Hello fellow scientists! I'm currently trying to construct a vector for agrobacterium-mediated transformation into rice. Our construct aims to overexpress two proteins (protein A and protein B), each driven by an ubiquitin promoter and nos terminator. We have succeeded in cloning two vectors with each protein individually, but we want to put them into the same vector for transformation, a vector that will be about 20 kb.
Our problem seems to be that, for some reason - potentially toxicity - protein B will not successfully insert into the full vector and be replicated by E. coli. Indeed, getting protein B into its original vector took many attempts with DH5a colonies growing slowly on the selection plates, then not at all in liquid culture. We have run backbone only controls to verify that our antibiotic isn't bad, or that our LB is off. Furthermore, our first attempt at cloning the full vector with both proteins succeeded, but the protein B actually turned out to be flipped (we are forced to use just one restriction digest site), and non-expressible with its stop codon adjacent to the promoter, which suggests that the assembly strategy works. We also tried using a different strain (DH10-B), but all 20 picked colonies were self-ligation products (we are using more insert than vector to try and account for this). To me, it seems like the best explanation would be that the ubiquitin promoter has leaky expression in E. coli, and that protein B is toxic.
So, my questions for you all are: 1. Are there other possible explanations for the reduced to complete lack of growth of DH5a with the insert vs. backbone only? 2. Do you have any other strategies that we should consider for cloning the fully assembled vector?
Thank you in advance for your help!
I should add that when inserting protein B into the full vector, we are now amplifying it with the promoter and terminator, so it's impossible to have the same issue with our first "successful" assembly; any vectors that receive the insert will have the expressible protein.
If protein B is toxic then it is likely to be difficult to do what you want. It may not be that the ubiquitin promoter is leaky in E. coli, but really any multi copy plasmid is going to have some random transcription. I can detect expression of some genes that are in reverse orientation to the promoter.
A few suggestions, 1) try using a lower copy number plasmid so that will reduce the overall amount of expression of protein B. 2) try inserting repressor binding site upstream of gene B (like the lac operator) and have ample repressor in the strain. That might reduce random transcription across gene B.
1) If the sequence of protein B has repetitive elements, or ones that are homologous to other sequences elsewhere in the plasmid, then recombination within the plasmid can occur in the bacteria leading to the loss of the antibiotic resistance gene. This would make the transformed bacteria grow more slowly. If this is the issue, you can resolve it by using a recombination deficient strain of E coli (like NEB Stable), and by growing the bacteria at 32C instead of 37C.
2) Gibson assembly is a simple and very efficient cloning method that works well with a single restriction site, and it will always ensure correct orientation of the insert. NEB's manual on Gibson assembly is a good place to learn about it.
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