PANC 1.FH11
Contents
1. PANC 1 mini Tn5 package Ed 1 2006 Biomedal Sik User s manual Avda Am rico Vespucio 5 E Planta 1 M dulo 12 Parque Cient fico y Tecnol gico Cartuja 93 41092 SEVILLA Espa a Spain Tel 34 954 081 276 Tel Fax 34 954 081 279 B O m e da Web www biomedal com e Email info biomedal com ot NOTES PANC 1 mini Tn5 package A colletion od Delivery System pUT mini Tn5 plasmids carrying antibiotic selection markers and donor and helper strains For research use only Contents Product Description 2 Vectors 2 1 1 pUTmini Tn5 vectors 3 About mini Tn5 System 4 Vectors Maps 5 Restriction analysis 5 1 pUTmini Tn5 Cm Restriction analysis 5 2 pUTmini Tn5 Km Restriction analysis 5 3 pUTmini Tn5 Sm Sp Restriction analysis 5 4 pUTmini Tn5 Tc Restriction analysis 6 Protocol 6 1 Cloninig and transformation of donor strain 6 2 Transfer of the DNA into the recipient strain 6 3 Selection of exconjugants N Troubleshooting 8 Reagents and recipes 9 References 10 Related products 11 Short protocol Oo O Oo 0 RN 21 22 MY Biomedal 1 Product Description Mini Tn5 system is the generic name of a system derived of mini Tn5 transposon vectors that can be used in the analysis construction and manipulation of complex phenotypes in a wide range of Gram negative bacteria This system includes cloning vectors pUTmini Tn5 and strains for mating experi2. from Tn 5 Vig Biomedal pUTmini Tn5 Sm Sp 10 Related products pUTmini Tn5 Sm Sp is a 7386 basepair plasmid that contains the minitransposon mini Tn5 Sm Sp cloned in the pUT backbone Mini Tn5 Sm Sp PRODUCT DESCRIPTION Cat No carries as selection marker the add gene that confers streptomycin and E coli DH5a Apir Propagation of pUT plasmids B5 3233 spectinomycin resistante E coli CC118 Apir Propagation of pUT plasmids BS 3235 STRAINS E coli S17 1 Apir Propagation of pUT plasmids BS 3234 pUTmini Tn5 Tc E coli DH5a pRK2013 Mating helper strain BS 3236 E coli DH5a pRK2073 Mating helper strain BS 3263 pUTmini Tn5 Tc is a 7477 basepair plasmid that contains the minitransposon Amplification by PCR selection mini Tn5 Tc cloned in the pUT backbone Mini Tn5 Te carries as selection PRIMERS pUToriR6K of positive clones and PR 3280 marker the tet gene that confers tetracycline resistance sequencing of cloned fragments pUT mini Tn5 Cm Mini transposons derivates CV 3223 vectors repeat insertion events pUT mini Tn5 Km Mini transposons derivatives CV 3224 3 About mini Tn5 System vectors repeat insertion events pUT mini Tn5 Sm Sp Mini transposons derivatives CV 3225 vectors repeat insertion events Mini transposons derivatives vectors provide a straightforward tool to clone and insert foreign genes stably into the ch
3. l Magnesium phosphate MgSO4 7H20 1M solution mini In5 Tc 1 2258 omits le Dissolve 24 6 g of MgSO 7H20 in 100 ml of deionised water O termini 2239 2258 Deel terili T4 gene transcription terminators inverted VIO CON e cms repeats 64 188 2050 2174 tet coding region Tc resistance 489 1676 3 ANTIBIOTICS AND ADDITIVES pUTmini In5 Tc oriR6K bla 7477 bp R6K origin of replication 2351 2731 5 Not RP4 origin of tranfer 2735 4461 O nT Gnigin At 2735 el Ampicillin 100 mg ml stock solution N bla coding region ampicillin resistance N 4921 5768 tnp coding region transposase 5932 Dissolve 1 g ampicillin in 9 ml of deionised water mobRP4 7372 Add deionised water to a 10 ml volume final Filter sterilizes the solution with a 0 22 u filter Store the stock solution at 20 C Biomedal Cat No RS 3217 Y Biomedal 7 Troubleshouting PROBLEM Loss of pUT plasmids Low or zero yield of exconjugants All exconjugants are resistant to ampicillin or others B lactam antibiotics omeda Plasmid derivatives cannot be maintained into delivery Apir strains Selection conditions are inappropriate Zero or low frequency of RP4 mediated transfer The exconjugant phenotype is not due to an authentic transposition events SOLUTIONS Using of alternative donor strains It is advisable to make and assay with the target cells before the matting plating is carried out
4. Km 1 2356 termini 1 19 831 849 reverse complement O termini 2338 2356 km coding region kanamycin resistance 857 1648 T4 gene 32 transcription terminators 64 188 2148 2272 inverted repeats R6K origin of replication 2448 2829 mobRP4 origin of tranfer 2833 4559 bla coding region ampicillin resistance 5018 5876 tnp coding region transposase 6031 7458 bla bla mobRP4 tnp pUTmini Tn5 Cm 8686 bp O oriR6K cat II Not pUTmini Tn5 Km 7575 bp mobRP4 Not yO oriR6K Vig Biomedal 8 Reagents and recipes pUTmini Tn5 Sm Sp A Media and solutions SEQUENCE REFERENCE POINT 1 MEDIA mini Tn5 Sm Sp 1 2167 termini 1 19 A vin af O termini 2149 2167 LB per liter liquid T4 gene transcription terminators inverted UTmini Tn5 Sm S repeats 64 188 1959 2083 10 gtryptone Pm SSP aad coding region Sp Sm resistance 5 g yeast extract bla 7386 bp Y Notl 443 1450 10g NaCl O R6K origin of replication 2260 2640 3 i mobRP4 origin of tranfer 2644 4370 y ee 5 oriR6K e e ah Dissolve in 950 ml of deionised water p 4830 5687 Adjust pH to 7 0 with 5 N NaOH TPPA tnp coding region transposase 5842 Add deionised water to 1 final volume iag 7269 Autoclave and let cool to below 55 C LB agar plates Exactly like liquid LB but adding 15 g agar after adjusting the pH pUTmini Tn5 Tc 2 MATING SOLUTION SEQUENCE REFERENCE POINT
5. because the minimal inhibitory concentration of the toxic compound used as marker can vary considerably among genera and strains Properties of target cells such as restriction system surface exclusion phenomena may lead to low frequency or zero transfer If the target strain is unable to act as a recipient for RP4 the pUT plasmid cannot be introduced at high frecuency Also some changes in the mating protocol can be introduced For example avoid the pregrowth of the recipient strains on antibiotic medium prior to mating since traces of the antibiotic present in the suspension or accumulated by the recipient cells can kill donor cells Shorter times Lower temperatures 30 C To E coli recipient strains use LB medium with 0 1 M citrate to avoid the recipient strain infection by Apir phage 5 Restriction analysis 5 1 pUTmini Tn5 Cm Restriction analysis RESTRICTION ENZYMES THAT DO NOT CUT pUTmini Tn5 Cm Aat ll BbvC EcoICR Nru Sbf v Acc 51 BhBl EcoN Nsi She 5 Afl Il Blp Fse Pme Srf O Ale Bpl FspA PshA Swa e Apa BsiW Kpn PspOM Zra 2 Asc BstB Mlu Psr a AsiS Bsu36 Nco Rsr Il Bae Cla Nde Sac En E a y RESTRICTION ENZYMES THAT CUT ONCE pUTmini Tn5 Cm a Age BssH Dra Ill Pac Sph Bel BssS Drd Pml Stu Bmt BstAP EcoR V Pvu Xba Bpul0 BstE II Mfe Sca Xcm 9 Bsax BstX Msc SexA Xmn BsrG Bs
6. necessary These genes can be present in a host strain or can be provided by way of a helper plasmid When it is used the chromosomally integrated conjugal transfer functions of a specific host strain such us E coli SM10 Apir or E coli S17 1 Apir BIOMEDAL Cat No BS 3234 into which the transposon vector DNA is transformed the transfer is accomplished by means of biparental mating The transfer can also be achieved by triparental mating mediated by a helper plasmid In the triparental mating the conjugatable suicide minitransposon donor is introduced into the recipient strain by a helper plasmid which self mobilises from its own host into the donor strain and provides the genes neccesary for the conjugal transfer of the miniTn5 from the donor host strain to the recipient provided the vector plasmids contain the specific recognition site for mobilisation Since some Apir RP4 strains like E coli SM10 Apir or E coli S17 1 Apir cannot stably maintain the pUT plasmids we recommend other strains to propagate this vectors without problems such as DH5a Apir In this case to mobilize the delivery plasmid directly from the donor strain into target cells a triparental mating with a helper strain such as E coli DH5a pRK2013 BIOMEDAL Cat No BS 3236 or E coli DH5a pRK2073 BIOMEDAL Cat No BS 3263 is necessary NOTE e To propagate pUTmini Tn5 in Ipir strains you can use the TSS solution to perform the transformation Triparen
7. see Troubleshouting Vig Biomedal 3 After the filtering remove carefully the drained membrane on the agar RESTRICTION ENZYMES THAT CUT ONCE pUTmini Tn5 Tc surface of an LB plate cell side up You can help yourself with sterile tweezers preferably with curved tips Age Bsg Drd Mfe Psh Al Sex Al Xcm Alo Bss HII Eco NI Msc Psi Sna Bl Xho Bbs Bss SI EcoRV Notl Pvu Sph Xmn NOTE Bcl Bst Z171 Fsp Al Nru Pvu Il Tth 1111 Air bubbles should be avoided between the filter and agar surface Bpu 101 Dra Ill Hpa Pml Sca Xba 4 Incubate the plates at 30 37 C for 8 18 hours NOTE Several alternatives to mating protocol are possible according to individuals needs Strains ratios donor recipient helper temperatures time of mating and culture medium can be changed to suit specific requeriments of the recipients 6 Protocol f Biparental mating 1 Clonin t f ti i If you use E coli SM10 Apir or E coli SM17 1 Apir like donor strains to Cloning drid iransiormoton of donar sirain set up the biparental mating you must follow the same protocol that when use triparental mating but without helper strain You must mix equal To insert your DNA fragment in the chromosome of the recipient strain volumes 5 10 ul of overnight cultures of the donor and recipient strain first of all you must clone it into the pUTmini Tn5 vector We recommend that in 5 ml of 10 mM MgSO4 and proceed as described abov
8. A added do not must be more than 15 ul of volume 10 of the competent cell volume Mix gently and place the transformation reaction on ice for 30 minutes 9 Heat shock Incubate the tubes by immersing in a 42 C water bath for exactly 45 seconds Immediately replaces the tubes on ice 10 Add 1 ml of LB medium to each tube Incubate at 37 C for the time required to the resistance antibiotic expression typical 60 min 11 For each transformation reactions we recommend diluting the cells 1 10 and plating out 100 ul of the 1 10 dilutions 100 ul of the transformation undiluted cells and the rest of the cells concentrated for centrifugation onto LB plates containing the appropriate selection antibiotics Allow the plates to completely absorb any excess media 12 Incubate the plates overnight at 37 C 12 14 hours The delivery system employed for all mini Tn5 transposons is the pUT plasmid The backbone pUT includes the R6K origin of replication sequence the RP4oriT origin of transfer sequence the bla gene sequence ampicillin resistance and the tnp gene sequence The inp is a mutant tnp gene of IS50p Tn5 transposon that lacks Not sites and which encodes for the transposase needed for transposition of the mini Tn5 elements tnp carries a single mismatch that changed the GCG codon specifying Ala 168 of the tnp gene to the Ala codon GCC This change eliminate the Not site without changing the structure of the tnp product 4
9. The tnp gene is presented in cis but external to the mobile element Due to the loss of the tnp gene after insertion minitransposons are stably inherited and do not favour DNA rearrangements or others forms of genetic inestability Plasmids having the R6K origin of replication require the R6K specified replication protein z and can be maintained only in host strains producing this protein lt also carry the origin of transfer oriT of plasmid RP4 which results in its efficient conjugal transfer to recipient strain from donor strains expressing RP4 conjugative functions 5 Delivery plasmids are thus mantained stably in Apir lysogens or in E coli strains with the pir gene recombined in their chromosome and can be mobilised into target strains through RP4 transfer functions However once transferred it is unable to replicate in recipients that lack the z protein The pUT plasmids are suicide vectors since the oriR6K is functional only in the presence of the pir gene and they cannot replicate in the recipient cells which have not the x protein Delivery of the donor plasmids into selected host bacteria is accomplished through mating with the target strain One important feature of mini Tn5 elements is that as a result of the loss of the transposase cognate inhibitor along the pUT system after transposition transposase function is not maintained in target cells a single recipient strain can be used for repeated insertion events with differe
10. coli plasmid chromosome shuttle system Journal Bacteriology 182 p 842 847 MY Biomedal Chloramphenicol 25 mg ml stock solution Dissolve 125 mg chloramphenicol in 5 ml of ethanol Store the stock solution at 4 C Biomedal Cat No RS 3218 Kanamycin 25 mg ml stock solution Dissolve 0 25 g of kanamycin in 9 ml of deionized water Bring up to a 10 ml final volume with water deionized Filter sterilizes the solution with a 0 22 u filter Store the stock solution at 20 C Biomedal Cat No RS 3219 Streptomycin 10 mg ml stock solution Dissolve 0 1 g of streptomycin in 9 ml of deionised water Bring up to a 10 ml final volume with water deionised Filter sterilizes the solution with a 0 22 u filter Store the stock solution at 20 C Biomedal Cat No RS 3221 Tetracycline 25 mg ml stock solution Dissolve 200 mg tetracycline in 5 ml water deionised 5 ml ethanol mix Store the stock solution at 20 C Biomedal Cat No RS 3220 A Vectors Maps pUTmini Tn5 Cm mini In5 Cm 1 3467 termini 1 19 O termini 3449 3467 T4 gene transcription terminators inverted repeats 64 188 3259 3383 cat II coding region Cm resistance 1452 2090 R6K origin of replication 3560 3940 mobRP4 origin of tranfer 3944 5670 bla coding region ampicillin resistance 6130 6987 tnp coding region transposase 7139 8581 pUTmini Tn5 Km SEQUENCE REFERENCE POINT miniTn5
11. e you clone your fragment in pUC1 8Not auxiliary plasmid prior the cloning in pUT plasmid The protocol should be as follows 1 Excise the DNA fragment from pUC1 8Not plasmid by means of Not digest 6 3 Selection of exconjugants 2 Digest the pUTmini Tn5 vector transfer vector with Not We recommend to treat the digested plasmid with alkaline phosphatase to prevent re ligation The selection of clones with the minitransposon inserted in the 3 Ligate your DNA fragment and pUTmini Tn5 backbone chromosome is a critical step The procedure is as follows 4 Transform the ligation reactions into Apir cells Select the transformants by plating onto LB ampicillin 100 ug ml antibiotic that the pUTmini Tn5 1 If you use filter system confers resistance the minimal inhibitory concentration in each case plates Resuspend the filter with the mating mix in 5 ml of 10 mM MgSOa Incubate at 37 C overnight 2 If you use drop system Recover the matting mix on the plate using a sterile inoculating loop and Y NOTE resuspend in 5 ml of 10 mM MgSO4 e You can use the TSS solution BIOMEDAL Cat No RS 3215 RS 3216 to perform the transformation l NOTE e This suspension can be kept at 4 C for several weeks MY Biomedal 6 2 Transfer of the DNA into the recipient strain To transfer the DNA cloned in pUTmini Tn5 plasmid into the recipient strain the genes that code for mobilisation and chromosomal transfer tra and mob genes are
12. l Srampruenies 20 BOA Apa Bpl BstBl EcoNI Mlul Olil PshAl Sbfl Swal Kanamycin 25 75 ug ml Asc Bsa XI BstXl Eco RV Ncol Pacl Psp OMI Sof Streptomycin 50 100 ug ml Spectinomycin 50 100 ug ml RESTRICTION ENZYMES THAT CUT ONCE pUTmini Tn5 Sm Sp Tetracycline 2 5 15 ug ml Age Bsg Bst Z17 Nhel Sca Xba Alo Bsm Drd Not Sex Al Xho Bbs Bss SI Hpa Pml Sna Bl Bip Bst API Mfe l S i ia 3 Incubate the plates at the optimal growth temperature of the recipient Bpo TOI Ber EIl Mge eval i strain until colonies become visible 5 4 pUTmini Tn5 Tc Restriction analysis 4 Confirm that the acquisition of the selected phenotype is due to an authentic transposition event and not to integration of the delivery plasmid RESTRICTION ENZYMES THAT DO NOT CUT pUTmini Tn5 Tc in a recipient replicon or its illegitimate replication in the new host The Aar Il Boei Bsa XI Bst Ell Fsel Nsil Psp OMI Sof insertion can be contirmed by Southern blot or PCR analysis Other Acc 65 BbyCl BsiWl BstXl Kpn Oli Psr Spe procedure consists to grow the exconjugants onto medium containing B Afl 1l Bfr BI Bsp El Bsu 36l Mlul Paci Rsr Il Srf lactam antibiotic because authentic transposition results in the loss of the Apa Blp Bsr Gl Cla Ncol Pme Sac Stu portion of the delivery plasmid containing bla gene and the exconjugants Asc Bpl Bst Bl Ecl 13611 Ndel Ppul10l Sbf Swa will be sensitive to ampicillin and other B lactams
13. ments donor and helper strains The main feature of the cloning vectors of this system is their ease for insertion of one or more segments of heterologous DNA in the chromosome of the strain of interest by means of a mating between the donor strain and the recipient strain This system can be used for insertional mutagenesis promoter probing and analysis of transcriptional terminators KIT COMPONENTS CAT NO KT 3260 PRODUCT QUANTITY STORAGE CAT NO pUTmini Tn5 Cm 8 ug 4 C or 20 C CV 3223 pUTmini Tn5 Km 8 ug 4 C or 20 C CV 3224 pUTmini Tn5 Sm Sp 8 ug 4 C or 20 C CV 3225 pUTmini Tn5 Tc 8 ug 4 C or 20 C CV 3226 Sequences CD 1CD o e NOTE e All components of the Kit are shipped at room temperature Upon arrival store the kit components according to the directions in the table above e The plasmid is supplied dryed CD Rom supplied with the kit contains vectors sequences NOTES Vig Biomedal 11 Short protocol A Cloning and transformation of donor strain 1 Ligate the DNA fragment with pUTmini Tn5 vector prior digestion with Not 2 Transform the ligation reactions into host strain Apir cells and select the transformants by plating onto selective LB plates Incubate overnight at 37 C B Transfer of the DNA into the recipient strain triparental mating Filter system 1 Mix the 10 50 ul of overnight cultures of the donor recipient and helper strains 1 1 1 mixtures of cultures o
14. name AINS 7 chromosome of Gram negative bacteria target The modular nature of the Streptomycin Test culture RS 322 mini transposons facilitates the construction of mobile elements la carte Tetracycline Test culture RS 3220 Hybrid transposons bearing the DNA segment of interest can be easily CENT Potassium Tellurite Selection of transformants RS 3222 assembled with the help of the specialised cloning vector as pUC18Not or SOLUTIONS TSS Competent cells preparation RS 3215 pUC185Sfi vector RS 3216 Vig Biomedal NOTE e Alternatively you can use exponential growing cells obtained in the same day of the transformation directly from fresh colonies l IMPORTANTI While the culture is growing place on ice or at 4 C LB medium that you will use in step 6 4 When the culture reaches the desired OD gg divide the cultures in microcentrifuge tubes 1 ml tube and place on ice for 5 minutes 5 Centrifuge at 12000 r p m for 30 seconds at room temperature 6 Discard the medium and resuspend gently the pellets in 75 ul of cold LB medium Place on ice for 5 minutes 7 Add 75 ul of 2xTSS solution and mix gently Replace on ice for 5 minutes IMPORTANTI Invert the tube of TSS solution several times before use Now the cells are ready to perform the transformation TRANFORMATION PROCEDURE STANDARD PROTOCOL 8 Add 1 50 ng of DNA supercoiled DNA to the competent cells obtained in step 7 We recommend adding 5 10 ul of DNA the DN
15. ng vector because of the modular nature of the constructions Use of cleavage sites of the rare cutters Not and Sfi it s convenient that the vector has an array of unique cloning sites The pUTmini Tn5 plasmids are transferable to a variety of bacteria 9 References 1 De Lorenzo V Herrero M Jakubzik U Timmis K Mini Tn5 Transposon Derivatives for Insertion Mutagenesis Promoter Probing and Chromosomal Insertion of Cloned DNA in Gram Negative Eubacteria Journal Bacteriology 172 p 6568 6572 2 Prentki P Binda A Epstein A Plasmid vectors for selecting 15 promoted deletions in cloned DNA sequence analysis of the omega interposon Gene 103 p 17 23 3 De Lorenzo V Timmis K N Analysis and Construction of Stables Phenotypes in Gram Negative Bacteria with Tn5 and Tn10 Derived Minitransposons Methods in Enzimology 235 p 386 405 4 Herrero M De Lorenzo V Timmis K N Transposon vectors Containing Non Antibiotic Resistanse Selection Markers for Cloning and Stable Chromosomal Insertion of Foreign Genes in Gram Negative Bacteria Journal Bacteriology 172 p 6557 6567 5 De Lorenzo V Herrero M S nchez J Timmis K N Mini transposons in microbial ecology and environmental biotechnology FEMS Microbiology Ecology 27 p 211 224 6 Boyd D Weiss D S Chen J C Beckwith J Towards single Copy gene expression systems making gene cloning physiologically relevant Lamda Inch a simple Escherichia
16. ntially marked minitransposons Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers Vig Biomedal Ease for insertion of one or more segments of the heterologous DNA in the chromosome of the strain of interest any heterologous DNA segments can be inserted into the chromosome of target cells after a few simple genetic manipulations Conditional replication to allow selection for integration into the chromosome suicide plasmids The mini Tn5 would be able transpose from the plasmid to the chromosome but when succesive cell divisions occur the plasmid vector would be lost to the population Insert foreign DNA stably into the chromosomes Wide range of inserted fragments size pUTmini Tn5 plasmids allow to clone from small fragments until large size fragments Heterologous DNA of 12 Kb cloned in mini Tn5 derivatives are transposed at frequencies in the range of those observed with insert lacking transposon 3 In other system the range of sizes is more restricted For example lambda Inch vectors system allow to insert fragments a total of about 7 Kb 6 Inserted DNA segments remain stably inherited and produce no burden to the carrier strain Posibility of multiple insertions in the same strains only limited by the available selection markers as a result of the loss of the transposase function which is not maintained in target cells Easy modification of the cloni
17. on LB plate without dispense it Let it dry Go to step 4 NOTE You can use alternative culture medium instead of LB to favour recipient strain viability When you use the filter system if too many donor recipient or helper cells are used and the membrane clogs it is better to refilter a more diluted mix e If you need only few insertions 10 ul drops of cultures of donor recipient and helper strains can be mixed and spotted on an LB plate which is then dried and incubated for several hours before the mating mixture is streaked out on selective medium go to step 4 MY Biomedal
18. romosomes of a variety of Gram pUT mini Tn5 Tc aS aU sels em CV 3226 negative bacteria such E coli Klebsiella Salmonella Proteus Vibrio Bortedella Terre ee v 2 o Actinobacillus der da a Rhodobacter ena VECTORS PY mini Ino le M oe anspor cea ie Aca genes and S Peudomongds ajent a eae P y 3 pUT lacZl Mini transposons derivatives CV 3256 generation of insertion mutants insertion mutagenesis and in vivo fusions with vectors With a reporter gene reporter genes promoter probing Mini transposons cloning vectors have the pUT lacZ2 A CV 3257 advantages of natural transposons but lack their disadvantages vectors withia reporter gene pUT phoA Mini transposons derivatives CV 3258 All pUTmini Tn5 plasmids carry a backbone common pUT backbone vectors with a reporter gene and a mobile element variable mini Tn5 The mobile element mini Tn5 pUT luxAB Mini trangposons derivatives CV 3259 elements consists of an Sfi cassette containing a selection marker and a single vectors with a reporter gene Not site outside of the cassette that can be used for cloning foreign DNA pUC18Not Auxiliary plasmid for cloning CV 3282 fragments Flanking these two features are the 19 basepair and O termini of pUC18NotSfi Auxiliary plasmid for cloning CV 3248 Tn5 transposon In this vectors any heterologous DNA segment can be cloned Apicillin Test culture RS 3217 bo Pa Chloramphenicol Test culture RS 3218 Edd the dl of a pel radia Pa n a el ANTIBIOTICS Ka
19. tZ17 Nhe SnaB 5 2 pUTmini Tn5 Km Restriction analysis RESTRICTION ENZYMES THAT DO NOT CUT pUTmini Tn5 Km Aat ll Bbs Bsi WI Cla Fsp Al Pac Psp OMI Spel Afl Il Bbv CI Bsp El Ecl 136ll Mlu PfI MI Psr Srf Apa Bfr Bl Bsr Gl Eco NI Nde Pme Sac Stu Asc Blp Bst Ell Eco RV Nsi Pou 101 Sbf Swa Bae Bsa XI Bst XI Fse Oli Psh Al Sgt M Biomedal RESTRICTION ENZYMES THAT CUT ONCE pUTmini Tn5 Km 2 Plate 100 500 ul of this suspension on selective LB agar medium with tetracycline The tetracycline concentration must be adequate to ensure Acc 651 Bsg r a P de maintenance of the delivery plasmid but you must determine the minimal a a e Pr A on inhibitory concentration of antibiotic s x Bpl Bst Z171 Nco Psi Sna BI Bpu 10l Bsu 36l Nhe Pvu Sph NOTE When the marker carried by the minitransposon is an antibiotic resistance determinant optimal concentrations of antibiotics to the selection can vary among strains Must be determined the minimal inhibitory concentration MIC in each case Can be orientatives the concentrations which appears in the next table 5 3 pUTmini Tn5 Sm Sp Restriction analysis RESTRICTION ENZYMES THAT DO NOT CUT pUTmini Tn5 Sm Sp Aat Il Bae Bsi WI Bsu 36l Fse Nde PfI MI Psr Spe ANTIBIOTIC ORIENTATIVE MIC Acc 651 BbyCl BspEl Clal Fsp Al Nrul Prel Rsr Il Srf Chi henicol 5 50 ug ml Afl Il BfrBI BsrGl Ecl136ll Kpnl Nsil PpulOl Sacl Stu
20. tal Mating 1 Grow overnight cultures in LB of each of the three strains donor strain recipient strain and helper strain a Inoculate a single colony of donor strain DH5a Apir carrying the desired pUT derivative plasmid in LB medium containing 100 ug ml ampicillin and the adequate concentration of the antibiotic of selection to ensure maintenance of the delivery plasmid Incubate with shaking at 37 C overnight b Grow the recipient strain Inoculate a single colony under the same conditions at different temperature if necessary but preferably without selection see Troubleshooting c Inoculate a single colony of E coli DH5a pRK2013 or E coli DH5a pRK2073 if you use pUTmini Tn5 Km in LB medium preferably without selection 2 Mix the 10 50 ul 108 cells of overnight cultures of each of the three strains 1 1 1 mixtures of cultures overnight in 5 ml of 10 mM MgSO4 vortex for a few seconds transfer to a 5 ml disponsable syringe and filter throught a Millipore membrane 13 25 mm diameter type HA 0 45 um or equivalent placed on a reusable filter case NOTE To remove antibiotics from donor strain culture an additional washing step of the culture is necessary IMPORTANT Alternatively mix the 10 50 ul of overnight cultures of helper strain donor strain and recipient strain centrifugate the mix and discard the supernatant Resuspend the pellet in 20 ul of LB medium or 10 mM MgSO4 using vortex and place it
21. tors All pUTmini Tn5 vectors included in this kit contain a mini Tn5 element carrying an antibiotic resistance cassettes flanked by and O termini terminal repeat sequences of Tn5 The cassettes conferring resistance to tetracycline streptomycin spectinomycin and chloramphenicol are derived from Q interposon and the cassette conferring resistance to kanamycin contains the kanamycin resistance interposon originally isolated from Tn5 1 These four mini Tn5 carry 156 nt inverted repeats consisting of the T4 phage gene 32 transcriptional terminators strong transcriptional terminator flanking the antibiotic resistance gene as well as synthetic translational stop codons in all three reading frames 1 2 Selection for resistance to Antibiotic selection marker Replication Origin R6K Transfer Origin RP4oriT Antibiotic Resistance Ampicillin selection marker Host strain E coli Apir strains Copy number Low UTmini In5 Cm pUTmini Tn5 Cm is a 8686 basepair plasmid that contains the minitransposon mini In5 Cm cloned in the pUT backbone Mini Tn5 Cm carries as selection marker the cat Il gene that confers chloramphenicol resistance pUTmini Tn5 Km pUTmini Tn5 Km is a 7575 basepair plasmid that contains the minitransposon mini Tn5 Km cloned in the pUT backbone Mini Tn5 Km carries as selection marker a kanamycin resistance gene Mini Tn5 Km has one more end at the left extreme of the kanamycin resistance gene that itself originates
22. vernight in 5 ml of 10mM MgSO 4 vortex for a few seconds transfer to a 5 ml disponsable syringe and filter through a Millipore membrane or equivalent 2 Remove carefully the drained membrane on the agar surface of an LB plate cell side up avoiding bubbles formation 3 Incubate the plates at 30 37 C for 8 18 hours Drop system 1 Mix the 10 50 ul of overnight cultures of the donor recipient and helper strains 1 1 1 mixtures of cultures overnight centritugate the mixture and discard the supernatant Resuspend the pellet in 20 ul of LB medium and place on a LB plate Let to dry the drop on the plate 2 Incubate the plates at 30 37 C for 8 18 hours C Selection of exconjugants 1 To filter system Resuspend the filter with the mating mix in 5 ml of 10 To drop system Recover the mating mix on the plate and resuspend in 5 ml of 10 mM MgSO4 2 Plate 100 500 ul of this suspension on selective LB agar medium containing tetracycline 3 Incubate the plates at the optimal growth temperature of the recipient strain until colonies become visible 4 Confirm that the acquisition of the selected phenotype is due to an authentic transposition event and not to integration of the delivery plasmid in a recipient replicon or its illegitimate replication in the new host by Southern blot PCR analysis or growing the exconjugants onto medium containing B lactam antibiotic 2 Vectors and strains 2 1 Vectors 2 1 1 pUT mini Tn5 vec
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