Part:BBa_K4706000:Design
pSB1C30YIpHR-HO Chromosomal integration at HO loci
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 3049
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 3055 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 3049
Illegal BglII site found at 2500
Illegal XhoI site found at 1533
Illegal XhoI site found at 2425 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found at 3049
Illegal suffix found at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found at 3049
Plasmid lacks a suffix.
Illegal XbaI site found at 3064
Illegal SpeI site found at 2
Illegal PstI site found at 16 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Design Notes
To design our own YIp (Yeast integrative plasmid) we needed to combine our initial Backbone with HO Homology Regions from the Open yeast collection. For facilitating the cloning process we uses Homology based cloning with the use of NEB Builder
The initial step in the assembly of all constructs is the addition of the corresponding homologous regions ( ScHR3p-HO/Ba_J435241 and ScHR5p-HO/BBa_J435242 Homology region) into the plasmid backbone pSB1C30. Thus we also have the Build phase in this cycle twice as we needed to perform two insertions and confirm that each of those was sucessfull.
How to build it youself from scratch:
Gel standard from this page Duesseldorf 2023:
As we were conducting homology based cloning, linearization pSB1C30 and BBa_J435242 was necessary first and foremost.
Each of the two fragments has a 40bp homology to each other and were assemled as previously shown via homology based cloning (NEBuilder).
The resulting intermediate will look like this:
After yielding transformants, we perfomed a PCR to confirm if our insert was present. We also loaded the plasmid itself on-to a gel to confirm aproximate total size via gel electrophoresis.
Gelelectrophoresis of PCR and insert check. In our internal documentation we refered to pSB1C30YIpHR-HO5' as CD006.
As we were conducting Homology based cloning, we again needed to linearize our intermediate plasmid and [BBa_J435241]
Each of the two fragments had 40bp homology to each other and were assemled as previously told vie Homology Based cloning (NEBuilder).
This is the resulting intermediate plasmid:
After yielding transformants we perfomed a PCR to confirm if our insert was present. We also loaded the Plasmid itself on the gel to confirm aproximate total size.
Gelelectrophoresis of PCR and insert check. In our internal documentation we refered to pSB1C30YIpHR-HO as CD007.
As it has been shown that linear DNA fragments have a much higher recombination frequency than if left in a circular plasmid, we decided that we wanted to linearize our Backbone via PCR in our design, not with restriction enzymes, as to not degrade potential cloning capabilities of the backbone.
Source
All direct sources are from the iGEM distribution/Open Yeast collection itself: pSB1C30; BBa_J435241; BBa_J435242