Difference between revisions of "Team:KU Leuven/Research/Basic Part"

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.