Difference between revisions of "Part:BBa E0033"
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===Application Submitted by Team Michigan 2016=== | ===Application Submitted by Team Michigan 2016=== | ||
Alpha complementation is a flexible tool that can be applied for a wide variety of uses where colorimetric detection is desirable. Our team's project design this year proposes to use the in vitro transcription and translation of the alpha fragment as a reporter for proximity dependent ligation. This design involves a pair of ssDNA probes containing aptamer sequences specific to two sites on a target protein. In addition to containing aptamer sequences, these probes also code for the lacZ alpha fragment, but can only express it after a ligation reaction has occurred to join the probes together. The rate of this ligation reaction would be drastically increased when the target biomarker is present to hold the probes in close proximity to one another; therefore expression of the alpha fragment signals the presence of the target protein. The newly produced alpha fragment then complements with the omega fragment (the protein product of the lacZ delta M15 mutation) to form fully functional beta galactosidase which can cleave X-gal to form a blue colorimetric output. | Alpha complementation is a flexible tool that can be applied for a wide variety of uses where colorimetric detection is desirable. Our team's project design this year proposes to use the in vitro transcription and translation of the alpha fragment as a reporter for proximity dependent ligation. This design involves a pair of ssDNA probes containing aptamer sequences specific to two sites on a target protein. In addition to containing aptamer sequences, these probes also code for the lacZ alpha fragment, but can only express it after a ligation reaction has occurred to join the probes together. The rate of this ligation reaction would be drastically increased when the target biomarker is present to hold the probes in close proximity to one another; therefore expression of the alpha fragment signals the presence of the target protein. The newly produced alpha fragment then complements with the omega fragment (the protein product of the lacZ delta M15 mutation) to form fully functional beta galactosidase which can cleave X-gal to form a blue colorimetric output. | ||
| + | https://static.igem.org/mediawiki/parts/4/4b/Proximity-Dependent_Ligation_Diagram_small.png | ||
| + | https://static.igem.org/mediawiki/parts/8/85/MSBT_Expression_Diagram_small.png | ||
For more information on this application see http://2016.igem.org/Team:Michigan | For more information on this application see http://2016.igem.org/Team:Michigan | ||
Revision as of 10:11, 28 October 2016
LacZ alpha fragment; complements matching N-terminal deletion mutant (lacZ-omega)
To greatly reduce the number of bases, this is only a portion of the LacZ gene. It can be used as a detection system with N-terminal deletion mutants of lacZ. Combination of lacZ-alpha with such a deletion mutant protein restores enzyme activity that can be assayed colorimetrically or more sensitively with chemiluminescent substrates.
Application Submitted by Team Michigan 2016
Alpha complementation is a flexible tool that can be applied for a wide variety of uses where colorimetric detection is desirable. Our team's project design this year proposes to use the in vitro transcription and translation of the alpha fragment as a reporter for proximity dependent ligation. This design involves a pair of ssDNA probes containing aptamer sequences specific to two sites on a target protein. In addition to containing aptamer sequences, these probes also code for the lacZ alpha fragment, but can only express it after a ligation reaction has occurred to join the probes together. The rate of this ligation reaction would be drastically increased when the target biomarker is present to hold the probes in close proximity to one another; therefore expression of the alpha fragment signals the presence of the target protein. The newly produced alpha fragment then complements with the omega fragment (the protein product of the lacZ delta M15 mutation) to form fully functional beta galactosidase which can cleave X-gal to form a blue colorimetric output.
For more information on this application see http://2016.igem.org/Team:Michigan
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 93
Illegal XhoI site found at 144 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]