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.
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
Hello all.
I just noticed that some pTi vectors are described as having resistance to ampicillin on GitHub, particularly on the Molecular Diagnostics Toolkit in the platemap for the MDT v1.1 plate.
Can someone fix this information?
Thank you.
Natalia
Thank you very much for spotting and raising this issue!
This has now been corrected here.
Please do let us know if there are any other discrepancies - you can let us know here and/or log them as an issue on our Reclone GitHub repo directly.
If you’re working more closely on this work, I’d be happy to give you access as a contributor so that you can directly maintain the GitHub repo on the parts relevant to your own research. Let me know
Thank you again!
Yan Kay
Ahh yes, I thought your name seemed familiar - Marko has mentioned you. I was wondering if it was you who had recently joined the Reclone meetings Welcome, and thanks for joining us in setting up more hubs and nodes around the world!
I hope the information I passed to Marko about the collections was helpful to you, and thanks for sending over the photos of the plates.
For adding you to the GitHub repo - can I double-check if you’re ok with me adding you using the email address associated with your account here? I couldn’t find you when I searched on GitHub. Else, feel free to use the forum to directly message or email me the email you want to use.
