Team:Wisconsin-Madison

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Expression, Purification, and Quality Control of Gibson Enzymes.

Gibson assembly is a common cloning technique that was pioneered in 2009 by Daniel Gibson. The technique utilizes three enzymes: a thermostable DNA ligase, a non-thermostable exonuclease, and a thermostable DNA polymerase to simultaneously join multiple DNA fragments with 20-40 base pair overlaps. The Gibson Assembly is significantly simpler to perform than prior conventional methods, as it is carried out in a single, isothermal step, and has greater flexibility than previous cloning methods using restriction enzymes. Because of its simplicity and versatility, it has quickly become a common technique in molecular biology. The cost of the necessary enzymes, however, is very high and can be inhibitory for teams who lack financial resources.

The gibson assembly reaction mixture is composed of Taq ligase, T5 exonuclease, a thermostable DNA polymerase, the DNA fragments and a buffer. The mixture is incubated at 50 degrees Celsius for one hour. The T5 exonuclease chews back at the 5’ end of the DNA to create sticky overhangs that allow the complimentary fragments to anneal. Being non-thermostable, however, the exonuclease becomes heat inactivated rather quickly, and after annealing, at which point the DNA polymerase fills in the gaps created by the exonuclease. The Taq ligase then bonds the adjacent segments together.

We have developed protocols for the purification of hexahistidine-tagged recombinant taq ligase, as well as T5 exonuclease in BL21 E. Coli using a Ni-NTA column. In addition to this, we have developed a protocol for making a gibson master mix using lab purified enzymes, as well as an assay using a red fluorescent protein to test the functionality of a gibson reaction mix.

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        <p class = "classtheoverview"> <strong> Expression, Purification, and Quality Control of Gibson Enzymes. </strong></p>
-       <p align="left" class = "classtheinlinecontent2"> A powerful method for the production of novel metabolites is the expression of heterologous enzymes in a bacterial host. A common challenge when using non-native genes in metabolic engineering is determining if they are being properly expressed. To address this issue, we have constructed a BioFusion-compatible system for testing the translation of a gene of interest. This system couples the translation of the target gene to a fluorescent reporter gene; fluorescence will only be detected when the target gene is entirely translated. This construct enables synthetic biologists to quickly determine if a gene is being expressed without the need for costly antibodies or analytical instruments (e.g. mass spectrometry). Currently, we are utilizing this cassette to optimize the expression of limonene synthase, an enzyme that catalyzes the production of limonene, a monoterpene with potential as a renewable jet fuel.</p>
+       <p align="left" class = "classtheinlinecontent2"><pre>    </pre>Gibson assembly is a common cloning technique that was pioneered in 2009 by Daniel Gibson. The technique utilizes three enzymes: a thermostable DNA ligase, a non-thermostable exonuclease, and a thermostable DNA polymerase to simultaneously join multiple DNA fragments with 20-40 base pair overlaps. The Gibson Assembly is significantly simpler to perform than prior conventional methods, as it is carried out in a single, isothermal step, and has greater flexibility than previous cloning methods using restriction enzymes. Because of its simplicity and versatility, it has quickly become a common technique in molecular biology. The cost of the necessary enzymes, however, is very high and can be inhibitory for teams who lack financial resources.
+<br><pre>    </pre>The gibson assembly reaction mixture is composed of Taq ligase, T5 exonuclease, a thermostable DNA polymerase, the DNA fragments and a buffer. The mixture is incubated at 50 degrees Celsius for one hour. The T5 exonuclease chews back at the 5’ end of the DNA to create sticky overhangs that allow the complimentary fragments to anneal. Being non-thermostable, however, the exonuclease becomes heat inactivated rather quickly, and after annealing, at which point the DNA polymerase fills in the gaps created by the exonuclease. The Taq ligase then bonds the adjacent segments together.
+<br><pre>    </pre>We have developed protocols for the purification of hexahistidine-tagged recombinant taq ligase, as well as T5 exonuclease in BL21 E. Coli using a Ni-NTA column. In addition to this, we have developed a protocol for making a gibson master mix using lab purified enzymes, as well as an assay using a red fluorescent protein to test the functionality of a gibson reaction mix.</p>
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