Protocols for This Method

Protocols for this method:

Note: See our previous manuscript for detail process

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Note: The protocols are using bifunction(Red + I-SceI)helper plasmidpREDTKI (kanR) and ampicillinresistance 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 otherderivative 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 Methods6(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 platescontaining glucose(5g/L), ampicillin , kanamycin, apramycin / spectinomycin/ chloramphenicol.

(antibioticsconcentration:25, 50 or 100 mg/L)

1.2

Transform donor plasmid into intermediate strain with bifunctional helper plasmid

Co-transform donor plasmid and bifunctional helper plasmid into intermediate strain without bifunction helper plasmid

Spread E. coli cells onto LB platescontaining 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. (OD600about 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 and20 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 plasmidcould 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 fromplate A :

(1) Ampicillin , chloramphenicol/apramycin / spectinomycin sensitive phenotype.

Streak on 3or4 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

Retain the bifunctional helper plasmid for further modifications