Difference between revisions of "Template:McGill iGEM 2024/Rep Gate Design"
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This part is created from two annealed ssDNA strands, Rep[X]-t and Rep[X]-b.  
 
This part is created from two annealed ssDNA strands, Rep[X]-t and Rep[X]-b.  
  
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To create the part, a fluorophore-quencher (FQ) pair was covalently linked 5’ and 3’ to the top and bottom oligonucleotides respectively. This linkage is on the 5’ and 3’ non-sticky ends. In McGill iGEM’s design, Rep-t-F, Rep-b-Q are annealed in a 1:1 stoichiometric ratio. The fluorophores used are:
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To create the part, a fluorophore-quencher (FQ) pair was covalently linked 5’ and 3’ to the top and bottom oligonucleotides respectively. This linkage is on the 5’ and 3’ non-sticky ends. In McGill iGEM’s design, Rep-t-F, Rep-b-Q are annealed in a 1:1 stoichiometric ratio.  
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A custom synthesis of fluorophore-linked oligonucleotides was performed by Jathavan Asohan at Sleiman Lab, McGill Department of Chemistry.
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To anneal oligonucleotides, both Rep[x]-t and Rep[x]-b strands are HPLC purified and diluted to a concentration of 100uM. 4.5uL volumes of top strand and bottom strand are added with 1uL of 10xMg1xTE added, and strands are heated to 95ºC and allowed to cool 1ºC/min. Reaction volumes can be scaled up as necessary. No downstream purification was done.
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All fluorophore displacements are done at a 200nm concentration in 12.5mM Mg+ ion concentration.

Revision as of 02:47, 13 September 2024

This documentation is shared across Rep1, Rep2, Rep3, Rep4:

The diagnostic results of these modules, which allows the identification of infection-causing bacterial species and associated antimicrobial resistance markers, is reported as a level of fluorescence that increases over time. As such, a general fluorescent reporter for the diagnostic is developed which triggers upon the terminal strand displacement reaction. This reporter part can also be adapted to report the performance of the CasX and SDR amplifier modules through the use of a DNA translator gate.

This reporter gate consists of a simple 5nt toehold domain with a 15nt branch migration domain. This part is created from two annealed ssDNA strands, Rep[X]-t and Rep[X]-b.

To create the part, a fluorophore-quencher (FQ) pair was covalently linked 5’ and 3’ to the top and bottom oligonucleotides respectively. This linkage is on the 5’ and 3’ non-sticky ends. In McGill iGEM’s design, Rep-t-F, Rep-b-Q are annealed in a 1:1 stoichiometric ratio.

A custom synthesis of fluorophore-linked oligonucleotides was performed by Jathavan Asohan at Sleiman Lab, McGill Department of Chemistry.

To anneal oligonucleotides, both Rep[x]-t and Rep[x]-b strands are HPLC purified and diluted to a concentration of 100uM. 4.5uL volumes of top strand and bottom strand are added with 1uL of 10xMg1xTE added, and strands are heated to 95ºC and allowed to cool 1ºC/min. Reaction volumes can be scaled up as necessary. No downstream purification was done.

All fluorophore displacements are done at a 200nm concentration in 12.5mM Mg+ ion concentration.