Difference between revisions of "Part:BBa K678070"

Latest revision as of 10:23, 20 September 2011

p68

p68 is a plasmid that can be used to assay promoter activity in Aspergillus nidulans. It contains the lacZ gene, the TtrpC terminator, a USER cassette, an argB marker cassette for selection, an ori for propagation in E. coli, and an ampR resistance cassette. It also contains up- and down stream regions for targeting to a specific site in the chromosome called IS1 situated 202 bp downstream of AN6638 and 245 bp upstream of AN6639. For homologous recombination to occur gene-targeting substrates have to contain these large homologous sequences around 1500 bp to ensure the targeted integration (1).

To insert a promoter by USER cloning p68 should be digested with AsiSI 2-8 hours and afterwards nicked with Nb.BstI for 1-3 hours. Instructions on how to perform the USER cloning and transformation of A. nidulans can be found [http://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#USER%20cloning here].

DTU-Denmark-2 2011 Plasmid map of p68. After digestion of the USER cassette a promoter of interest can be inserted.

Usage and Biology

This plasmid was successfully used to characterize the A. nidulans promoters PalcA and DMKP-P6. Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 6337
Illegal EcoRI site found at 11000
Illegal XbaI site found at 1257
Illegal XbaI site found at 7126
Illegal XbaI site found at 11325
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 6337
Illegal EcoRI site found at 11000
Illegal NotI site found at 1296
Illegal NotI site found at 11282
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 6337
Illegal EcoRI site found at 11000
Illegal BglII site found at 3225
Illegal BglII site found at 8299
Illegal BglII site found at 10490
Illegal BamHI site found at 6407
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 6337
Illegal EcoRI site found at 11000
Illegal XbaI site found at 1257
Illegal XbaI site found at 7126
Illegal XbaI site found at 11325
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 6337
Illegal EcoRI site found at 11000
Illegal XbaI site found at 1257
Illegal XbaI site found at 7126
Illegal XbaI site found at 11325
Illegal NgoMIV site found at 7043
Illegal AgeI site found at 8847
Illegal AgeI site found at 8877
Illegal AgeI site found at 10254
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 2122
Illegal BsaI site found at 8680
Illegal BsaI site found at 9852
Illegal BsaI site found at 10446
Illegal BsaI site found at 10642
Illegal BsaI site found at 10813
Illegal BsaI.rc site found at 3137
Illegal BsaI.rc site found at 6757
Illegal SapI.rc site found at 2793

References

(1) Hansen, Bjarne G.; Bo Salomonsen; Morten T. Nielsen; Jakob B. Nielsen; Niels B. Hansen; Kristian F. Nielsen; Torsten B. Regueira; Jens Nielsen; Kiran R. Patil; and Uffe H. Mortensen; “Versatile enzyme expression and Characterization system for Aspergillus, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case.” American Society for Microbiology, 2011, 3044-3051.

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K678070 short</partinfo>
-p68 is a plasmid that can be used to assay promoter activity in ''Aspergillus nidulans''. It contains the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K678026 '' lacZ''] gene, the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K678036 TtrpC] terminator, a USER cassette, an ''argB'' marker cassette for selection, an ori for propagation in E.coli, and an ''ampR'' resistance cassette. It also contains up- and down stream regions for targeting to a specific site called IS1 situated 202 bp downstream of AN6638 and 245 bp upstream of AN6639 (5). For homologous recombination to occur gene-targeting substrates have to contain these large homologous sequences around 1500 bp to ensure the targeted integration (5).
+p68 is a plasmid that can be used to assay promoter activity in ''Aspergillus nidulans''. It contains the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K678026 '' lacZ''] gene, the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K678036 TtrpC] terminator, a USER cassette, an ''argB'' marker cassette for selection, an ori for propagation in ''E. coli'', and an ''ampR'' resistance cassette. It also contains up- and down stream regions for targeting to a specific site in the chromosome called IS1 situated 202 bp downstream of AN6638 and 245 bp upstream of AN6639. For homologous recombination to occur gene-targeting substrates have to contain these large homologous sequences around 1500 bp to ensure the targeted integration (1).
 To insert a promoter by USER cloning p68 should be digested with AsiSI  2-8 hours and afterwards nicked with Nb.BstI for 1-3 hours. Instructions on how to perform the USER cloning and transformation of ''A. nidulans'' can be found [http://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#USER%20cloning here].
@@ Line 15: / Line 15: @@
 <partinfo>BBa_K678070 SequenceAndFeatures</partinfo>
+===References===
+(1) '''Hansen, Bjarne G.; Bo Salomonsen; Morten T. Nielsen; Jakob B. Nielsen; Niels B. Hansen; Kristian F. Nielsen; Torsten B. Regueira; Jens Nielsen; Kiran R. Patil; and Uffe H. Mortensen'''; “Versatile enzyme expression and Characterization system for ''Aspergillus'', with the ''Penicillium brevicompactum'' Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case.” American Society for Microbiology, 2011, 3044-3051.
 <!-- Uncomment this to enable Functional Parameter display
 ===Functional Parameters===
 <partinfo>BBa_K678070 parameters</partinfo>
 <!-- -->