So this doesn’t quite count as “synbiofun” since it didn’t work, so it’s not that fun. But figure I may as well post negative data here when we have it…
Based on this paper, I felt compelled to test some of the tricks they had published on that improved recombination efficiency. First up was DNA sequences that may help with nuclear targeting / import. Tried the NFkB DTS (DNA nuclear-targeting sequences) since that seemed to perform the best for them.
Didn’t exactly reproduce what they did, since I wanted to 1) Use my own construct that we normally use for recombination reactions, and 2) insert the sequence at a convenient location that I could put in with a single molecular cloning step and didn’t get in the way of any other elements we already had in the plasmid.
We cloned the sequences into the “G1180C_AttB_ACE2(del)-IRES-mScarletI-H2A-P2A-PuroR” backbone, where we could use the percentage of mScarlet fluorescent cells to tell us if recombination efficiency increased. Because of the repetitive nature of the NFkB DTS sequence, we ended up getting two different clones with the intended sequence: clone D had the indicated NFkB DTS sequence plus and additional repeat (for 5 repeats total), while clone E had the NFkB DTS sequence missing a repeat (for 4 repeats total).
Sarah recombined these into landing pad HEK 293T cells, and these were the results of red+ cells.
Negative control: 0.002%
G1180C (unmodified): 17.4%
Clone D (5 repeats): 4.78%
Clone E (3 repeats): 17.8%
So yea. Really didn’t seem to do anything. Not sure why Clone D is worse, although this is an n=1 experiment. If we really wanted to continue this, we would probably need to re-miniprep the plasmids to make sure it’s nothing about that specific prep. That said, nothing about the above results makes me optimistic that this will actually help our current system, so this avenue is likely going on ice.