pUT lacZ2.FH11
Contents
1. Biomedal Cat No RS 3217 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 sterilize the solution with a 0 22 y filter Store the stock solution at 4 C Biomedal Cat No RS 3219 8 References 1 De Lorenzo V Timmis K N 1994 Analysis and Construction of Stables Phenotypes in Gram Negative Bacteria with Tn5 and Tn10 Derived Minitransposons Methods in Enzimology 235 p 386 405 2 De Lorenzo V Herrero M Jakubzik U Timmis K N 1990 Mini Tn5 transposon Derivatives for Insertion Mutagenesis Promoter probing and Chromosomal Insertion of Cloned DNA in Gram Negative Eubacteria Journal of Bacteriology 172 p 6568 6572 3 De Lorenzo V Timmis K N 1994 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 1990 Transposon vectors Containing Non Antibiotic Resistance 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 1998 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 2000 Towards single Copy gene expression syst2. protein 1 and can be maintained only in host strains producing this protein Apir strain lt also carry the origin of transfer oriT of plasmid RP4 which results in 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 strain cells through RP4 transfer functions Delivery of the donor plasmids into selected host bacteria is accomplished through mating with the target strain MY Biomedal 2 General usages Applications of pUT lacZ2 Mini Tn5 vectors are used in the analysis construction and manipulation of complex phenotypes in a wide range of Gram negative bacteria The main virtue of these cloning vectors 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 a donor strain and a recipient strain As cloning vectors are useful to stably maintain a recombinant phenotype in the recipient strain Mini transposons derivative vectors provide a straightforward tool to clone and insert foreign genes stably into the chromosomes of a variety of Gram negative bacteria such E coli Klebsiella Salmonella Brucella Proteus Vibrio Bortedella Actinobacillus Rhizobium Acinetobacter Rhodobacter Agrobacterium Alcaligenes and several Pseudomonad
3. transform the donor strain with pUTlacZ2 vector The protocol should be as follows 1 Transform S17 1 Apir or DH5a Apir cells with pUTlacZ2 2 Select the transformants by plating onto LB ampicillin 100 ug ml kanamycin 25 ug ml plates Incubate at 37 C overnight NOTE You can use the TSS solution to perform the transformation Biomedal Cat No RS 3215 RS 3216 amedal 6 Troubleshooting PROBLEM Loss of pUT plasmids Low or zero yield of exconjugants All exconjugants are resistant to ampicillin or others B lactam antibiotics 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 perform assay with the target cells before the matting plating is carried out 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 s
4. 216 1 2 pUT mini Tn5 vectors features All pUTmini Tn5 plasmids carry a common backbone pUT backbone and a variable mobile element mini Tn5 The mobile element mini Tn5 elements consists of an Sfi cassette containing a selection marker and a single Not site outside of the cassette that can be used for cloning foreign DNA fragments Flanking these two features are the 19 base pair and O termini of Tn5 transposon In this vectors any heterologous DNA segment can be cloned within the boundaries of a mini Tn5 element and finally inserted into the chromosome of Gram negative bacteria target 3 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 tnp is a mutant tnp gene of IS50pR Tn5 transposon and encodes for the transposase needed for transposition of the mini Tn5 elements tnp carries a single mismatch that changed the GCG codon specitying 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 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 cause DNA rearrangements or others forms of genetic instability Plasmids having the R6K origin of replication require the R6K specified replication
5. Apir Propagation of pUT plasmids BS 3235 STRAINS E coli S17 1 Apir Propagation of pUT plasmids BS 3234 E coli DH5a pRK2013 Mating helper strain BS 3236 E coli DH5a pRK2073 Mating helper strain BS 3263 Amplification by PCR selection PRIMERS pUToriR6K of positive clones and PR 3280 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 vectors repeat insertion events pUT mini Tn5 Sm Sp Mini transposons derivatives CV 3225 vectors repeat insertion events pUT mini Tn5 Tc Mini transposons derivatives CV 3226 vectors repeat insertion events VECTORS PUT mini Tn5 Tel Mini transposons derivatives CV 3227 vectors repeat insertion events pUT lacZ1 Nig tes Peso Ns derivatives CV 3256 vectors with a reporter gene pUT lacZ2 Mini transposons derivatives CV 3257 vectors with a reporter gene pUT phoA Mini transposons derivatives CV 3258 vectors with a reporter gene pUT luxAB Mini transposons derivatives CV 3259 vectors with a reporter gene pUC18Not Auxiliary plasmid for cloning CV 3282 pUC1 8NoiSfi Auxiliary plasmid for cloning CV 3248 Apicillin Test culture RS 3217 Chloramphenicol Test culture RS 3218 ANTIBIOTICS Kanamycin Test culture RS 3219 Streptomycin Test culture RS 3221 Tetracycline Test culture RS 3220 oe Potassium Tellurite Selection of transformants RS 3222 an SOLUTIONS TSS Competent cells preparation RS 3215 RS 3
6. LE 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 Web www biomedal com e Email info biomedal com Ed 1 2006 Biomedal S L pUT lacZ2 vector User s manual Biomedal NOTES pUT lacZ2 vector Delivery System pUTmini In5 vector carrying a minitransposon containing a lacZ gene to generate transcripcional translational fusions For research use only Contents 1 Product Description 2 1 1 pUTlacZ2 vector 2 1 2 pUTmini Tn5 vectors features 3 2 General usages Applications of pUT lacZ2 4 3 Advantages of mini Tn5 Vectors vs other chromosome integration systems 5 4 pUT lacZ2 Map 5 5 Protocol 6 5 1 Transformation of donor strain 6 5 2 Transfer of the DNA into the recipient strain 7 5 3 Selection of exconjugants 9 5 4 B galactosidase assay 10 6 Troubleshooting 11 7 Reagents and recipes 12 8 References 13 9 Related products 14 10 Short protocol 15 MY Biomedal 1 Product Description 1 1 pUT lacZ2 vector pUT lacZ2 is a 10 4 Kb plasmid that is included in the collection of pUTmini Tn5 derived vectors This vector contains the minitransposon mini Tn5lacZ2 cloned in the pUT backbone Mini Tn5lacZ2 carries a lacZ sequence downstream of the Tn5 end of the transposon Since the termini have neither transcriptional terminators nor stop codons in several of the possib
7. by Southern blot or PCR analysis Another procedure consist of growing the exconjugants on medium containing B lactam antibiotic because authentic transposition results in the loss of the portion of the delivery plasmid containing bla gene and the exconjugants will be sensitive to ampicillin and other B lactams see Troubleshooting MY Biomedal
8. ems making gene cloning physiologically relevant Lamda Inch a simple Escherichia coli plasmid chromosome shuttle system Journal Bacteriology 182 p 842 847 MY Biomedal 7 Reagent and Receipes 1 MEDIA LB per liter liquid 10 g tryptone 5 g yeast extract 10g NaCl Dissolve in 950 ml of deionised water Adjust pH to 7 0 with 5 N NaOH Add deionised water to 1 final volume Autoclave and let cool to below 55 C LB agar plates Exactly like liquid LB but adding 15 g agar after adjust the pH 2 MATING SOLUTION Magnesium phosphate MgSO 7H 20 1M solution Dissolve 24 6 g of MgSO 7H O in 100 ml of deionised water Autoclave sterilize 3 B GALACTOSIDASE ASSAY REAGENTS Z Butter 16 1 g NagHPO47H2O 0 75 g KCI Dissolve in 950 ml of deionised water Adjust pH to 7 0 Add deionised water to 1 final volume Do not autoclave Store at 20 C in 50 ml aliquots Add 135 ul of B mercaptoethanol 50 ml Z buffer before use it ONPG Dissolve 0 8 g ONPG in 200 ml of Z buffer Store at 20 C in 10 ml aliquots 3 Advantages of mini Tn5 Vectors vs other chromosome integration system 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 chrom
9. le frames this vector is designed for generation of type II transcriptional translational lacZ fusions 1 Mini Tn5 lacZ2 creates carboxy terminal gene fusions with the interrupted chromosomal gene and lacZ when inserted in the proper reading frame These fusions begin with an lacZ moiety at codon 9 which is separated from the interrupted gene by a 49 base pair linker sequence composed of the 19 base pair end of Tn5 and an additional 30 base pairs 2 The minitransposon also carries as a selection marker a gene that confers kanamycin resistance upstream of O termini Selection for resistance to Kanamycin Replication Origin R6K Transter Origin RP4oriT Antibiotic Resistance Ampicillin Kanamycin Host strain E coli Apir strains Copy number Low pUT lacZ2 8 ug 4 C or 20 C CV 3257 NOTE The plasmid is supplied dried 10 Short Protocol A Transformation of donor strain 1 Transform 17 1 Apir or DH5a Apir cells with pUTlacZ2 2 Select the transformants by plating onto LB ampicillin 100 tg ml kanamycin 25 ug ml plates Incubate at 37 C overnight B Transter 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 overnight in 5 ml of 10mM MgSO vortex for a few seconds transfer to a 5 ml disposable syringe and filter through a Millipore membrane or equivalent 2 Remove careful
10. lper strain e Inoculate a single colony of donor strain DH5a Apir carrying the desired pUT vector or derivative plasmid in LB medium containing 100 ug ml ampicillin and 25 ug ml of kanamicyn the adequate concentration to ensure maintenance of the delivery plasmid Incubate with shaking at 37 C overnight e Grow the recipient strain Inoculate a single colony under the same conditions at different temperature if necessary but preferably without selection see Troubleshooting e Inoculate a single colony of E coli DH5a pRK2073 in LB medium preferably without selection MY Biomedal 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 10mM MgSO vortex for a few seconds transfer to a 5 ml disponsable syringe and filter through a Millipore membrane 13 25 mm diameter type HA 0 45 um or equivalent placed on a reusable filter case NOTE e pRK2073 plasmid confers streptomycin spectinomycin resistance e To remove antibiotics from donor strain culture an additional washing step is necessary IMPORTANTI Alternatively mix the 10 50 ul of overnight cultures of helper strain donor strain and recipient strain centrifuge the mix and discard the supernatant Resuspend the pellet in 20 ul of LB medium or 10 mM MgSO4 using vortex and place it on LB plate without dispersing it Let it dry Go to step 4 NOTE e When you use the filter system if to
11. ly 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 centrifuge 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 exconjungants 1 To filter system Resuspend the filter with the mating mix in 5 ml of 10 mM MgSO To drop system Recover the mating mix on the plate and resuspend in 5ml of 10 mM MgSO 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 MY Biomedal 9 Related Products PRODUCT DESCRIPTION Cat No E coli DH5a Apir Propagation of pUT plasmids_ BS 3233 E coli CC118
12. o 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 3 After the filtering remove carefully the drained membrane on the agar surface of an LB plate cell side up You can help yourself with sterile tweezers preferably with curved tips NOTE e You can use alternative culture medium instead of LB to favour recipient strain viability e Air bubbles should be avoided between the filter and agar surface 4 Incubate the plates at 30 37 C for 8 18 hours NOTE e 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 Biparental mating If you use E coli SM10 Apir or E coli SM17 1 Apir as donor strains to set up the biparental mating you must follow the same protocol as when using triparental mating but without helper strain You must mix equal volumes 10 50 ul of overnight cultures of the donor and recipient strain in 5 ml of 10 mM MgSO and proceed as described above 5 3 Selection of exconjugants The selecti
13. on of clones with the minitransposon inserted in the chromosome is a critical step The procedure is as follows 1 lf you use filter system Resuspend the filter with the mating mix in 5 ml of 10 mM MgSO If you use drop system Recover the matting mix on the plate using a sterile inoculating loop and resuspend in 5 ml of 10 mM MgSQ NOTE This suspension can be kept at 4 C for several weeks 2 Plate 100 500 ul of this suspension on selective LB agar medium with kanamycin The kanamycin concentration must be adequate to ensure maintenance of the delivery plasmid but you must determine the minimal inhibitory concentration of antibiotic NOTE When the marker carried by the minitransposon is an antibiotic resistance determinant optimal concentrations of antibiotics to the selection can vary among strains The minimal inhibitory concentration MIC must be determined in each case The concentrations in the table below can be used as a guideline Chloramphenicol 5 50 pg ml Kanamycin 25 75 ug ml Streptomycin 50 100 ug ml Spectinomycin 50 100 ug ml Tetracycline 2 5 15 ug ml 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 The insertion can be confirmed
14. osome suicide plasmids The mini Tn5 would be able to 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 the cloning of variable 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 1 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 cloning vector because of the modular nature of the constructions Use of cleavage sites of the rare cutters Not and Sfi for cloning convenience the vector has an array of unique cloning sites The pUTmini Tn5 plasmids are transferable to a variety of bacteria Vig Biomedal A pUT lacZ2 map EcoRI Cal 5 Protocol 5 1 Transformation of donor strain Notl To insert mini Tn5 lacZ2 into the chromosome of a recipient strain first of all you must
15. s 1 This vector also simplifies the generation of insertion mutants insertion mutagenesis in vivo fusions with reporter genes promoter probing and analysis of transcriptional terminators 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 differentially marked minitransposons Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers pUT lacZ2 is included into mini Tn5 derivatives vectors that have been adapted for promoter probing by means of generation of type ll gene fusions transcriptional translational with target genes This vectors do not have transcriptional terminators within the and O terminal sequences of Tn5 and the insertion of the minitransposon in the appropriate orientation downstream of chromosomal promoters may active the expression of a promoterless gene placed within the mobile unit 1 pUT lacZ2 is a vector designed to generate random gene fusions with lacZ as reporter gene 4 ANTIBIOTICS AND ADDITIVES Ampicillin 100 mg ml stock solution Dissolve 1 g ampicillin in 9 ml of deionised water Add deionised water to a 10 ml volume final Filter sterilize the solution with a 0 22 u filter Store the stock solution at 20 C
16. sation and chromosomal transfer tra and mob genes are necessary These genes can be present in a host strain or can be provided by way of a helper plasmid When employing host strains such as E coli SM10 Apir or E coli S17 1 Apir the transformed transposon DNA is transfered via 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 necessary 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 To mobilize the delivery plasmid directly from the donor strain into target cells in this case a triparental mating with a helper strain such as E coli DH5a pRK2013 or E coli DH5a pRK2073 is necessary NOTE To propagate pUTmini Tn5 in Apir strains you can use the TSS solution to perform the transformation Biomedal Cat No RS 3215 RS 3216 Triparental mating 1 Grow overnight cultures in LB of each of the three strains donor strain recipient strain and he
17. trains 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 5 M citrate to avoid the recipient strain infection by Apir phage JOPeA ZZI0 nd M Biomedal 5 4 B galactosidase assay pUT lacZ2 carries lacZ gene as reporter You can monitor expression levels by B gal activity 1 Take 1 ml pellet from induced and uninduced expression culturers with a known ODgog Resuspend the pellet in 1 ml of Z buffer 2 Mix 100 ul of sample 900 ul of Z buffer 2 drops of chloroform and 1 drop of 0 1 SDS 3 Vortex the tubes for 10 seconds 4 Equilibrate samples to room temperature for 15 minutes to evaporate the chloroform 5 Add 200 ul of ONPG in Z buffer to each tube 6 After a yellow color develops stop the reaction by adding 500 ul of 1 M of Na CO 1 M solution to each tube following the same timed addition order Record in minutes the incubation time 7 Read the absorbance of the samples at OD 499 8 Determine the Miller units of expression by means of the formula OD4y9 x 1000 Miller units x 1000 tnin x OD gq9 x dilution min 5 2 Transfer of the DNA into the recipient strain To transter the minitransposon into the recipient strain the genes that code for mobili
Download Pdf Manuals
Related Search