2. Marco Referencial
2.4 Normas nacionales
2.4.4 Decreto 4829 de 2011
For phage transduction, lysates were first prepared from the P1cml,clr100 lysogen (E. coli
strain that harbours the virus in its chromosome), followed by phage titration and then phage transduction. P1cml,clr100 is a mutant of P1 that harbours the gene for
chloramphenicol resistance (cml) and carries a mutation that makes P1 thermo-inducible
(clr100) (Miller, 1972).
6.4.1.3.1 Preparation of P1 lysates
A freezer stock of E. coli CSH128 (lysogen for P1cml,clr100) was used to inoculate 3 mL
LB-chloramphenicol medium (12.5 μg.mL-1 final concentration) and incubated at 28 ºC
overnight. Three aliquots of the overnight culture (2x 200 μL and 1x 500 μL) were added to three separate sterile glass flasks containing 10 mL LB broth supplemented with MgSO4
(100 mM), and then incubated at 28 ºC (shaking at 190 rpm) to OD600 ~ 0.7. To make
lysates, 1x 200 μL and 500 μL cultures were transferred to a 40 ºC shaking water bath for 5 min, shifted to 40 ºC for 45 min (with shaking) and then moved to 37 ºC for an additional hour (with shaking). The second 200 μL culture served as a control and was left to incubate at 28 ºC during this period. To collect the P1 lysate, all of the cultures were centrifuged (2,400 xg, room temperature for 10 min) and the supernatant was filtered into sterilised glass bottles using a syringe (0.22 μm, 33 mm filters; Merck). To store the samples, 600
μL and 900 μL of chloroform was added to the 2x 200 μL and 500 μL supernatant samples, respectively, mixed and placed at 4 ºC.
6.4.1.3.2 Phage titration
To decide which dilution of P1 lysates to use for phage transduction, phage titering was performed. The lysate from the 200 μL culture (not the control) was serially diluted (10-1,
10-2, 10-3, 10-4, 10-5, 10-6) in LB broth. One hundred μL aliquots of a fresh
E. coliDH5α-E
overnight culture were transferred into six separate microcentrifuge tubes containing 50 μL of CaCl2 (100 mM final volume). One hundred μL of the 10-2 to 10-6 lysate dilutions were
Meanwhile, ‘LB hard agar’ (2 %) supplemented with CaCl2 (5mM final
concentration) was poured into six regular petri dishes to fill ¼ of the plate and left to set at room temperature. The cultures (250 μL total volume) were pipetted onto the centre of six separately prepared agar plates. Three mL of ‘LB soft agar’ (0.6 %) was then pipetted on top of the cells (and covered the centre of the ‘LB hard agar’) and left to set at room temperature. The plates were incubated at 37 ºC overnight. The lysate dilution used for P1 phage transduction was selected based on the plate that contained the highest phage dilution with the most plaques (indicative of cell lysis).
6.4.1.3.3 Phage transduction
To package the DNA of interest into P1 phage, an overnight culture (5 mL) of the strain carrying the desired DNA [E. coli BW25113 ΔyejG knockout strain or E. coli MDS42 yejG
(HTD)] was prepared. CaCl2 (5 mM final concentration) was added to the culture before
pipetting 300 μL of culture into four individual microcentrifuge tubes. For phage adsorption, 10 μL, 30 μL and 100 μL of aP1 lysate (10-2 dilution) was added only to three
of the tubes. No phage was added to the fourth culture as it served as a control. The tubes were incubated at 37 ºC for 20 min (no shaking).
The cultures were transferred into four separate glass flasks containing 10 mL LB broth supplemented with CaCl2 (5mM final concentration). The cultures were incubated at
40 ºC (with shaking) for b4 h; this step allows for growth to occur after ~ 1 h followed by
lysis at 2-3 h. To select which sample to use for downstream experiments, the cultures containing phage were compared to the control culture. The culture with the smallest volume of P1 phage that shows lysis (i.e., less turbidity) was selected. The culture was centrifuged (2,325 xg room temperature for 10 min). The supernatant (containing the phage with packaged target DNA) was filtered into glass bottles using a syringe. To kill cells that may have filtered through, 600 μL of chloroform was added to the filtered supernatant and stored at 4 ºC.
For the P1 phage transduction step, the following experiments were performed. Three 15 mL Falcon tubes containing CaCl2 (5 mM final concentration) were labelled as
sample, cell control and phage control. To the sample and cell control tubes, 1.3 mL of the recipient overnight culture (naïve E. coli BW25113 or E. coli MDS42) was added. To the
BW25113 ΔyejG knockout strain or E. coli MDS42 yejG (HTD)] was added. The tubes
were incubated at 37 ºC for c15 min with no shaking (infection step).
The cells were centrifuged (2,325 xg room temperature for 3 min) and the pellets were washed three times with 1mL LB, supplemented with sodium citrate (25 mM). The pellets were resuspended in 1 mL LB-sodium citrate and incubated at 37 ºC for 2 h (with shaking) (expression step). The cells were centrifuged once again (2,325 xg, room temperature for 10 min) and resuspended in 150 μL LB broth. Aliquots (20 μL and 130
μL) were spread on LB agar selection plates supplemented with sodium citrate (5 mM final concentration) and incubated at 37 ºC overnight. For the experiments involving the
fusA(G1478T) mutation and the kanR marker, agar plates containing 18 μg.mL-1
tobramycin and 30 μg.mL-1 kanamycin were used for selection, respectively.
If colonies appeared on the agar selection plates, PCR (Appendix I.8) was performed using the yejG.for and rev primers (Table I.3, Appendix I.5) to confirm whether recombination had been successful: PCR screening (Appendix I.8) using yejG.for and rev primers (Appendix I.3) was performed on E. coli BW25113 colonies and PCR
discrimination (Section 6.4.2) using fusA_m.for and rev primers (Appendix I.3) was performed on E. coli MDS42 colonies.
In an attempt to optimise this experiment a few of the steps were altered:
aFor phage adsorption 5 μL, 50 μL and 80 μL of P1 lysate (10-2 dilution) were also used in
separate experiments.
bFor the lysis step, the cultures were incubated at 40 ºC (with shaking) for 6 and 8 h. cThe infection step was increased from 15 min to 20 and 30 min.