Part:BBa_K4947036:Experience

Experience

We amplified the initial parts using high-fidelity PCR with primers designed to anneal at each end. We then DpnI-digested and purified these amplicons. Subsequently, we performed Golden Gate assembly using NEBridge® Golden Gate Assembly Kit (which was also donated in-kind) and their specified protocol to build plasmids using this part. We electroporated TransforMax EC100D pir+ electrocompetent E. coli with the assembled DNA, and plated on selective media. Then, we ran diagnostic colony PCR that amplified parts of the plasmid to check for the presence of successful junctions, which indicate successful assembly (Figure 1). Of the colonies that had positive results, some were inoculated, plasmid-purified (using QIAGEN mini-prep kit and protocol), and sent for whole plasmid sequencing, a service purchased from Plasmidsaurus. Finally, whole plasmid sequencing results confirmed success or failure. This is the general procedure we recommend for using and characterizing this part, as it was successful for us.

Applications of BBa_K4947036

This device is crucial to start production of daidzein. It is also an important device for production for isoliquiritigenin, an expensive and understudied intermediate. This composite part specifically is important for optimal daidzein production, when being produced recombinantly by E. coli. Take a look at the rest of our wiki (https://2023.igem.wiki/yale/index.html) for how this device connects to human health, economics, and more!

User Reviews

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