Difference between revisions of "Part:BBa K2105000"
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The lacZ delta M15 mutation encodes a form of beta-galactosidase lacking residues 11-41. This mutation causes it to be inactive until complemented by the lacZ alpha fragment. The lacZ delta M15 mutant is constitutively expressed in DH5 alpha cells. When the delta M15 mutant and the alpha fragment undergo alpha complementation, they are able to cleave the substrate X-gal to produce a blue product. | The lacZ delta M15 mutation encodes a form of beta-galactosidase lacking residues 11-41. This mutation causes it to be inactive until complemented by the lacZ alpha fragment. The lacZ delta M15 mutant is constitutively expressed in DH5 alpha cells. When the delta M15 mutant and the alpha fragment undergo alpha complementation, they are able to cleave the substrate X-gal to produce a blue product. | ||
| − | This can be demonstrated by performing a | + | This can be demonstrated by performing a double transformation [1] of BBa_K2105000 and a plasmid containing BBa_E0033 (lacZ alpha fragment). This is best performed in BL21 cells or similar because DH5 alpha cells already constitutively express the lacZ delta M15 mutant. The two plasmids should have differing antibiotic selection markers to allow for the selection of only colonies containing both plasmids using double antibiotic agar plates. The plates should also contain IPTG and X-gal to induce protein production and provide a substrate for LacZ. The plate should show noticeably blue colonies within 16-20 hours, demonstrating that the alpha and omega fragments are capable of complementation in vivo to produce the functional enzyme. |
| + | 1. [https://static.igem.org/mediawiki/2015/2/2b/TU_Eindhoven_Protocols_Double_Transformation.pdf] | ||
Revision as of 09:38, 28 October 2016
LacZ delta M15 mutation
The lacZ delta M15 mutation encodes a form of beta-galactosidase lacking residues 11-41. This mutation causes it to be inactive until complemented by the lacZ alpha fragment. The lacZ delta M15 mutant is constitutively expressed in DH5 alpha cells. When the delta M15 mutant and the alpha fragment undergo alpha complementation, they are able to cleave the substrate X-gal to produce a blue product.
This can be demonstrated by performing a double transformation [1] of BBa_K2105000 and a plasmid containing BBa_E0033 (lacZ alpha fragment). This is best performed in BL21 cells or similar because DH5 alpha cells already constitutively express the lacZ delta M15 mutant. The two plasmids should have differing antibiotic selection markers to allow for the selection of only colonies containing both plasmids using double antibiotic agar plates. The plates should also contain IPTG and X-gal to induce protein production and provide a substrate for LacZ. The plate should show noticeably blue colonies within 16-20 hours, demonstrating that the alpha and omega fragments are capable of complementation in vivo to produce the functional enzyme.
1. [1]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]