Difference between revisions of "Part:BBa K510000"

Revision as of 15:32, 21 September 2011

pUC18Sfi-miniTn7BB-Gm

pUC18Sfi-miniTn7BB-Gm is a vehicle vector for the minitransposon miniTn7BB-Gm. This transposon is a part of the [http://2011.igem.org/Team:UPO-Sevilla/Foundational_Advances/MiniTn7/Overview miniTn7 BioBrick toolkit], a set of plasmids harboring Tn7 derivatives that can be used for integration of BioBricks in single copy at a conserved target (the attTn7 site), in multiple bacterial genome.

The main features of miniTn7BB-Gm are: (i) wild-type Tn7R and Tn7L ends, enabling transposition in vivo when the Tn7 transposase is expressed in trans, (ii) prefix and suffix with unique EcoRI, XbaI, SpeI and PstI restriction sites, fully compatible with Assembly Standard 10, (iii) annealing sequences for standard primers to facilitate amplification and sequencing of cloned BioBricks, (iv) transcriptional terminators flanking the multi-cloning site to efficiently insulate cloned BioBricks from vector transcription, (v) a selectable gentamycin (Gm) resistance cassette flanked by duplicated SphI and NcoI sites to facilitate marker replacement, (vi) flipase recognition elements (FRTs) flanking the gentamycin resistance cassette, to allow clean excision of the marker in strains bearing transposon insertions, and (vii) SfiI sites flanking the complete transposon to facilitate cloning into the appropriate plasmid vectors. A schematic of miniTn7BB-Gm showcasing the aforementioned features is shown in the figure below.

pUC18Sfi is a pUC18-derived cloning vector harboring an ampicillin resistance marker, a high copy number mutant pMB1 replication origin and the pUC18 multi-cloning site flanked by two SfiI restriction sites. Because of its high copy number and its ability to replicate in any E. coli strain and in other enterobacteria, pUC18Sfi is suitable for maintenance of miniTn7 transposon derivatives, and for simple plasmid preparation and genetic manipulation. pUC18Sfi can be used to deliver transposons by transformation in bacteria in which it does not replicate (i.e., non-enteric bacteria), but not in the enterics.

As a BioBrick cloning vector, pUC18Sfi-miniTn7BB-Gm is fully compatible with Assembly Standard 10.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal prefix found in sequence at 4367
Illegal suffix found in sequence at 1
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 4367
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 4373
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 4367
Illegal BglII site found at 3051
Illegal BglII site found at 3322
Illegal BglII site found at 3608
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found in sequence at 4367
Illegal suffix found in sequence at 2
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found in sequence at 4367
Illegal XbaI site found at 4382
Illegal SpeI site found at 2
Illegal PstI site found at 16
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1681
Illegal SapI.rc site found at 2763

@@ Line 5: / Line 5: @@
 pUC18Sfi-miniTn7BB-Gm is a vehicle vector for the minitransposon miniTn7BB-Gm. This transposon is a part of the [http://2011.igem.org/Team:UPO-Sevilla/Foundational_Advances/MiniTn7/Overview miniTn7 BioBrick toolkit], a set of plasmids harboring Tn7 derivatives that can be used for '''integration of BioBricks''' in single copy at a conserved target (the attTn7 site), in '''multiple bacterial genome'''.
-The structure of the mini-Tn7BB-Gm transposon is shown in the figure below. '''Tn7R''' and '''Tn7L''' are the right and left ends of Tn7 transposon, respectively, required for recognition of the transposase machinery and transposition. '''FRT''' is the Flp recombinase target site for excision of the resistance once the transposon is inserted in the genome of a living organism. The gentamycin resistance cassette ('''Gm''') is flanked by restriction sites in order to facilitate the generation of variants with different antibiotic resistance markers. The BioBrick cloning site ('''BCS''') includes the prefix and suffix restrictions sites used in assembly standard 10, and it is the place where BioBricks are inserted for genome integration.
+The main features of '''miniTn7BB-Gm''' are: (i) wild-type '''Tn7R and Tn7L ends''', enabling transposition in vivo when the Tn7 transposase is expressed in trans, (ii) '''prefix and suffix''' with unique EcoRI, XbaI, SpeI and PstI restriction sites, fully compatible with Assembly Standard 10, (iii) annealing sequences for '''standard primers''' to facilitate amplification and sequencing of cloned BioBricks, (iv) '''transcriptional terminators''' flanking the multi-cloning site to efficiently insulate cloned BioBricks from vector transcription, (v) a selectable gentamycin (Gm) '''resistance cassette''' flanked by duplicated SphI and NcoI sites to facilitate marker replacement, (vi) '''flipase recognition elements''' (FRTs) flanking the gentamycin resistance cassette, to allow clean excision of the marker in strains bearing transposon insertions, and (vii) SfiI sites flanking the complete transposon to facilitate cloning into the appropriate plasmid vectors. A schematic of miniTn7BB-Gm showcasing the aforementioned features is shown in the figure below.
-This plasmid can be used to maintain the miniTn7-Gm transposon in E. coli and other enterics and may be used for transposition into the genomes of non-enteric bacteria, in which it is non-replicative. As a BioBrick cloning vector, pUC18Sfi-miniTn7BB-Gm is fully compatible with''' Assembly Standard 10'''. The complete transposon is flanked by SfiI restriction sites to facilitate subcloning in other vectors
+'''pUC18Sfi''' is a pUC18-derived cloning vector harboring an ampicillin resistance marker, a high copy number mutant pMB1 replication origin and the pUC18 multi-cloning site flanked by two SfiI restriction sites. Because of its high copy number and its ability to replicate in any E. coli strain and in other enterobacteria, pUC18Sfi is suitable for '''maintenance''' of miniTn7 transposon derivatives, and for simple plasmid preparation and '''genetic manipulation'''. pUC18Sfi can be used to deliver transposons by transformation in bacteria in which it does not replicate (i.e., non-enteric bacteria), but not in the enterics.
+As a BioBrick cloning vector, pUC18Sfi-miniTn7BB-Gm is fully compatible with''' Assembly Standard 10'''.
 [[Image:Mini-Tn7-Gm1.png|700px|center]]