Difference between revisions of "Team:KU Leuven/Research/Methods"

Theory
To be able to create patterns, two different cell types called A and B will interact with each other. In order to achieve the desired behavior, the cells used in the experiments were derived from K12 Escherichia coli strains with introduction of specific knockouts. Cell type A has a deletion of tar and tsr, whereas in cell type B both tar and cheZ are knocked out. The Keio collection is composed of a set of precisely defined single-gene deletions of all nonessential genes in E. coli K-12. The targeted genes were replaced by a kanamycin resistance cassette. The kanamycin cassette is enclosed between two FRT sites making excision possible using FLP recombinase (reference 1). FLP recombinase triggers an intramolecular recombination between FRT repeats in the chromosome. Since both the antibiotic resistance gene and the plasmid replication region are surrounded by two FRT sites both are to be eliminated (Figure 1, step 1).

A genetic procedure for moving selectable mutations of interest called the P1 transduction was used. Since the packaging of the bacteriophage P1 is rather inaccurate, it will on occasion package the DNA of its bacterial host instead of its own phage chromosome. This implies that the lysate contains either packaged phage or bacterial DNA. After infection of a second host with this lysate, a transfer of parts of the chromosome from the donor strain into the receiver strain will take place. Those DNA pieces can then recombine using the FRT sites and hereby be incorporated permanently into the chromosome of the new strain. Here, the recombination was triggered by selection on kanamycin. (reference 2)

In general, we used three steps to obtain our double knock-outs (Figure 1). In the first step, the kanamycin cassette of our tar knock-out strain was removed by flippase, coded on plasmid PCP20. Afterwards, the temperature sensitive plasmid was removed by growing the cells overnight at 42°C. In a third step, the tar knock-out strain is infected by lysate originating from our tsr and cheZ knock-out strains. After selection on kanamycin plates, we obtained our double knock-outs. These knock-outs were confirmed by PCR. For more information, please check our result page.

Figure 1

P1 transduction. Click to enlarge

Protocol

1. Preparation of lysate starting from stock plate of phage

1. Make an overnight culture of E. coli MG1655.

2. Take 500 µL overnight culture and add the phage P1. Incubate overnight at 37°C.

3. Take single plaques of the P1 stock plate and bring this in a sterile Eppendorf tube together with 200 µL of mQ.

4. Overnight extraction while shaking at 37°C.

5. Add 0.01, 0.1, 10 and 100 µL of extraction to 500 µL of a stationary phase culture of E. coli MG1655. Vortex and plate out.

6. Add LB soft agar containing 10 mM MgSO₄ and 5 mM CaCl₂ and incubate at 37°C.

7. Choose the plate with the best lysates.

8. Sterilize your spoon using a Bunsen burner, cool it down with water and wash it with 100% ethanol.

9. Cut out a plaque from the soft agar and put this in a 10 mL syringe.

10. Press the content of the syringe in an Eppendorf tube and centrifuge for 10 minutes at 14000 rpm.

11. Take 650 µl and bring this in a new Eppendorf tube.

12. Extraction with 30 µL of CHCl₃.

13.Vortex vigorously.

14. Store lysate at 4°C.

2. Preparation of the lysate of donor strain

1. Firstly, centrifuge the lysate to ensure the chloroform is at the bottom of the Eppendorf tube. Then add 0.1, 1, 10 and 100 µl of lysate to 500 µL stationary phase overnight culture of the donor strain.

2. Add LB soft agar containing 10 mM MgSO₄ and 5 mM CaCl₂. Incubate this at 37°C.

3. Sterilize your spoon in a Bunsen flame, cool it down with water and wash with 100% ethanol.

4. Centrifuge the Eppendorf tubes 10 minutes at 14000 rpm.

5. Transfer 650 µL into a new Eppendorf tube.

6. Extract with 30 µL of CHCl₃.

7. Vortex vigorously.

8. Store the lysate at 4°C.

3. Transduction to acceptor strain

1. Concentrate 500 µL of stationary phase overnight acceptor strain culture five times in LB with 10 mM MgSO₄ and 5 mM CaCl₂.

2. Add 0.1, 1, 10 and 100 µL of donor strain lysate to 100 µL acceptor strain.

3. Incubate 30 minutes at 37°C.

4. Plate out on selective medium and incubate overnight.

5. Plate out lysate-only to check for contamination as well.

Theory
The Gibson assembly as described by Gibson et al., is a rapid DNA assembly method which assures directionional cloning of fragments in one single reaction. For the Gibson assembly to happen, three essential enzymes are needed: a mesophylic nuclease, a thermophylic ligase and a high fidelity polymerase. For this reaction, we used NEBuilder. In the first step of this reaction, the exonuclease rapidly cleaves off the 5’ DNA ends. The exonuclease is unstable at 50°C and gets degraded. In the second step, the designed sequence overlaps anneal and the polymerase starts filling in the gaps. In the final step, the ligases covalently joins both ends. After this, the plasmid is ready to be transformed. This text was based on the IDT website as seen on 13/09/2015

Figure 1

Gibson assembly reaction and its essential components E.coli

Materials

Protocols

Protocol

1. Prepare selective media (LB with 0.25% agar (2,5 g/L) in Petri dishes (85 mm dia).

2. Apply 1.5 µL of the diluted cell suspensions from mid-log-phase cultures (~2×105 cells/µL (OD=0.5)) to the center of the plates, and let dry in air for 15 min.

3. Incubate at 37 °C for 10 h.

Theory

N-acyl homoserine lactones (AHL) are small diffusible molecules used for bacterial cell-to-cell signaling in Gram-negative bacteria. Chromobacterium violaceum is a Gram-negative bacteria which produces the violet pigment violacein as a result of sensing AHL. AHL is produced by the autoinducer synthase CviI and released in the environment. When a quorum has been reached, the AHL diffuses back into the bacteria and binds to the transcriptional regulator CviR. This activates the expression of specific genes which lead to violacein.

In our project, the mutant C. violaceum CV026 is used to quantify the amount of OHHL, a specific type of AHL which is produced by luxI. This strain is deficient CviI and therefore requires exogenous addition of AHL to produce violacein. The idea is to achieve a standard curve where the C. violaceum CV026 is induced with different amounts of AHL. In this standard curve we also take into account how long we grow the bacteria and make a correction for the optical density. In the end, we obtained an absorbance value divided by the optical density.

To begin quantifying the samples, the samples are grown until a certain optical density. Then the cells are spun down whereafter the C. violaceum CV026 is added. After incubating them for several hours, the C. violaceum CV026 and violacein are spun down and the supernatant is removed. The pellet is resuspended in dimethyl sulfoxide whereafter the cells are spun down. Because the violacein prefers to solve in dimethyl sulfoxide, we used the supernatans to measure the absorbance at 585 nm.

Protocol

Make a standard curve

The goal is to measure the absorbance of violacein versus the concentration of OHHL around 0.04 mM.

1. Make a OHHL stock solution of 10 mM.

2. Make a dilution series of OHHL in LB medium. Take into account that this will further be diluted when adding the C. violaceum CV026.

3. Add C. violaceum CV026 in this way that the volume of the cells is 10% of the end volume.

4. Incubate this for 18 hours at 30 degrees in a shaking incubator (200 rpm).

Preparation of media

1. Incubate Chromobacterium violaceum CV026 for 16-18h.

2. Take 1 mL of the sample that you would like to quantify. Measure the OD(600 nm) and centrifuge till the cells are down (max speed 15000 rpm, 10 min). If you want to include the amount of AHL inside the cell, you should lyse the cells in advance.

3. Inoculate the C. violaceum CV026 to OD(600 nm) = 0.1 in air-lid culture tubes containing LB and LB supplemented with 1 mL supernatans of your sample at 27°C (150 rev/min agitation) for 24 h in a shaking incubator.

Quantification

1. Centrifuge 1 ml culture (10 min at 13000 rev/min) to precipitate the insoluble violacein.

2. Discard supernatant (culture) and add 1 ml of dimethyl sulfoxide to the pellet.

3. Vortex the solution vigorously for 30 s to completely solubilize violacein and centrifuge at 13 000 rev/min for 10 min to remove the cells.

4. Add 200 µl of the violacein-containing supernatants to 96-well flat-bottomed microplates.

Read the absorbance with a microplate reader at a wavelength of 585 nm.

References

1. iGEM ETH Zurich (2013). Modeling part overview [online]

2. Choo, J.H., Rukayadi, Y. and Hwang, J.K. (2005).Inhibition of bacterial quorum sensing by vanilla extract. Letters in Applied Microbiology. [online]

Theory

The chemiluminescence detection assay of Kugimiya and Fukada (2015) was used as foundation to quantify the amount of leucine produced by IlvE. In this technique, leucine-tRNA synthetase in combination with a luminol chemiluminescence reaction was used to recognize leucine. In the article, they mention a selective quantification from 1 to 20 µM leucine. In our protocol, the hints given in the article were used for optimization. The suppliers of the enzymes were different, so it is possible that another range of quantification can be obtained. Below, the reaction equations can be seen. In the first step, leucine-tRNA synthetase (LeuRS) is activated by ATP to form leucyl-AMP with the formation of a side product pyrophosphate. The formation of pyrophosphate is further used to detect the amount of leucine. After the addition of inorganic pyrophosphatase, this enzyme hydrolyses pyrophosphate to phosphate. When pyruvate oxidase and pyruvate is added to phosphate, it results in the formation acetyl phosphate and hydrogen peroxide. In the fourth reaction, the hydrogen peroxide in combination with luminol and horseradish peroxidase leads to the formation of light. This luminescence is detected with the luminometer. Figure xxx From Kugimiya and Fukada (2015) The idea is to grow the bacteria on a minimal medium without leucine. These bacteria will be spun down and the supernatants will be further investigated on the presence of leucine. The luminescence originated from the bacterial sample will be compared with our standard curve. First, a big range is used for the standard curve (0 to 100 μM). When we notice in which range the bacteria produce leucine, a more narrow standard curve will be generated.