Difference between revisions of "Team:Imperial College/Measurement"
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<p>During PixCell project we discovered that conventional DNA assembly methods not always adapt to rapid troubleshooting of some construct parts. As our initial construct was open for improvement to improve our model we decided to create a library of transcription factors and promoters and try creating new constructs with them. So, we started looking for a more modular method that would allow us to easily vary 1 or 2 components in a construct keeping everything else the same in order to build our library. We found that BASIC was the perfect technology for this.</p> | <p>During PixCell project we discovered that conventional DNA assembly methods not always adapt to rapid troubleshooting of some construct parts. As our initial construct was open for improvement to improve our model we decided to create a library of transcription factors and promoters and try creating new constructs with them. So, we started looking for a more modular method that would allow us to easily vary 1 or 2 components in a construct keeping everything else the same in order to build our library. We found that BASIC was the perfect technology for this.</p> | ||
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<p>BASIC assembly can be divided in four simple steps. First linkers are prepared and ligated to the corresponding parts. Every part contains a BASIC prefix and suffix, linkers bind to prefix or suffix, and each linker has a homologue linker that binds to the other (prefix or suffix). At the end of this step all parts have a prefix and a suffix linker. Then parts with linkers are purified with magbead purification. Purified parts are assembled at 50ºC for 45min, each prefix linker will bind to its homologue suffix linker and this will give an order to the construct, so it is a one-step assembly. Finally, constructs are transformed into the desired strain and selected. </p> | <p>BASIC assembly can be divided in four simple steps. First linkers are prepared and ligated to the corresponding parts. Every part contains a BASIC prefix and suffix, linkers bind to prefix or suffix, and each linker has a homologue linker that binds to the other (prefix or suffix). At the end of this step all parts have a prefix and a suffix linker. Then parts with linkers are purified with magbead purification. Purified parts are assembled at 50ºC for 45min, each prefix linker will bind to its homologue suffix linker and this will give an order to the construct, so it is a one-step assembly. Finally, constructs are transformed into the desired strain and selected. </p> | ||
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Revision as of 17:39, 17 October 2018
BASIC Method
During PixCell project we discovered that conventional DNA assembly methods not always adapt to rapid troubleshooting of some construct parts. As our initial construct was open for improvement to improve our model we decided to create a library of transcription factors and promoters and try creating new constructs with them. So, we started looking for a more modular method that would allow us to easily vary 1 or 2 components in a construct keeping everything else the same in order to build our library. We found that BASIC was the perfect technology for this.
BASIC is a modular assembly method in which the different construct parts are made modular linking them to pairs of oligos called linkers. Each linker assembles a part to the previous and subsequent parts. This way it allows to build complex genetic circuits and multipart combinatorial assemblies. Besides, linker oligos are large enough to allow encoding an RBS sequence inside, this enables RBS troubleshooting of a construct trying different combinations of RBS without having to change the full structure of the assembly. Furthermore, BASIC includes linkers capable of fusing proteins, making it the best method capable of rapid assembly and characterisation of protein fusion libraries.
Developed in 2015 in Baldwin’s lab at Imperial College London no one had used BASIC in iGEM before. From PixCell we want to introduce this versatile technique to the iGEM community and make it compatible with BioBrick assembly, for which we created pSB1C3_BASIC vector (with BioBrick and BASIC sites). Using this method, we were able to assemble a library of 48 different constructs in 3 days, using 6 different transcription factors and 8 promoters.
BASIC assembly can be divided in four simple steps. First linkers are prepared and ligated to the corresponding parts. Every part contains a BASIC prefix and suffix, linkers bind to prefix or suffix, and each linker has a homologue linker that binds to the other (prefix or suffix). At the end of this step all parts have a prefix and a suffix linker. Then parts with linkers are purified with magbead purification. Purified parts are assembled at 50ºC for 45min, each prefix linker will bind to its homologue suffix linker and this will give an order to the construct, so it is a one-step assembly. Finally, constructs are transformed into the desired strain and selected.
