Experiments












QSMiD

Quorum Sensing system for Microplastics Detection

Content

Transformation

After receiving the gene synthesized by Synbio Technologies, 2μg of the powder in the vial was dissolved with 20μL of MilliQ water, forming a solution with a concentration of 100μg/μL. It is kept at -20°C. Bacterial tranformation was performed in order to produce multiple copies of DNA for experiments.

  • 1. Mix 1μL of the plasmid DNA with 200μL of E. coli DH5α cells.
  • 2. Put the mixture in ice for 30 seconds.
  • 3. Incubate at 42°C for 90 seconds.
  • 4. Put in the ice again for 1-2 minutes.
  • 5. Incubate at 37°C at 200rpm for 1 hour.
  • 6. Discharge until 200μl of liquid is left, and pipette up and down
  • 7. Spread plate

pET plasmid expressing LasR sensing module and pUC57 plasmid expressing pLasRL or pLasR3 were co-transformed into BL21C41(DE3)pLysS. In the transformation, 50 ng of each plasmid DN A was used in 1:1 modules ratio. In 2:1 and 3:1 modules ratio, 100 ng and 150 ng of plasmid expr essing sensing module were co-transformed with 50 ng of plasmid expressing reporting module res pectively. The resulting engineered biosensors were named as pET21b-LasR-pUC57-pLasRL-EGFP, pET21b-LasR-pUC57-pLasR3-EGFP, pET23b-LasR-pUC57-pLasRL-EGFP and pET23b-LasR-pUC57-pLasR3-EGFP.

Each plasmid contains an antibiotic-resistant gene on it, whereas the antibiotic resistant genes of the 2 modules are different. The 2 antibiotics of the corresponding antibiotic-resistant gene were applied, indicating that only bacteria with both plasmids present can survive. The antibiotics we used were carbenicillin in pET plasmids and kanamycin in pUC57 plasmids. The resulting biosensor cells were selected in LB medium with both 100mg/ml carbenicillin (C) and 50mg/ml kanamycin (K).



Bacterial DNA extraction (mini-prep)

  • 1. Centrifuge 1.5 mL of bacterial culture to form a pallet.
  • 2. Discard supernatants.
  • 3. Add 250 μL of the Resuspension Solution to resuspend the pellet. Vortex to ensure complete resuspension.
  • 4. Add 250 μL of Lysis Buffer to lyse cells. Invert the tube 4-6 times. Incubate for 2 mins (incubation time should not exceed 5mins to avoid denaturation of supercoiled plasmid DNA).
  • 5. Add 350 μL of Neutralization Buffer to neutralize the lysate. Invert the tube 4-6 times.
  • 6. Centrifuged for 5 mins to pellet cell debris and chromosomal DNA.
  • 7. Transfer supernatant to the GeneJET spin column by decanting or pipetting.
  • 8. Centrifuge for 1 minute. Discard the flow-through and place the column back into the same collection tube.
  • 9. Add 500 μL of Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Re-insert the column into the same collection tube.
  • 10.Repeat step 9
  • 11.Discard the flowthrough and centrifuge for an additional 1 min to remove the residual Wash Solution.
  • 12.Transfer the GeneJET spin column into a fresh 1.5mL microcentrifuge tube. Add 30 μL of Milli-Q water to the center of the GeneJET spin column membrane to elute the plasmid DNA. Incubate for 2 minutes at room temperature and centrifuge for 2 min.
  • 13.Discard the column and store the purified plasmid DNA at -20°C.



Restriction enzyme digestion:

If there is only 1 enzyme, replace enzyme 2 with H2O Incubate mix at 37°C for 30 min



PCR

  • 1. Before the replication cycle had begun, the DNA samples were heated at 95°C for 5 minutes, and then the cycle began.
  • 2. Reaction mixture was exposed to a high temperature of 95°C for 30 seconds to denature.
  • 3. The samples were annealed at a lower temperature of 55°C for 30 seconds.
  • 4. The temperature was then raised back up slightly to 72°C for 1 minute, completing one cycle of DNA duplication.
  • 5. The cycle was conducted 30 times.
  • 6. After the cycle, the DNA were incubated at 72°C for 5 minutes.
  • 7. The DNA are stored at 4°C.



Gel electrophoresis

1% Gel
  • 1. For each 100mL of gel, 1g of agarose powder is added to 100mL of 1XTBE buffer.
  • 2. The mixture is then microwaved until the powder dissolved completely.
  • 3. The mixture is then cooled down to room temperature under running water.
  • 4. 1µL of midori green dye is added to the mixture.
  • 5. The mixture is poured into the gel tank and a comb is inserted.
  • 6. After the gel solidifies, it is put into the gel tank which is filled with 1X TBE buffer.
  • 7. The comb is removed carefully to prevent the formation of air bubbles in the wells.
  • 8. DNA ladder and samples are loaded into the wells.
  • 9. A electric field is applied with anode at the side that the DNA samples run to.
Loading buffer is added to the DNA samples before loading. DNA samples should be put in icebath to prevent degradation of samples.

Preparation of N-acylhomoserine lactone (AHL) stock solutions

We have ordered 3 synthetic AHL molecules from commercial companies, named N-butyryl L-homoserine lactone (C4HSL), N-3-oxo-decanoyl L-homoserine lactone (C10HSL) and N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL). They were first dissolved to produce a stock solution of 1x10-2M, by using the following equation:



1x10-2M is the stock solution for AHL. We carry out serial dilution and make AHL concentrations of 1x10-3M, until 1x10-11M.

When incubate AHL in bacterial cultures, we add AHL to bacterial culture in the ratio of 1:99 AHL to bacteria. The final concentration will be 100 times lower than the concentration of the AHL solution added.

AHL induction

Starter culture

  1. For starter culture, a bacterial colony is picked from the bacterial plate and transferred into 1mL LKC medium.
  2. Shake at 37°C at 230 rpm overnight.

Bacterial subculture

  1. Carry out subculture by adding starter culture to LKC in the ratio of 1:999 starter culture to LKC in a 50 ml falcons. The final volume of the subculture is determined by the amount of culture we needed for the AHL induction experiment.
  2. The falcon is put to shake at 33°C at 210 rpm.
  3. It is allowed to grow for around 3 hours until the bacterial concentration reaches 0.3 OD600.
  4. The culture in the falcon is aliquoted to different 15ml culture tubes, with AHL induced and no AHL added as a negative control. This is when 0h starts.
  5. After collecting 300μL sample, the bacteria were put back into the shaker at 210 rpm.
  6. Collection of samples takes place every half an hour until 3 hours.
  7. At each time point, 1mL of culture is transferred to a cuvette for each culture tube, and bacterial concentration OD600 is measured using a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer. 50μL of the sample is collected to be observed under a fluorescence microscope.

Washing cells with PBS (phosphate buffer saline)

PBS is an isotonic solution often used to wash cells to remove impurities.

  1. Centrifuge at 14,000 rpm for 1 minute to form a pellet.
  2. Discard the supernatant and add 500µL of 1x PBS to each Eppendorf
  3. Vortex and centrifuge for 1 minute.
  4. Repeat steps 2 and 3.
  5. Add 200µL of 1x PBS to resuspend the cells, then pipette up and down to mix.

200uL of samples are added into a well on a 96 well plate, where the relative fluorescence unit (RFU) is measured by the microplate reader.

Modifications of protocol

Construct

We used different constructs to compare the use of different pET vectors 21b and 23b or the effect of different inducible promoters in pUC57-EGFP in the EGFP production of biosensors.

From interviews with professors, we acknowledged that in a situation where there is more AHL than LasR protein, the LasR protein formed may not be sufficient to form LasR-AHL complexes with AHL. LasR becomes a limiting factor. Thus, we tried to modify the constructs by using ratios of 2:1 and 3:1 (sensing module:reporting module) for constructs to test the effect of different ratios on the fluorescence signal exhibited by the constructs.

AHL concentration

We used various concentrations of AHL to investigate whether different AHL concentrations would lead to any correlation between fluorescence data. AHL concentrations we used ranged from 1x10-3M to 1x10-11M in cocktail.

Autofluorescence (primary fluorescence)

It is the fluorescence of naturally occurring substances. They can be detected under a fluorescence microscope and thus may affect our results.

We have changed dissolved AHL solutions with DMSO, originally carrying out serial dilution with DMSO to using MilliQ water. PBS is diluted without mixing precipitation calcium chloride when diluting. Both changes can reduce autofluorescence from other substances.

Shaking speed

We changed bacterial shaking speed from 210 rpm to 250 rpm to increase the rate of bacterial growth.

Addition of IPTG

We discovered that IPTG can activate pET vectors and promote translation. We added 7 μL IPTG per 3mL culture which results in concentration of 0.5 mM. Accordingly, we changed starting OD600 to 0.6 OD600 as it is the optimum value to best utilize IPTG.

OD value

We started our experiments at 0.3 OD600. We later discovered that OD 0.3 was not optimal for AHL induction as the rate of cell growth was too slow. OD 0.5 is farther along the exponential phase of growth. The bacterial exponential growth phase is towards 1 OD600 and thus 0.5 OD600 is more optimal than OD 0.3. We later introduced the addition of IPTG, thus changing starting OD600 from 0.5 to 0.6 which is the optimum OD600 for IPTG to work most efficiently.

Negative experiments

We designed four negative experiments to investigate the autofluorescence of substances throughout the experiment.

We measured the RFU of PBS, milliQ and DMSO of different concentrations, LKC medium and LCM medium. Such negative experiments show the fluctuations of autofluorescence in these substances so as to compare with fluorescence levels the construct produces. It shows the effect of DMSO in increasing autofluorescence and that it will affect fluorescence value measured in the construct . Results also show that LKC has high RFU and may affect the result during quantifying EGFP from the construct.

Fluorescein

We also used a fluorescence chemical named fluorescein. We obtained the optimal concentration by serial dilution and then measured its RFU under the microplate reader in order to calibrate it. Fluorescein can act as a positive control as it shows the most optimal situation where constant fluorescence is emitted.

  1. Get the stock reagent tube with Fluorescein calibrant from the Measurement Kit. It is in powder form. Spin down to make sure the pellet is at the bottom.
  2. Transfer 1.0 mL of Phosphate Buffered Saline (PBS) to stock reagent tube Fluorescein calibrant. Resuspend pipetting up and down a few times, and vortex for 30 seconds. This is now the reconstituted Stock Fluorescein Solution 10X with a concentration of 100 μM in PBS.
  3. Obtain a tube to make the working concentration solution 1X Fluorescein.
  4. Dilute the Stock Fluorescein Solution 10X with 1X PBS to make a 1X reference working solution with a concentration of 10 uM. E.g. dilute 100 μL of 10X fluorescein stock into 900 μL 1X PBS.

    5. Serial Dilution
  5. Prepare a 96 well microplate.
  6. Transfer 100.0 μL of PBS to wells A2:B12 of calibration plate. Column 12 has the blanks.
  7. Transfer 200.0 μL of Fluorescein 1X solution to wells A1 and B1 of calibration plate.
  8. Perform a series of 10 2-fold dilutions from the first column of the calibration plate using PBS or Water (already prefilled) as diluent to a final volume of 200.0uL in wells A1:B11 of the calibration plate. For each of the ten steps transfer 100uL from the initial column into the next one and pipette up and down 3X to ensure the dilution is mixed homogeneously before the next transfer.
  9. Discard 100.0μL from wells A11:B11 of calibration plate. This step ensures that all wells contain the same volume (100μL).
  10. Transfer 100.0μL of PBS to wells A1:B12 of calibration plate. This will bring all wells to volume 200μL. Pipette up and down 3X to ensure the dilution is mixed homogeneously.

J364000

We measured fluorescence of Part: J364000 which is a GFP expressing constitutive device for the 2017 iGEM InterLab study. This construct can constantly produce the GFP protein. We can quantify the fluorescence of this gene using the microplate reader. It can act as a positive control as it shows the most optimal situation where constant fluorescence is emitted. We measured the RFU value of J364000 under a microplate. We added 200uL of control LCM into wells A1-C1. We added 200uL culture of colony 1 of J364000 and colony 2 into wells A2-C2 and A3-C3 respectively.

Microplate

Experiments show that the fluorescence emitted from the EGFP protein produced after washing cells was not up to standard; and that LKC used in washing cells with 1x PBS had higher autofluorescence. We then modified the experiment protocol where we directly measure RFU and OD value of constructs in the microplate reader. Subculture protocol is the same as previously mentioned. After subculture, add 2μL subculture and 2ml LKC/LCM.Then, add 20μL AHL + 5μL IPTG into the culture. Then, extract 200 μL from the culture and add into microplate wells. Add positive control into wells A-C:9-12. Add negative control into wells A-C:1-4. Reserve one row for control which consists of control LKC in wells D1-8 and LCm in wells 9-12. The rfu data obtained will include autofluorescence from impurities or fluorescence from other solutions used during experiment. We can obtain the rfu produced by the construct by minusing the rfu from the construct to the control.

pSB1C3-LasR-LasRL-EGFP

pSB1C3-LasR-LasRL-EGFP is a recombinant plasmid that contains both our sensing and reporting module. It consists of a promoter followed by RBS and LasR gene and a terminator to produce LasR protein. This is followed by the pLasRL inducible promoter with an RBS, the EGFP gene and a terminator. LasR protein binds with AHL molecules to form a LasR-AHL complex. Then, the complex binds with a pLasRL promoter with RBS (Part: B0034) to activate the EGFP gene to express EGFP. Transformation, Bacterial DNA extraction, restriction enzyme digestion, PCR, gel electrophoresis, was carried out in the same way as the former constructs. Transformation 1μL of the pSB1C3-LasR-pLasRL-EGFP was transformed into the host cell E. Coli strain DH5-alpha, C41(DE3) by heat shock method. The resulting biosensor cells were selected in LB medium containing 34mg/ml chloramphenicol (CM).

Starter culture

  1. For starter culture, a bacterial colony is picked out from the bacterial plate and transferred into 1mL LCM.
  2. Shake at 37°C at 230 rpm overnight.

Subculture:

  1. Carry out subculture by adding starter culture to LCM in the ratio of 1:499 starter culture to LKC in a falcon. The final volume of the subculture is determined by the amount of culture we needed for the AHL induction experiment.
  2. The falcon is put to shake at 37°C at 230 rpm
  3. It is allowed to grow for 3 hours until the bacterial concentration reaches 0.6 OD600.
  4. The culture in the falcon is aliquoted to different culture tubes. Different concentrations of AHL are added. This is when 0h starts.
  5. After collecting the sample, the bacteria were put back into the shaker at 250 rpm
  6. Collection of samples takes place every hour until 3 hours or assigned time points.
  7. At each time point, 1mL of culture is transferred to a cuvette for each culture tube, and bacterial concentration OD600 is measured using a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer.

AHL induction

Collect 300 uL of solution from culture tube to undergo cell washing protocol.

  1. Centrifuge at 14,000rpm for 1 minute to form a pellet.
  2. Discard the supernatant and add 500µL of 1x PBS to each Eppendorf
  3. vortex and centrifuge for 1 minute.
  4. Repeat steps 2 and 3.
  5. Discard supernatants. Add 200µL of 1x PBS and pipette up and down to resuspend the cell

Samples are added into a well on the microplate, where the relative fluorescence unit (RFU) is measured by the microplate reader. Blank: 1x PBS