经典大肠杆菌基因敲除方法--之详细操作步骤

streptomycin50 µg/ml LB50 µg/ml LB- 1057bppIJ780 Fig.7 vph viomycin30 µg/ml DNA+ 1497bppIJ781 vph viomycin30 µg/ml DNA- 1622bpTable 1: Disruption cassettes containing different resistance markers with and without oriT: All disruption cassettes werecloned into the Eco RV site of pBluescript SK II (+) allowing the isolation of a Eco RI/Hind III fragment for use as template forthe PCR reaction. The size of the cassettes includes the 19 bp and 20 bp primer site (see section 2: “primer design”) which areidentical in all disruption cassettes. The resistance genes with or without oriT are flanked by FRT sites (FLP recognitiontargets) which allows FLP-mediated excision of the cassette (see section 7: “FLP-mediated excision of the disruptioncassette”).Fig. 1: Flowchart of gene disruption by PCR-targeting

Protocol (see Flowchart Fig. 1)Purification of the PCR template (resistance (-oriT) cassette)Using whole plasmids as templates for the PCR can result in a high proportion of antibiotic-resistant transformants withoutgene disruption. This is caused by traces of CCC DNA that compete with the linear PCR fragment and result in the occurrence

of false positive transformants. Using gel-purified disruption cassettes as templates prevents the occurrence of false ~ 10 µg plasmid DNA (see Table 1) with 50 U Eco RI (Roche) and50 U Hin dIII (Roche) in 1 X buffer B (Roche) in a 100 µl reaction. A 2938 bp vector fragment and a fragment 14 bp larger than the size of the cassette given in Table 1 should be the digest on a 20 x 20 x 0.25 cm (100 ml) 1% TAE (1x) agarose gel at5V/cm for 2 - 3 h in 1x TAE runs exhaust the buffer capacity and destroy the gel unless the buffer is out the cassette band from the gel and purify using the Qiagen gelextraction kit. The purified fragment is stored in 10 mM (pH 8) at aconcentration of 100 ng / µl at –20°e of plasmid DNA is tested by using 1µl (100 ng) of purified cassetteDNA to transform highly competent E. coli DH5α cells (108/µg). Plate on LBagar containing 100 µg/ml carbenicillin. If any transformants appear, repeatsteps of long PCR primersFor each gene disruption, two long PCR primers (58 nt and 59 nt) are required. Each has at the 5′end 39 nt matching the olor sequence adjacent to the gene to be inactivated, and a 3′sequence (19 nt or 20 nt) matching the right or left end ofthe disruption cassette (all cassettes have the same “right” and “left” ends). The precise positioning of the 39 nt sequence asindicated in Fig. 2 is important for creating in-frame deletions by FLP recombinase-induced excision of the resistance marker(see section 7).The 5′- 39 nt sequence of the forward primer (upstream primer; Fig. 2) must be from the coding strand of the gene of interestand its 3’ end must be in the correct reading frame with respect to the replaced gene. The 5′- 39 nt sequence of the reverseprimer (downstream primer; Fig. 2) must be from the complementary prevent unwanted recombination, a BlastN search is performed comparing each39 nt sequence with the “real cosmid” (sequences at the Sanger Centre Homepage in thefolder /doc/ /pub/S_coelicolor/cosmid_inserts and on the CD in thefolder /S_coelicolor/cosmid inserts). The perfect match should be found but no other matches >30 bp. If necessary, the 39 ntsequence is shifted in 3 nt steps until the above criteria are .2: Designing PCR primers for making an 6

(20bp + 19bp priming sequence + 42bp FLP core recombination site (see Fig.3); no in frame STOP)in-frame deletion(the example illustrates a complete deletion)39 nt from sense strand ending in ATG or GTG start codon39 nt from anti-sense strand ending in Stop codon58 nt downstream primerFLP recombinase (BT340)PCR amplification of the extended resistance cassetteAll PCR amplifications are performed using the Expand high fidelity PCR system according to the manufacturer’s instructions(Roche). Reaction conditions:Primers (100 pmoles/µl) 0.5 µl each 50 pmoles eachTemplate DNA (100 ng/µl) 0.5 µl 50 ng ≈ 0.06 pmoles1µlx5Buffer(10x)dNTPs (10 mM) 1 µl each 50 µM eachDMSO (100 %) 2.5 µl 5%DNA polymerase (2.5 U/µl) 1 µl 2.5 UnitsWater 36 µl

50µlvolumeTotalCycle conditions:1. Denaturation: 94°C, 2 min2. Denaturation: 94°C, 45 sec3. Primer annealing: 50°C, 45 sec 10 cycles4. Extension: 72°C, 90 sec5. Denaturation: 94°C, 45 sec6. Primer annealing: 55°C, 45 sec 15 cycles7. Extension: 72°C, 90 sec8. Final extension: 72°C, 5 min5 µl of the PCR product is used for analysis by gel electrophoresis. The expected sizes are 78 bp larger than the sizes of thedisruption cassettes listed in Table 1 (because of the 2 x 39 bp 5′-primer extensions). The remaining 45 µl of the PCR productis purified using the Qiagen PCR purification kit according to the manufacturer’s instructions. The PCR product is finally elutedfrom the columns with 12 µl of water (~200 ng/µl).Introduction of S. coelicolor cosmid clone into E. coli BW25113/pIJ790 (λ RED recombination plasmid) by electroporationpIJ790 contains the resistance marker cat (chloramphenicol resistance) and a temperature sensitive origin of replication(requires 30°C for replication). E. coli BW25113/pIJ790 overnight at 30°C in 10 ml LB (Luria-Bertanimedium; Sambrook et al., 1998) containing chloramphenicol (25 µg/ml).ate 100 µl E. coli BW25113/pIJ790 from overnight culture in 10 mlSOB (Hanahan, 1983) containing 20 mM MgSO4 (add 200 µl of 1M stock to10 ml SOB) and chloramphenicol (25 µg/ml). for 3-4 h at 30°C shaking at 200 rpm to an OD600 of ~ r the cells by centrifugation at 4000 rpm for 5 min at 4°C in a SorvallGS3 rotor (or equivalent). medium and resuspend the pellet by gentle mixing in 10 ml ice-cold10 % fuge as above and resuspend pellet in 5 ml ice-cold 10 % glycerol,centrifuge and decant. Resuspend the cell pellet in the remaining ~ 100 µl10 % 50 µl cell suspension with ~ 100 ng (1-2 µl) of cosmid DNA. Carry outelectroporation in a 0.2 cm ice-cold electroporation cuvette using a BioRad GenePulser II set to: 200 Ω, 25 µF and 2,5 kV. The

expected time constant is4.5 – 4.9 ately add 1 ml ice cold LB to shocked cells and incubate shaking for1h at 30° onto LB agar containing carbenicillin (100 µg/ml), kanamycin(50 µg/ml) and chloramphenicol (25 µg/ml).te overnight at 30°er one isolated colony into 5 ml LB containing antibiotics as in (9)te overnight at 30°C. This culture will be used as a pre-culture forgenerating competent cells to be transformed with the extended resistance targeting of the S. coelicolor cosmidE. coli BW25113/pIJ790 containing a S. coelicolor cosmid is electro-transformed with the extended resistance cassette. Theexample described uses the apramycin – oriT disruption cassette from pIJ773. Table 1 lists alternative cassettes and theirresistance determinants. 8. Spread onto LB agar containing carbenicillin (100 µg/ml), kanamycin (50 µg/ml) and apramycin (50µg/ml). If no further gene disruptions will be made on this cosmid, incubate overnight at 37°C to promote the loss of pIJ790. (Iffurther disruptions are planned propagate overnight at 30°C and include chloramphenicol (25 µg/ml) so that pIJ790 isretained).7. Immediately add 1 ml ice cold LB to shocked cells and incubated shaking 1 h at 37°C (or 30°C if further gene disruptionswill be made on the same cosmid; see below).6. Mix 50 µl cell suspension with ~ 100 ng (1-2 µl) of PCR product. Carry out electroporation in a 0.2 cm ice-coldelectroporation cuvette using a BioRad GenePulser II set to: 200 Ω, 25 µF and 2,5 kV. The expected time constant is 4.5 – 4.9ms.5. Centrifuge as above and resuspend pellet in 5 ml ice-cold 10 % glycerol,centrifuge and decant. Resuspend the cell pellet in remaining ~ 100 µl 10 % glycerol.4. Decant medium and resuspend the pellet by gentle mixing in 10 ml ice-cold 10% glycerol.3. Recover the cells by centrifugation at 4000 rpm for 5 min at 4°C in a Sorvall GS3 rotor (or equivalent).2. Grow for 3-4 h at 30°C shaking at 200 rpm to an OD 600 of ~ 0.4.1. Inoculate a 10 ml SOB (without MgSO 4) culture containing carbenicillin (100µg/ml), kanamycin (50 µg/ml) and chloramphenicol (25 µg/ml) with 1% of the overnight culture of E. coli BW25113/pIJ790 andthe S. coelicolor cosmid. Add 100 µl 1M L-arabinose stock solution (final concentration is 10 mM, induces red genes).If no colonies are obtained after 16 h growth at 37°C, repeat the experiment starting with a 50 ml SOB culture instead of 10ml culture for generatingelectrocompetent cells. Try to concentrate the cells as much as possible byremoving all of the remaining 10% glycerol. Resuspend the cell pellet in 50 µl10% glycerol and use for 12 – 16 h growth at 37°C different colony-sizes are observed. Cultivating for longer time results in an increasedbackground of small colonies, which arefalse positives. It is important to note that at this stage wild-type and mutant

cosmids exist within one cell. The transformation with a PCR product and itsintegration in the cosmid DNA by homologous recombination will not occur inall copies of the cosmid molecules in one cell. One copy of a cosmid containingthe incoming resistance marker is sufficient for resistance to this ly, the larger the size of a colony, the more copies of mutagenisedcosmids are present. Inoculating a large colony in 5 ml LB liquid culturescontaining carbenicillin (100 µg/ml), kanamycin (50 µg/ml) and apramycin(50 µg/ml) result in a growth at 37°C to a cell density (OD600 ~ 0.1 – 0.3) within3-4 h (E. coli BW25113 without pIJ790 grows very fast). After 6 h plasmid DNAcan be isolated and tested by restriction analysis and/or PCR using the primersdescribed analysis with a primer pair (test primers) priming just ~ 100 bp outside the region affected by homologous recombinationwill generate the expectedfragment after gene disruption, but will usually also generate the wild-typefragment, caused by remaining wild-type copies within the same will be lost during the subsequent transformation step into themethylation-deficient E. coli host ET12567 containing the non-transmissibleplasmid pUZ8002 (this is not a problem anyway because wild-type copies lackthe oriT).Notes on viomycin selection: selecting for viomycin R depends critically on the amount of salt in the medium; more viomycinis required at higher saltconcentrations. For a clean selection of E. coli clones, use DNA agar or 2xYTagar containing 30 µg/ml viomycin (see Kieser et al., 2000).For multiple gene replacements, choose an oriT-containing disruption cassette for the first knock-out, and a cassette withoutoriT and different resistance markers for further gene gene disruption is confirmed by restriction analysis and/or PCR. Cosmid DNA of transformants is isolated from a 6 h,37°C, 5 ml LB culture containing carbenicillin (100 µg/ml), kanamycin (50 µg/ml) and apramycin (50 µg/ml). Alkaline lysisfollowed by phenol/chloroform extraction produces cosmid DNA suitable for restriction CCC DNA end the cell pellet from 1 ml culture by vortexing in 100 µl solution I(50 mM Tris/HCl, pH 8; 10 mM EDTA).ately add 200 µl solution II (200 mM NaOH; 1% SDS) and mix byinverting the tubes ately add 150 µl solution III (3 M potassium acetate, pH 5.5) and mixby inverting the tubes at full speed in a microcentrifuge for 5 min at room ately extract supernatant with 400 µl phenol/chloroform, vortex 2 minand spin at full speed in a micro centrifuge for 5 min.

er the upper phase and add 600 µl 2-propanol. Leave the tubes on icefor 10 as above and wash the pellet with 200 µl 70% as above and leave the tube open for 5 min at room temperature to drythe pellet. Resuspend the pellet in 50 µl 10mM Tris/HCl (pH 8) and use 10 µl for restriction ng the phenol/chloroform extraction step results in degradation of the cosmid DNA. Use of miniprep-columns withoutincluding a phenol/chloroformextraction is not cation of positive transformants by PCR requires an additional pair of 18 – 20 nt test primers which anneal 100 – 200 bpupstream and downstream of the 39 bp recombination region. (These primers can also be used later to verify the FLP-mediated excision of the resistance cassette.)Primers (100 pmoles/µl) 0.2 µl each 20 pmoles eachTemplate DNA (~50 ng/µl) 1 µl 50 ng1µlxBuffer5(10x)dNTPs (10 mM) 1 µl each 50 µM eachDMSO (100 %) 2.5 µl 5%DNA polymerase (2.5 U/µl) 1 µl 2.5 UnitsWaterµl36.150µlvolumeTotalCycle conditions:1. Denaturation: 94°C, 2 min2. Denaturation: 94°C, 45 sec3. Primer annealing: 55°C, 45 sec 30 cycles4. Extension: 72°C, 90 sec5. Final extension: 72°C, 5 min5 µl of the PCR product is used for gel electrophoresis.

Transfer of the mutant cosmids into StreptomycesIf the target Streptomyces for mutagenesis carries a methyl-sensing restriction system (as is the case for S. coelicolor and tilis), it is necessary to passage the cosmid containing an apramycin resistance-oriT cassette through a non-methylatingE. coli host. To achieve this, it is introduced by transformation into the non-methylating E. coli ET12567 containing the RP4derivative pUZ8002. The cosmid is then transferred to Streptomyces by intergeneric conjugation (see Table 2 for resistancemarkers). If the target Streptomyces for mutagenesis does not carry a methyl-sensing restriction system (as is the case for ns), common E. coli strains such as DH5α containing pUZ8002 can be used ption Name Replication Carb R Cml R Kan R Tet R S. coelicolorSupercos 1Carb R Kan Rcosmid clonesλ Red plasmid pIJ790 t s Cml RFLP recombinaseBT340 t s Carb R Cml RplasmidOriT- RP4 derivative pUZ8002 Kan ROriT+ RP4 derivative pUB307 Kan RNon-methylating E. coli ET12567 Cml R Tet RTable 2. Resistance markers of vectors, helper plasmids and strains (carbenicillin resistance (Carb R ), chloramphenicolresistance (Cml R), kanamycin resistance (Kan R), tetracycline resistance (Tet R), temperature sensitive replicon (t S)). SeeTable 1 for replacement e competent cells of E. coli ET12567/pUZ8002 grown at 37oC in LBcontaining kanamycin (25 µg/ml) and chloramphenicol (25 µg/ml) to maintain selection for pUZ8002 and the dam mutation,respectively. (ET12567 has a doubling time > 30 min.)High competence is required when Dam-methylated plasmids are introduced into a dam- orm competent cells with the oriT-containing cosmid clone, and selectfor the incoming plasmid only using apramycin (50 µg/ml) and carbenicillin (100 µg/ml) .ate a colony into 10 ml LB containing apramycin (50 µg/ml),chloramphenicol (25 µg/ml) and kanamycin (50 µg/ml). Grow overnight mphenicol S or Kanamycin S segregants arise frequently among transformants, so set up more than one culture. Thekanamycin selection is probably ineffectivebecause both the cosmid and pUZ8002 confer resistance (Table 2).ate 100 µl overnight culture into 10 ml fresh LB plus antibiotics asabove and grow for ~ 4 h at 37°C to an OD600 of the cells twice with 10 ml of LB to remove antibiotics that might inhibitStreptomyce s, and resuspend in 1 ml of washing the E. coli cells, for each conjugation add 10 µl (108)Streptomyces spores to 500 µl 2 × YT broth. Heat shock at 50°C for 10 min,

then allow to 0.5 ml E. coli cell suspension and 0.5 ml heat-shocked spores and spinbriefly. Pour off most of the supernatant, then resuspend the pellet in thec. 50 µl residual a dilution series from 10-1 to 10-4 each step in a total of 100 µl of out 100 µl of each dilution on MS agar + 10mM MgCl2 (withoutantibiotics) and incubate at 30°C for 16-20 y the plate with 1 ml water containing 0.5 mg nalidixic acid (20 µl of25 mg/ml stock; selectively kills E. coli) and 1.25 mg apramycin (25 µl of50 mg/ml stock). Use a spreader to lightly distribute the antibiotic solutionevenly. Continue incubation at 30°a-plate each MS agar plate with single colonies onto DNA platescontaining nalidixic acid (25 µg/ml) and apramycin (50 µg/ml) with andwithout kanamycin (50 µg/ml). Double cross-over exconjugants are kanamycin S and apramycin R. (DNA gives fast, non-sporulating growth.)cin S clones are picked from the DNA plates and streaked for singlecolonies on MS agar (promotes sporulation) containing nalidixic acid(25 µg/ml) and apramycin (50 µg/ml).m kanamycin sensitivity by replica-plating onto DNA plates containingnalidixic acid (25 µg/ml) with and without kanamycin (50 µg/ml).ed kanamycin sensitive strains are then verified by PCR and Southernblot analysis. Typically, ~ 10 % of the exconjugants are double cross-over recombinants. The frequency of double cross-overs depends onthe length of the flanking regions of homologous DNA on the cosmid. If < 1 kb is left on one side of the disrupted gene,obtaining kanamycin S double cross-over types directly on the conjugation plates may be difficult. It may be necessary tostreak out several exconjugants for single colonies on MS agar without antibiotics. After 3-5 days growth replica-plate ontoDNA with and without tration in AntibioticStock mg/mlµl for 1 ml overlayFinal conc. after floodingµg/mlMS, DNA µg/mlR2YE µg/mlApramycin 50 25 50 50 50 Kanamycin 50 100 200 50 200 Spectinomycin 200 25 200 400 400 Streptomcyin 10 25 10 10 10Viomycin 30 25 30 30 NANalidixic acid 25 in

0.3 M NaOH20 202525Table 3: Antibiotic concentrations for selection on S. coelicolor MS conjugation plates, DNA replica plates or R2YE protoplastregeneration plates (Note some small differences from Kieser et al ., 2000).FLP-mediated excision of the disruption cassetteThe disruption cassettes are flanked by FRT sites (FLP recognition targets). Expression of the FLP-recombinase in E. coliremoves the central part of the disruption cassette, leaving behind a 81 bp “scar” sequence which, in the preferred readingframe (bold in Fig. 3), lacks stop . 3: Sequence of the 81 bp “scar” sequence remaining after FLP-mediated excision of the disruption cassette. Thetranslation of the preferred reading frame is printed bold. The 20 and 19 nt priming sites are underlined and printed in colour.(Fig.2 explains the determination of the reading frame.)indicate stop codons,priming site (20 nt)priming site (19 nt)This allows the generation of (hopefully) non-polar, unmarked in-frame deletions and repeated use of the same resistancemarker for making multiple knock-outs in the same cosmid or in the same strain. E. coli DH5α cells containing the temperaturesensitive FLP recombination plasmid BT340 (Datsenko and Wanner, 2000; can be obtained from the E. coli Genetic StockCenter: CGSC Strain# 7629) are transformed with the mutagenised cosmid DNA (obtained in section 5). BT340 containsampicillin and chloramphenicol resistance determinants and is temperature sensitive for replication (replicates at 30°C). FLPsynthesis and loss of the plasmid are induced at 42°C (Cherepanov and Wackernagel, 1995). E. coli DH5α/BT340 overnight at 30°C in 10 ml LB containingchloramphenicol (25 µg/ml).Transforming E. coli BW25113/cosmid::apramycin (mutagenised cosmid) with the plasmid BT340 is not recommendedbecause the isolates after PCR targetingmay still contain copies of undisrupted cosmid DNA (see page 10, secondparagraph).ate 100 µl E. coli DH5α/BT340 from overnight culture into 10 mlLB containing chloramphenicol (25 µg/ml). for 3-4 h at 30°C shaking at 200 rpm to an OD600 of ~ 0.

r the cells by centrifugation at 4000 rpm for 5 min at 4°C in aSorvall GS3 rotor (or equivalent). medium and resuspend the pellet by gentle mixing in 10 ml ice-cold 10 % fuge as above and resuspend pellet in 5 ml ice-cold 10 % glycerol,centrifuge and decant. Resuspend the cell pellet in remaining ~ 100 µl 10% 50 µl cell suspension with ~ 100 ng (1-2 µl) of mutagenised cosmidDNA. Carry out electroporation in a 0.2 cm ice-cold electroporation cuvette using a BioRad GenePulser II set to: 200 Ω, 25 µFand 2,5 kV. The expected time constant is 4.5 – 4.9 ately add 1 ml ice cold LB to shocked cells and incubate shakingfor 1 h at 30° onto LB agar containing apramycin (50 µg/ml) andchloramphenicol (25 µg/ml).te for 2 d at 30°C (E. coli DH5α/BT340 grows slowly at 30°C).11.A single colony is streaked on an LB agar plate without antibiotics forsingle colonies and grown overnight at 42°C to induce expression of the FLP recombinase followed by the loss of two masterplates by streaking 20 – 30 single colonies with atoothpick first on a LB agar plate containing apramycin (50 µg/ml) and then on a LB agar plate containing kanamycin (50µg/ml). the masterplates overnight at 37°C. Apramycin S kanamycin R clonesindicate the successful loss of the resistance cassette and are furtherverified by restriction and PCR lly, ~ 10 % of the single colonies after non-selective growth lose the incoming resistance marker and the plasmidBT340 the same test primers as in section 5 (annealing ~ 100 bp upstream and downstream of the 39 nt primer sequence)should produce a PCR product of~ 300 bp (200 bp + 81 bp “scar”). PCR fragments can be sequenced using theamplification primers for ing resistance cassette inserts in S. coelicolor with the unmarked “scar” sequenceThe chromosomal apramycin resistance cassette insert in S. coelicolor is replaced by the “scar” sequence. This is achieved byhomologous recombination between the chromosome and the corresponding “scar cosmid” prepared in 7. The procedurediffers from section 6 because the cosmid lacks oriT, and the desired product is antibiotic sensitive. Therefore, it is necessaryto introduce the scar cosmid into Streptomyces by protoplast transformation, and then select for kanamycin resistantStreptomyces containing the entire scar cosmid integrated by a single crossover. Restreaking to kanamycin-free medium,followed by screening for concomitant loss of kanamycin resistance and apramycin resistance, then identifies the desiredStreptomyces ation of Streptomyces coelicolor 25 ml YEME medium to a baffled flask. Add ~ 0.1 ml spore suspension

and required growth factors. Incubate 36-40 h at 30°C in an orbital incubator es of S. lividans and S. coelicolor are ready for harvesting when they start to produce red culture broth into a 20 ml screw cap bottle and spin in the benchcentrifuge (~ 1000 x g, 10 min).Before centrifugation, examine the culture for contamination by unicellular bacteria, usually indicated by turbidity: theStreptomyces mycelium sedimentsquickly while unicellular contaminants remain suspended. In case of doubt, use the d the supernatant carefully; the pellet is easily the mycelium does not pellet add 5 ml sterile water to reduce the density of the medium and centrifuge end pellet in 15 ml 10.3% sucrose and spin in bench centrifuge asabove. Discard step mycelial pellet, without added liquid, can be stored frozen at –20°end mycelium in 4 ml lysozyme solution (1 mg/ml P buffer, filtersterilised); incubate at 30°C, 15-60 in and out of a 5 ml pipette three times and incubate for a further helps to free protoplasts from the mycelium so that they will pass through the cotton wool filter used in step9. At leastwith S. lividans, it is possible to obtain transformants with unfiltered material, but the washing (steps 9-10) is still needed toremove 5 m1 P buffer. Repeat step protoplasts through cotton wool (using a filter tube) and transfer to aplastic nt protoplasts gently by spinning in a bench centrifuge (~ 1000 x g, 7min).d supernatant and suspend protoplasts in 1 ml P this and any other steps when pelleted protoplasts are to be resuspended, resuspend in the remaining drop of liquid bytapping the side of the tube repeatedly with a finger until the protoplasts are dispersed to form a creamy suspension, then addthe suspending P buffer (otherwise the protoplast pellet is difficult to disperse).Avoid vortexing, which induces foaming and consequent lysis. To freeze the protoplasts for storage, place samples of theprotoplast suspension in small plastic tubes, close them and place them in ice in a plastic beaker. Place the beaker at –70°Covernight. Free the frozen protoplasts in their tubes from the ice and store at –70° thaw, shake the frozen tube under running warm water (i.e. freeze slowly, thaw quickly). To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in P buffer and in dilute detergent (~ 0.01% SDS) andplated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplastedunits.