1. In Silico

Ok, now that we have all the concepts out of the way, let's get into the details of the actual work you have to do!

 

Setup:

You have to ask yourself the important questions...


  • What do you want to do? 
  • Do you need a viral promoter? Constitutive? Or situational or cell site-specific
  • Do you need a full cassette? Or partial for silencing
  • What will your control be?
  • Did you see if your gene can be expressed in bacteria? Would it be an issue if a bacterial promoter was able to activate it?



 The more thinking you do now, the less troubleshooting later...


Components:


So you can use these techniques to clone whatever you please, but generally, if you want to express a gene, you will need the following components:


For your promoter - indicated by the green arrow

  • Do you intend to use a monocot/dicot promoter?
  • Is it inducible?
  • Is it cell-type specific
  • Be sure to match your desired level of expression to your promoter. You don't want something that is slightly toxic to be highly expressed

For your signal peptides - indicated by the yellow squares

  • Do you need to perform genome editing and would therefore need a nuclear localization signal?
  • Is it a gene that needs to be expressed in a particular organelle?
  • Has previous work shown better performance of a signal upstream or downstream of your cds, or both!

For your coding sequence - indicated by the blue square

  • Is this something that may need to be codon optimized for your plant?
  • If you run into accidental expression in bacteria, try adding an intron
  • Don't forget your start and stop codons!

For your terminator - indicated by the stop sign

  • This is more of a slow down than a guaranteed stop, so at least be aware of the risk of transcriptional leakage 
  • So chances are, you don't speak 9 languages, but you can see that the signs below indicate stop. It might take you a second to realize it though. You should think of your terminators like this, a sequence that is understandable even if not in your native tongue, but you may not understand them all!
https://www.frontsigns.com/blog/the-difference-of-world-traffic-signs/




Assembly:


You'll want to make sure you have everything situated and thought of, so you should use a software like Geneious Prime or APE to "stitch"(aka assemble) your cassette of components.

So, I recommend Geneious Prime if your university has access to it/can afford it. It is really user-friendly and has several features built-in for ease of cloning. It allows for easy sequencing comparisons and for automated RE digestions and even for type II cloning - this is something beyond the scope of the presentation, but I will likely update the blog if it gains traction so stay posted, but I mention it briefly in the E. coli module with gateway reactions. 

If you cannot use Geneious Prime, worry not! University of Utah's Dr. Wayne Davis developed a free software for plasmid/cloning work, it's called ApE (A plamid editor). It is a homebrew so it isn't as developed as Geneious Prime, but with a little practice, you can be a cloning pro in no time with it. They also have youtube videos showing how to use the software!





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