Creating " drop in" protein expression vector series based on pTi

Hi All

We have an open vector backbone for protein expression called pTi, developed by @FernanFederici @Isaac_N & @tfmatuteuc-cl :

At the moment this is an AF vector according to the Reclone Syntax, meaning that it only has the resistance markers, origin of replication and backbone parts. We’d like to create a series of vectors into which you can directly add CD coding sequences for expression using golden gate assembly with BsaI.

Current thinking is a concise series of vectors, building from a base vector with T7 promoter and His-Tag, that allow you to rapidly screen several solubility tags, periplasmic tags, promoters and different promoters.

Inspiration came from:
Correa, Agustín, et al. “Generation of a vector suite for protein solubility screening.” Frontiers in microbiology 5 (2014): 67.

Here is a draft starter list - any suggestions/comments/edits?

If anyone is sufficiently motivated by this idea to help with the cloning, please let us know :slight_smile:

Jenny

4 Likes

Hi Jenny, we have a large panel of E. coli expression plasmids with some 20 fusion partners, all constructed in consistent manner. Should be easy to lift them all off the same way and dump into an other plasmid backbone.
The list of (most) of the plasmids in this pExp series are here pEXP series of expression vectors – Hyvönen Group @ Biochemistry, Cambridge
They all have octa-His tag in the N-terminus, fusion partner, TEV site, cloning site for SLIC (but essentially for GoldenGate too) with BsaI sites and (an optional) C-terminal Strep-Tag. All empty constructs have stop codon after the 5’ BsaI site so easy to use for expression control.
Of the fusions we have, Bla, GB1, Sumo and MBP are the usually the most successful and they do express on their own at pretty ridiculous amounts.
As we just got the open collections from you, I have asked my RA to test couple of the fusions in pTi and couple of other backbones (incl different copy numbers) to do some comparisons (if anyone knows of a consistent comparison set between different plasmids, I’d love to hear).
(Sorry, did not have time to look at the document with details, head capacity limiting!)
Because of the way we (or rather Aleksei Lulla in my lab) made these, some of the fusions perhaps should be remade and synthetic clones, to avoid issues with distribution.

1 Like

Hi Jenny, we are moving more slowly than I had hoped with our Experiment.com project to move the Open enzymes ORFs into expression vectors. We now have all but 2 or 3 of the 81 ORF plasmids as glycerol stocks. We have pTi and pTiR in house thanks to Reclone. Right now we are limited to the parts that Scott gave us- parts from the E. coli expression kit from FreeGenes. We are about to start trying to Golden Gate Assembly ORFs into a pTi backbone with T7/lac0, RBS + Histag, TEV site, linker and a terminator at the end. It would be great to have a series of vectors like the one you described.

We are worried about the potential toxicity of enzymes that do work on DNA, although we bought the NEB® 5-alpha F’Iq cells and hope that will help. But being able to use different promoters and other tags would be great.

Let us know what we can do to help. We have idle hands and some funding to synthesize and clone stuff.

1 Like

Hello everyone, here are my “custom” parts that I use for my studies. They are in SnapGene (.dna) format. They are not exactly “Open” per se, but hope that they will be helpful. The backbones are exact clones of pET, p15A and pML backbones. Most of the parts are subcloned into pJET vectors, which I use directly without porting to the pOpen v3 vectors.

https://drive.google.com/drive/folders/13wSuSHHlZs0s6viSDrC6rey6lLkruYa5

Having said that, I think about a different approach - instead having a library of backbones, they also should be modularized. Like, having lacI, resistance, origin of replication, rop/bom, etc. as separate modules, such as the Open Yeast Collection kit. What are your thoughts on that?