After annotating the genome, I saw that there was a lot of overlap among the genes, which would make further engineering very difficult. Thus, the goal of my refactoring was to separate all of the genes and add new restriction enzymes on each side so that the indiviudal genes could be tinkered with by themselves. The main obstacle with this method is that the promoters are situated away from the the genes that they act on, and we don't know what the allowable distances should be. Thus, I left the promotors and moved around the genes and their rbs's instead. In any case, I feel that modifying the genes themsleves holds the most promise for future studies, so separating the genes is the most important aspect of the refactoring. As for more innovative refactoring, I would really like to add some sort of protein to gene 8 that we would like to amplify because the gene is copied so many times. However, as I do not know exactly what protein this should be nor what the DNA sequence is or where to place it, I did not include it in this refactoring. I hope, though, that we can figure out a good way to do this. Thus, overall, my refactored genome separates the genes, with the hope of being able to further engineer it in the future.
The part that I refactored is M30100.
|M13K07 from the unique Hpa1 site in gene 2 to the unique BamHI site in gene III.||There is a great deal of gene overlap in this region.|
|Altered Components||Refactoring (Modifications)|
|gene 2, gene 10, rbs gene 10||The rbs for gene 10 was in gene 2 and gene 10 itself overlapped a lot with gene 2, so I separated the parts by copying the rbs gene 10/ gene 10 sequence and placing it after gene 2. Additionally, I added zero cutter restriction enzymes (AatII and ApaI) on each side of the rbs/gene complex so that the part could be easily separated.|
|gene 10, rbs gene 5||The above modification disrupts the rbs of gene 5, so I added the altered base pairs to fix the problem. I also added another cutter (BmtI) to provide the first site to separate gene 5.|
|gene 5, rbs gene 7||I separated the parts and added two cutters (EcoRI and KasI) to complete the gene 5 section and start the gene 7 one.|
|gene 7, gene 9, rbs gene 9||I separatd the gene 9 components from gene 7 and added two cutters (NcoI and NheI) to complete the gene 7 section and start the gene 9 one.|
|gene 9, gene 8, rbs gene 8||I separated the gene 8 components from gene 9 and added two cutters (SalI and SpeI) to complete the gene 9 section and start the gene 8 one. Finally, I added a cutter (XbaI) at the end of gene 8 to finish that section.|
|Promoter overlaps||Many of the promoters overlap with other functional regions of the genome, but we don't know what the ideal distance is between promoter and gene, so moving them could be very detrimental. On the other hand, I moved some genes (especially gene 10) a good distance away from its promoter, so that might also be problematic.|
|Add a needed protein to gene 8||Gene 8 is copied many times, so if we could add a protein to gene 8 it might be possible to easily and quickly obtain large quantities of the protein.|