At the moment, as I mentioned in my last post. I'm trying to design a gene to get synthesised. I'm using GeneDesigner from 2.0, and I now have about 100 saved copies of my gene in various incarnations.
My first design was simple enough...the operon (a set of genes one after the other) surrounded by the biobrick prefix and suffix
For this design I used the whole vio operon, including all the bits at the beginning and end that weren't part of the gene. I was scared of cutting anything out, in case we got our nice designed gene back and it didn't make any product at all.
However, when I looked at the sequence I found that the genes within the operon were out of line. Each amino acid (the blocks that make up proteins) is coded for by three bases, for example the sequence AAAGGG will make two amino acids. AAA = lysine and GGG = glysine. However on my operon this was out of alignment; instead of getting XXX / AAA / GGG / XXX, I was getting XXA / AAG / GGX
To counter this I re-designed it, putting all the genes as separate blocks and double checking that they all made the correct proteins:
The end result was a little cramped, but it meant all the proteins were being produced as displayed, along with the prefix, suffix, and a ribosome binding site at the start of the gene.
So I showed it to my supervisor. Who looked at it, and then looked at me, and then said in a very kind voice, "Why do you *need* all that stuff around the genes?"
The thing is I'm a little afraid of taking it out. Just in case there's some sort of importance to it. But in terms of genetic engineering, and further gene manipulation, it's more useful to have each gene smartly laid out, with it's own ribosome binding site. We also want to add a promoter (at the suggestion of the ever-wonderful DNA2.0) which is a kind of START site for the gene, and allows it to be turned into mRNA (which is then turned into proteins). As the operon product has some antibiotic properties (if they're expressed at too high a level they can kill the bacteria), we want it under an inducible promoter as well, so we can turn this operon on and off by adding different chemicals to the bacteria.
So here is my plan at the moment:
Each gene is preceded by a ribosome binding site (rbs and B0034 are the same thing, I just don't know how to relabel things in GeneDesigner). The operon is preceded by a promoter and the whole thing is enclosed within the suffix and prefix.
I'm showing it to my supervisor today. I really hope it's good enough, I want to get this sequenced.
(as a point of interest, these are only a subset of the different versions currently on my computer. I also have variations on all of the above with codon optimisations, restriction sites removed and added, and different/differently placed ribosome binding sites)
EDIT (added after meeting)
I showed him the design, and this time it was pointed out that I was missing a gene...
But the rest of the design was good! So it looks like this will be the final product, bar a little fiddling about with the actual sequence:
Narrow-minded, short-sighted university administrators
3 hours ago in The Phytophactor