Protocols for this method:
Note: See our previous manuscript for detail process
(http://dx.doi.org/10.1128/AEM.00313-14)
Note: The protocols are using bifunction(Red + I-SceI) helper plasmid pREDTKI
(kanR) and ampicillin resistance donor plasmid.
When using other bifunction helper plasmid, the antibiotics should be used depending
on the resistance of the helper plasmid.
When using bifunction helper plasmid with rhaB promoter, L-rhamnose (instead of
IPTG) should be used for inducing I-SceI.
Note: The cultivation time should be suitable for MG1655, W3110, BL21(DE3) and
other derivative strains. For other strains, the time needs to be adjusted.
Initial work:
1. Lambda-Red PCR targeting: integration of resistance gene(s) cassettes flanked by
I-SceI sites in or beside the modification site.
Note:
Both bifunctional (Red + I-SceI) helper plasmid or conventional Red helper plasmid (such as
pKD46) could be used in this step.
If conventional Red helper plasmid pKD46 is used, it should be subsequently cured.
If the bifunctional helper plasmid is used, it could be either retained or cured.
If the intermediate strain is expected to survive long-term preservation, curing the bifunctional
helper plasmid is recommended.
2. Donor plasmid construction.
Any method can be used to construct the donor plasmid, including traditional
restriction cloning, site-specific mutagenesis, Gibson Assembly (Nature
Methods 6 (5): 343–345) or artificial whole gene synthesis, etc.
Preparation:
(1) Culture the intermediate strain overnight for competent cell preparation.
(2) Extract donor plasmid (and bifuctional helper plasmid, if needed)
Note: Donor plasmid and bifunctional plasmid should harbor different resistance
markers.
(3) Materials for preparation of competent cell (according to the corresponding
protocol.)
(4) Test tubes with 4mL LB liquid.
(5) LB agar
(6) Stock solutions:
ampicillin, kanamycin, spectinomycin, apramycin, chloramphenicol, glucose,
L-arabinose, L-rhamnose, IPTG.
(7) Sterile water.
Step 1:
1.1
Inoculate the overnight seed culture into fresh LB medium containing kanamycin
(depending on the resistance of the helper plasmid), chloramphenicol, apramycin and
spectinomycin (depending on the marker used for introducing the I-SceI site into
genome).
Prepare competent cells for electroporation or heat shock transformation.
Prepare plates for the next step:
LB plates containing glucose(5g/L), ampicillin , kanamycin, apramycin /
spectinomycin/ chloramphenicol.
(antibiotics concentration:25, 50 or 100 mg/L)
1.2
Transform donor plasmid into intermediate strain with bifunctional helper plasmid
or
Co-transform donor plasmid and bifunctional helper plasmid into intermediate strain
without bifunction helper plasmid
Spread E. coli cells onto LB plates containing glucose, ampicillin , kanamycin and
chloramphenicol/ apramycin / spectinomycin
Step 2:
2.1
Inoculate several resulting colonies into test tubes containing 4 mL LB medium
containing 0.5% glucose and kanamycin
Cultivated at 30 ºC * 200 rpm for 6~8 h. (OD600>=1 )
2.2
Inoculate 40 microliters of seed culture into a test tube containing 4 mL LB medium
containing 10mM L-arabinose and 50μg/mL kanamycin (depending on the resistance
of the helper plasmid).
Cultivate under 30 ºC * 200 rpm for about 2 h. (OD600 about 0.1 ~ 0.5)
Add IPTG to a final concentration of 20 mM.
Cultivate 30 ºC * 200 rpm overnight.
Note: The cultivation time should be suitable for MG1655, W3110, BL21(DE3) and other
derivative strains. For other strains, the time needs to be adjusted.
Step 3:
3.1
Inoculate 40 microliters of culture (OD600>= 1) into another test tube containing 4
mL LB medium and 20 mM IPTG, 10mM L-arabinose and 50μg/mL kanamycin
(depending on the resistance marker of the helper plasmid).
Note:
Alternatively, in this step and the following step, arabinose-free medium with only L-rhamnose or
IPTG for inducing I-SceI and antibiotic for maintaining the helper plasmid could be used, to avoid
the potential deleterious effects caused by the prolonged expression of the lambda Red functions.
But a slightly decreased efficiency would be obtained.
Cultivated the test tube was 30 ºC * 200 rpm for another 6~8h. (to OD600>= 0.5)
Note: This step (6~8h cultivation) could be skipped, but it's recommended NOT to skip it.
Skipping this step might result in a lower marker eviction rate.
Prepare plates for the next step:
Plate A:
LB with 20mM L-rhamnose or IPTG, 10mM L-arabinose and 50μg/mL
ampicillin or kanamycin (depending on the resistance of helper plasmid)
Plate B1:
Plate A plus apramycin
Plate B2:
Plate A plus spectinomycin
Plate B3:
Plate A plus chloramphenicol
(depending on the marker used for introducing the I-SceI site into the genome)
3.2
Harvest the culture was ed, suspend in sterile water and dilute (10-1 to 10-4).
Spread the diluted culture on Plate A and Plate B, respectively.
(or Plate A, Plate B1 and Plate B2)
Incubate 30 ºC overnight.
Prepare plates for the next step:
LB plates with kanamycin
LB plates with ampicillin
LB plates with chloramphenicol, apramycin or spectinomycin.
Step 4
4.1
Expected result after overnight incubation:
Colonies appear on plate A.
Significantly fewer colonies appear on Plate B (or B1 and B2).
Analyze colonies appear from plate A :
(1) Ampicillin , chloramphenicol/ apramycin / spectinomycin sensitive phenotype.
Streak on 3 or 4 plates:
LB plates with kanamycin ,
LB plates with ampicillin ,
LB plates with chloramphenicol, apramycin or spectinomycin .
(2) Colony PCR.
4.2
Strains with the desired modification should have the following phenotype
(1) CmS, spcS, aprS, amps, and kanR
and
(2) Have the expected colony PCR bands
Follow-up work:
Colony PCR reconfirm (if necessary)
Sequencing the PCR fragments (if necessary)
Cure the bifunction helper plasmid via 42°C growth
or
Retain the bifunctional helper plasmid for further modifications