Cloning Fun

Hello! It has taken me a while to get underway with results from what I’ve been working on because so far I’ve just been doing a lot of cloning (which has been giving me some trouble).  I began by designing and ordering SUMO-deficient mutants of TR using GeneArt.  Then, I worked on cloning these mutants into m-Cherry so that I would be able to visualize the mutant once transfected.  Unfortunately, I have been having some trouble sub-cloning successfully and had to try multiple times to clone the mutants into m-Cherry.  I am 90% sure I have successfully cloned my mutants into m-Cherry as of now (from checking on a gel), this confirmation has just been put on pause for a while because of issues with sequencing.  However, I believe that the sequencer will be working again soon and I will be able to 100% confirm my clone.

It is crucial that the m-Cherry tagged mutants were cloned successfully because I conducted a transfection, with the small plasmid prep I have, into HeLa cells.  I was able to quickly view the slides from my transfection and it was very interesting because it looked like a majority of my transfected cells were whole-cell, meaning that the mutant was localized mainly throughout the cell as opposed to the nucleus.  This could potentially be really exciting because typically TR likes to remain in the nucleus, and previously our lab has found that it is hard to get TR out of it.  Therefore, one could hypothesize that SUMO plays a role in nuclear retention of TR!  All of this is very exciting, but I have been trying to not get my hopes up until I am able to confirm my clone.  As of now I am working on cloning the mutant into GFP so that I can have images in green as well as red.  I have already had one unsuccessful transformation with GFP, so hopefully it will also work soon!  Send good vibes to the cloning gods for me!

Comments

  1. Reuben Levy-Myers says:

    Hey, this project sounds really interesting since I also work with SUMO. I was wondering how you made TR SUMO-deficient. Also, do you have any ideas why SUMO seems to affect nuclear localization, my project also deals with SUMO-dependent nuclear localization and I was curious how SUMO nuclear localization in your system.

  2. Hey Reuben! Oh my goodness I am so excited to read more about your project since you are also working with SUMO! So the way you design the gene construct is through GeneArt, gene synthesis technology through Life Technologies. This gives you the ability to create pretty much any genetic mutant of anything you want. The SUMO sites for TR falls between two of the nuclear export signals within TR. So you modify TR’s sequence by eliminating the SUMO sites and thus you have a construct of TR that is SUMO-deficient.
    https://www.lifetechnologies.com/us/en/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis/geneart-gene-synthesis-frequently-asked-questions.html#vector
    Here is the FAQ page on the GeneArt website if you want to learn more about the details of how it works.
    Because the SUMO sites are near the NES of TR, I’m hypothesizing that SUMO plays a role in nuclear retention of TR-therefore the transfection appearing to be rather whole-cell would make sense!