The genome sequence of the liverwort Marchantia polymorpha has just been published in Cell. Interest in developing this evolutionarily basal plant as a model system has been increasing in recent years, and we are currently gearing up to see if we are able to successfully transform this plant with Agrobacterium. Figure 1 of the aforementioned Cell paper nicely illustrates where M. polymorpha fits within the basal plants.
In the process of designing improvements to some of the tools under development in the lab, as well as engineering a fix for the Modular Cloning kit Level 2 position 5 end linker debacle, I have been making use of the valuable information housed in the Registry of Standard Biological Parts. This remarkable resource provides a vast array of DNA sequences, many of which have been functionally validated, that can be readily adapted for your specific needs. If you are ever faced with a seemingly intractable problem, take a look in the iGEM inventory and see if your creativity isn’t sparked to devise a novel and creative solution or work-around.
I recently stumbled across NP-Sticky: Model-Guided Ligation Calculator, a very easy to use tool for calculating the free energy of binding of single strand overhangs of double stranded DNA. Being able to query candidate motifs for unfavorably strong cross-reactivity with non-target overhanging sequences will be useful for engineering a robust solution of the previously identified MoClo Level 2 end linker mediated binary vector self ligation issue.
In my previous post, I presented information to illustrate an issue that I have observed when using the MoClo system for building binary plasmids containing 5 transcriptional units via Type IIS restriction endonuclease mediated technology (Golden Gate). When I finished composing that post, I was left with a nebulous feeling, because I simply didn’t really have a sense for why this phenomenon was occurring. Things happen for a reason, and I think I have ferreted out the reason for why cloning with position 5 will be fraught with problems. To unequivocally determine if this is a position 5 endlinker specific issue (or if the binary self ligation could be mediated by other endlinkers), I conducted an experiment by conducting Golden Gate reactions containing the RK2 binary with an endlinker, with individual reactions for each of the 5 endlinkers I was testing.
The results are pretty shocking. There aren’t too many colonies on the plates spread with E. coli transformed with the reactions performed with endlinkers for positions 1 through 4, but the position 5 endlinker clearly displays the issue I have been experiencing. The inset clearly shows how abundant the white colonies are in this reaction, and are currently the bane of my existence for my current project where I am constructing a binary plasmid containing 5 transcriptional units; there is a tremendous amount of background colonies to be screened!
But why is this happening? The reason I conducted the endlinker experiment was because I considered the nucleotide structure of the various endlinkers, relative to the binary plasmid. BpiI digestion produces specific 4 nucleotide 5′ overhangs, which have been agreed upon by the community; an examination of these reveals the problem.
Position 7 has been omitted for clarity (the letters for position 7 are exactly the same as are found on the canthaxanthin synthesis operon; endlinker 7 is mainly used when accessing the Level 2i-1 of Golden Gate cloning, when one seeks to stack more than 7 transcriptional units into the binary). The problem I have been having likely is due to the fact that the motif for endlinker 5 is “TGTG”, which of all 6 endlinkers has the greatest degree of homology with the left border (LB) proximal BpiI released overhang.
This is a perfect match in the 5′ most two nucleotides, and the third nucleotide is the pyrimidine “T”, which is in the same functional class as the letter “C”. A perfect match of two letters, plus a similar base in the third position of the motif means that there is only one letter of the four in the endlinker 5 overhang which is a true mismatch. “TGTG” might not have been the best choice for an endlinker motif, in the context that the LB proximal BpiI site opens a motif that is receptive to “TGCC”. The solution to this problem is something I need to think about; there might be a position 5 dummy fragment that I could utilize in conjunction with the endlinker 6 fragment, but this is an experiment I need to conduct.