode when shaking, and it is recommended to shake 5 seconds currently before reading.

2.     Kinetics mode:

Choose the proper wavelength.

The type of the boards, the default is that we use the opaque, select lidded depending on whether you are cap the boards.

Read the area, select the areas you want to read.

Select the total reading time and the two reading interval, which the computer will automatically calculate the total number of later.

PMT & Optics without adjustment

    You’d better use the linear mode when shaking, and it is recommended to shake 5 seconds currently before reading.

 

XIII. Test the function of convertor with the liquid handling robot

Day 0

1. Preparation of autoinducer (Rpa signal molecules) solution at different concentration.

Dilute the autoinducers in an 8-well PCR strip with DMSO to make the final concentration in each tube (mol•L-1):

10-7  10-8  10-9  10-10  0 (DMSO & LB only)

Since that every well is set to contain 50ml samples, working solution concentration in 1ml LB medium is:

10-4  10-5  10-6  10-7  0 (DMSO & LB only),

Note:

• For every Homoserine Lactate autoinducer, it’s recommended to prepare all working solution at the same time to ensure the reproducibility of the experiments.

• The concentration gradient working solution should be stored at 4°C.

• Most Homoserine Lactate autoinducers have a non-polar tail, we choose DMSO as the solvent.

• DO NOT use solvent with high volatility such as Chloroform. Using such solvents will led to great variation in concentration.

Day 1

2. The night before testing, grow bacteria culture overnight for the signal receiver in M9 Media.

3. The night before testing, grow bacteria culture overnight for the signal convertor in LB media.

Day 2

4. Dilute all the signal convertor cultures to OD600 (Optical Density at 600 nm) of 0.05 with at least 50ml volume.

5. Let the bacteria grow for approximately 3-4 hours, monitoring OD600 to make sure it does not grow above 0.5. It usually takes about 3 hours for the bacteria to grow to the desired OD600 value.

6. When the OD600 of the samples reaches 0.5, distribute samples into 50ml centrifuge tubes with 10ml per tube.

7. Add 100 μL pre-diluted autoinducer concentration gradient working solution to all 5 tubes with robotic liquid handling system.

8. Set the sampling program of robotic liquid handling system. Every 2 hours after adding autoinducer until 8 hours stimulation, take 20μL sample from all 5 centrifuge tubes, 3 parallel samples at a time. The samples should be added to a 96-well plate from well A4 to well D12.

9. Dilute all the signal receiver cultures to OD600 of 0.05 with at least 20ml volume.

10. Let the bacteria to grow for approximately 3-4 hours, monitor OD600 to make sure it does not grow above 0.5.

Note:

• When adding autoinducer concentration gradient working solution to 50ml centrifuge tubes, the order should be adding the lower concentrated working solution first to minimize the interference of leftover solution.

11. When the OD600 of the signal receiver culture reaches 0.5, distribute samples into a 96-well plate used in step 8, 200μL per well.

12. Add pre-diluted autoinducer concentration gradient working solution to first three columns as the positive control to prove that the Las signal molecules exist in the liquid that induce LasR-GFP .

13. Put the plate in a multifunctional microplate reader and take regular measurements of OD600 and fluorescence.

For GFP use excitation frequency of 485nm and an emission frequency of 528nm