Patent Application: US-14062505-A

Abstract:
provided are multiple methods resulting in a site - specific , marker - less integration of a sequence of interest . the methods are based on the following : the genomic presence in a cell of a selectable or screenable gene x . as a result of a mutation , this gene x can be essentially sensitive or insensitive to a certain component or condition z or , as a result of a mutation in gene x , the host cell is made dependent on the presence of a certain component or condition z ; a plasmid on which the desired insertion is present , further harboring a truncated gene x and a selectable marker to select or screen for the presence or absence of vector sequences in the host cell ; positive selection of the final recombination step , avoiding complicated and time - consuming screening for the desired recombinants . preferably , both recombination steps are positively selected . when the starting host cell contains a mutant gene x on the genome , the final recombinant has a gene x without a mutation and the genome further only comprises the desired insertion or deletion . when the starting host cell contains an original gene x , the final recombinant bears a mutation in gene x and the genome further only comprises the desired insertion or deletion .

Description:
e . coli k - 12 strains jm109 was used for propagating plasmids and was grown and transformed by standard procedures ( sambrook et al . 1989 ). e . coli et12567 ( macneil et al . 1992 ) was used to isolate dna for transformation of plasmid dna to streptomyces coelicolor . transformants were selected in l broth containing 1 % ( w / v ) glucose , and ampicillin at a final concentration of 200 μg ml − 1 . l broth with 1 % glucose and 30 μg ml − 1 chloramphenicol was used to grow et12567 . streptomyces coelicolor a3 ( 2 ) m145 was obtained from the john innes centre strain collection . protoplast preparation and transformation were performed as described by kieser et al . ( 2000 ). sfm medium ( mannitol , 20 g 1 − 1 ; soya flour , 20 g 1 − 1 ; agar , 20 g 1 − 1 , dissolved in tap water ) was used to make spore suspensions . minimal medium ( mm ) and r2ye agar plates ( kieser et al . 2000 ) were used for selection experiments ; r2ye was also used for regenerating protoplasts and , after addition of the appropriate antibiotic , for selecting recombinants . for standard cultivation of streptomyces , yeme ( kieser et al . 2000 ) or tryptone soy broth ( difco ) containing 10 % ( w / v ) sucrose ( designated tsbs ) were used . liquid cultures to select for glucose utilization were performed in nmmp ( minimal medium ), with 1 % ( w / v ) mannitol or 1 % ( w / v ) glucose as the carbon source . as the basis for a recombination plasmid , we used pij2581 ( 5192 bp ; genbank accession x98363 ; van wezel and bibb , 1996 ), a construct based on pbluescript sk + ( strategene ), with bla as selectable marker in e . coli , and tsr as selectable marker in streptomyces . the plasmid has both cole1 and f1 (+) origins of replication , the latter allowing the production of single - stranded dna in the presence of helper phage ( sambrook et al . 1989 ). single - stranded dna increases the transformation efficiency in streptomyces ( hilleman et al . 1991 ). the plasmid lacks a streptomyces origin of replication and can , therefore , only be maintained by integration into the host genome through cloned homologous sequences . a 2080 bp sequence harboring all but the first 36 bp of glka , as well as 1162 bp of the downstream sequence , was amplified from the s . coelicolor m145 genome using the 30 - mer oligonucleotides glkx and glky . these oligonucleotides were designed so as to add smai and kpni sites to the beginning and the end of the dna fragment , respectively . the exact sequence inserted is shown in fig6 . this pcr fragment was subsequently cloned into pij2581 , digested with kpni and partially digested with smai , effectively removing the approximately 1150 bp glka gene . the resulting construct pmbs011 is shown in fig7 . the unique bcli site in pmbs011 is compatible with bamhi and bglii restriction sites and can , for example , be used for cloning inserts from pij2925 , which is a derivative of puc19 , carrying bglii restriction sites flanking the multiple cloning site ( janssen and bibb , 1993 ). the non - utilizable glucose analogue 2 - deoxy - glucose ( 2 - dog ) is lethal when introduced in bacterial strains that have an active glucokinase ( designated glucose kinase in streptomycetes ). strains harboring mutant glka genes fail to grow on glucose , but are resistant to 2 - dog . introduction of an active glucokinase or restoration of the wild - type gene by recombination restores full glycolysis and glucose utilization and renders the cells sensitive to 2 - dog . an alignment of several bacterial glucokinases is shown in fig8 . several highly conserved regions can be observed , designated sequences a - h in the figure ( overlined ). sequence a represents the p - loop ( atp - binding consensus sequence ). many site - directed mutants have been created in the s . coelicolor glka gene , resulting in glucose kinases that have lost the ability to phosphorylate glucose . mutational hotspots , where all mutations made so far result in enzymatic inactivity are , for example , sequences a , e and g . to create streptomyces coelicolor strains mutant for glucose kinase , these were grown on solid mmd plates , consisting of mm ( kieser et al . 2000 ) with 1 % ( w / v ) mannitol and 100 mm 2 - deoxyglucose , the latter compound being lethal for glk + strains . therefore , colonies that develop on this medium have to be glk − . colonies that were able to grow on mmd were selected and tested for glucose kinase activity . glucose kinase - deficient ( δglka ) strains were checked by pcr , which showed that the nature of the mutations varied from large deletions to point mutations . for the experiments described herein , a generated mutant glka harboring a small deletion corresponding to aa 257 - 262 ( see fig8 ) was used . marker - less insertion of the egfp gene into the s . coelicolor genome with positive selection construction of the insertion vector for demonstration of the integration method , using pmbs011 as integration vector , the gene for egfp ( enhanced green fluorescent protein ) was chosen to be inserted into the s . coelicolor genome . for this purpose , we amplified an approximately 1 kb dna fragment harboring the egfp gene and its rbs with oligonucleotides gfpx and gfpy , designed so as to provide bglii sites at either end . the pcr - amplified egfp gene - containing dna fragment was digested with bglii and inserted into bcli - digested pmbs011 . dna from the latter was isolated from e . coli strain et12567 ( mutant for several modification genes , including dam dcm ; mcneill et al . 1992 ) to allow digestion of the normally dam - methylated bcli site . after ligation , the dna was re - digested with bcli so as to guarantee the absence of vector without insert . all colonies tested contained the expected plasmid , which was designated pmbs012 . construct pmbs012 has the n - truncated but otherwise wild - type glka gene followed by the egfp gene , which , in turn , is followed by orfs 6e10 . 19 and the end of orf6e10 . 18 , which is oppositely oriented ( not indicated in fig7 ). this construct allows integration of the egfp gene behind glka on the s . coelicolor genome . the resulting recombinant genome should preferably harbor no heterologous sequences ( other than the desired 800 bp egfp gene flanked by the fused bcli - bglii sites ). an s . coelicolor glka mutant lacking the codons for amino acid residues 257 - 262 ( ivgggl , fig8 ) was transformed with pmbs012 and colonies were selected for resistance to thiostrepton . subsequently , recombinants were plated on mm plates with mannitol as the sole carbon source ( kieser et al .) and containing 100 mm 2 - deoxy glucose and 10 μg / ml thiostrepton . in this way , positive selection was achieved of recombinants in which recombination event 2 ( fig1 a ) has occurred through recombination in sequence y ( i . e ., downstream of the egfp gene ). this results in a complete but catalytically inactive mutant glucose kinase and a truncated wild - type copy , rendering the recombinant 2 - dog resistant . the other type of recombination ( event 1 in fig1 a ), results in recombinants with a wild - type and catalytically active glka gene , which , therefore , fail to grow on 2 - dog . three colonies were checked and found to have undergone the correct recombination event . the viable primary recombinants were streaked on mm plates with 2 - dog and subsequently replicated onto mm with glucose as the sole carbon source to allow recombination events to occur and spores harvested . these were used to inoculate a liquid nmmp culture ( kieser et al . 2000 ) with mannitol as the sole carbon source , grown until od 600 of 0 . 5 , washed twice in nmmp without carbon source , resuspended in nmmp with glucose as the sole carbon source , and grown until stationary phase was reached ( typically overnight ). only mycelium with a wild - type glucose kinase gene can utilize glucose and such recombinants must have arisen from a further recombination event through the homologous glka sequences , resulting in a wild - type glucose kinase gene followed by the egfp gene on the genome . the mycelium was plated on mm plates with glucose as the sole carbon source to select the population with a wild - type glka gene . colonies that appeared were tested for sensitivity to 2 - dog and thiostrepton . the majority of the colonies tested harbored a wild - type glka gene and were sensitive to thiostrepton . southern hybridization on genomic dna isolated from two independent colonies confirmed that these had the expected egfp insertion . thus , we succeeded in inserting a dna fragment into the genome of s . coelicolor m145 , without leaving any selectable marker or other undesired sequences behind , and with both recombination steps positively selectable , namely resistance to thiostrepton and 2 - dog ( step 1 ), and ability to grow on glucose ( step 2 ). dna sequencing confirmed the presence of the expected insert . thus , we believe that this is an important step forward in creating recombinant microorganisms , especially those which are notoriously hard to screen , such as actinomycetes . solving possible polar effects of insertions on the transcription of downstream genes it is possible that insertion of a dna sequence into the genome affects the transcription of downstream - located genes , resulting in so - called polar effects . for example , this occurs if , in the final situation in fig1 a , 2 and 3 , the inserted dna alters and / or blocks transcription of genes in sequence y and / or downstream of it ; similarly , in fig1 b , insertion of dna could affect transcription of gene x ( glka ) and possibly also of downstream - located genes . in such a case , it is desirable or , in the case of genes indispensable for growth or selection , essential to provide promoter sequences immediately 3 ′ of the inserted dna on the disruption construct to ensure proper transcription of downstream genes . in a more specific case , gene x and sequence y are also part of the operon , where on the genome they are immediately preceded by the operon promoter and followed by one or more genes that also depend on this promoter . in such a case , insertion of a plasmid by recombination through gene x or sequence y results in block of transcription of all downstream - located genes . this is lethal if one or more of the downstream - located genes is essential for growth . negative effects of the insertion can only be counteracted by making sure that two promoters are present on the plasmid , one promoter either upstream of the truncated gene x ( fig1 a , 2 , 3 , and 4 a ) or upstream of sequence y ( fig1 b and 4b ), and a second promoter , either between the cloned dna and the truncated gene x ( fig1 b and 4b ) or between the inserted dna and sequence y ( fig1 a , 2 , 3 , and 4 a ). in the case of a glucose kinase gene , it is likely that insertion of dna into the bcli site ( fig6 ) will block transcription of the downstream - located orf6e10 . 19 . however , from earlier experiments ( kelemen et al . 1995 ), it is known that deletion of this gene does not affect growth or morphology . this was confirmed by the wild - type phenotype of the final recombinant harboring the egfp gene between glka and orf6e10 . 19 on the genome . recombination events between mutation in gene x and the cloned dna there is a possibility of a recombination event upstream of the cloned dna but downstream of the site of mutation . this event is exemplified in fig5 and depicted as 1b . before proceeding with the second recombination step , this possibility needs to be ruled out , for example , by pcr of genomic dna of a few recombinants . the chance p b for this undesirable recombination to occur can , in our experience , be estimated by the formula : p b = ½ ( a / b ) 2 × 100 %. for example , with a ratio a : b = 2 : 1 , it follows that the chance of recombination through sequence b is approximately 13 %. in most cases , the experimenter will be able to choose the situation such that the ratio a : b is much larger , as in the cases listed in table 2 , so that p b becomes negligible . while in principle area b should be minimized , in practice , it follows that as long as the ratio a : b exceeds 2 : 1 , checking a few recombinants is sufficient to identify the correct recombinant to enter recombination step 2 . in the glka experiment described in the experimental section where the ratio a : b is around 5 : 2 , all three colonies checked had undergone recombination through area a . in the example of the use of the epr1 gene for recombination in plants , mutations all lie between nucleotide positions 100 - 300 , while the whole gene is more than 2000 bp long . in such a case , the chance of finding the desired recombination through area a is close to 100 %. janssen g . r . and m . j . bibb ( 1993 ) derivatives of puc18 that have bglii sites flanking a modified multiple cloning site and that retain the ability to identify recombinant clones by visual screening of escherichia coli colonies . gene 124 : 133 - 134 . kelemen g . h ., k . a . plaskitt , c . g . lewis , k . c . findlay and m . j . buttner ( 1995 ) deletion of dna lying closes to the glka locus induces ectopic sporulation in streptomyces coelicolor a3 ( 2 ). mol . microbiol . 17 : 221 - 230 . kieser t ., m . j . bibb , m . j . buttner , k . f . chater and d . a . hopwood ( 2000 ) practical streptomyces genetics . norwich , u . k . : john innes foundation . knoester m ., l . c . van loon , j . van den heuvel , j . hennig , j . f . bol and h . j . m . linthorst ( 1998 ) ethylene - insensitive tobacco lacks nonhost resistance against soil - 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