pTi plasmid backbone

Open DNA Collections Shared Reagents
21

Greetings Ahmed,

For DpnI, you need to use a strain without dam or dcm methylases, NEB has one strain (C2925), but that is more like a cloning strain. I succeeded in getting DpnI expression using this strain (albeit in smaller quantities I would normally get by using BL21). A better strain, which is JM110, is available around (https://www.addgene.org/49763/) that is more suitable for protein expression.

After this, you either can use a DE3 lysogenization kit to integrate DE3 module (which has T7 pol under Lac promoter control) so that you can utilize T7-based systems, or use a E. coli promoter. What I did was to clone DpnI after the tac promoter (a chimera of trp and lac promoters) to do expression.

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22

Dear EJorgensen,

I apologize for the delayed reply. I’ve been dealing with some medical issues since my last message, and I’ve also settled in Egypt during this period.

I’m still very much interested in sharing the pTi plasmid, and I believe we could collaborate and exchange our results. Over the next few weeks, I plan to resume cloning methyltransferase enzymes using a different Anderson promoter (from iGEM) in the pOpen vector. Hopefully, I’ll achieve some promising results this time.
Best wishes for both of us:)
Atef