I'm currently a sophomore in course 20. When I'm behaving myself, I'm allowed to work in the Endy Lab at MIT. I will be documenting my UROP projects here. I will put my 20.109 work directly below.
M13 Engineering Ideas
||Extract from gene II.
||Extract gene X.
|| Add some base pairs between V and VII to allow for a restriction site.
||Separate from gene IX.
||Change the GTG to ATG Start?
|| Add some base pairs between III and VI and VI anda I to allow for restriction sites.
||Separate from genes IV and XI.
||Separate from genes I and IV.
||Separate from genes I and XI.
| M13 ORI 1 & 2
|| Are two ORIs necessary? If so, can they be consolidated?
Extractions, separations, etc. include ensuring that each gene has its own promoter, RBS, terminator, and any other pertinent regulatory sequences.
In addition to separating the genes, it would also be wise to develop or utilize/modify an existing system like Biobricks to build the genome so that convenient restriction sites exist between the genes so they can be easily removed or replaced.
I used the following annotation to locate and design changes to the genome.
|| Possible to remove bps 6600 - 7100 upstream of KanR? Does this DNA have functional significance?
||2.27.07 Ligation and Transformation Data
|DNA Ligation Sample
||Expected Number of Transformants
||Observed Number of Transformants
| Control Plasmid
| BKB with cocktail but no ligase
| BKB with ligase and cocktail
| BKB with insert and ligase and cocktail #1
| BKB with insert and ligase and cocktail #2
2.27.06 Ligation and Transformation Data
We didn't determine the concentration of the prepped DNA from the ligation reactions, so we cannot calculate the transformation efficiency of these transformations. We do know, however, that 5 ng of vector was used to transform the control plasmid, so efficiency = (1184 colonies)/(5*10^-3 micrograms of DNA)= 5.92*10^6 colonies/microgram of DNA.
The control plasmid transformation serves as a positive control. It should work. If this transformation didn't work (along with some or all of the others), we might suspect an issue with the competency of the cells or with the transformation process. The appearance of colonies on this plate, however, demonstrates that this is not the case. The cells are indeed competent and our protocol worked.
The backbone with and without ligase mixed with the killcut cocktail serve as negative controls. They should not work. If a significant number of colonies were to show up on these plates, this would first mean that the restriction endonucleases weren't cutting. This could be because they had expired or because the sites no longer existed in the DNA. Assuming the latter for the ligated vector, this would mean that the vector sequence was somehow modified during the initial digest. For the linearized vector, the appearance of colonies would suggest that the DNA somehow became ligated and lost restriction sites, or the cell accepted cut vector.
The controls do not provide a definitive diagnosis of the problem should the experimental ligations fail, but they provide starting points for the inquiry into the failure.