Part:BBa_K3014006:Design
ptRNA_backbone
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2140
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 2146 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2140 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found at 2140
Illegal suffix found at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found at 2140
Plasmid lacks a suffix.
Illegal XbaI site found at 2155
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal AgeI site found at 503 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Design Notes
In advance to the design of the plasmid, Dr. Josef Altenbuchner was consulted as an external expert, due to his long-year experience in plasmid design and the work with Vibrio natriegens at the Institute for Industrial Genetics (University of Stuttgart). Besides that, we also consulted the iGEM team Marburg 2018, as winners of last year’s grand prize with their exceptional foundational progress with Vibrio natriegens. As proposed by Dr. Altenbuchner we chose a two-plasmid system, one plasmid to raise the cellular rare tRNA concentrations and one plasmid for protein expression. By using a two-plasmid system, we want to maintain the possibility for the experimenter to use an expression plasmid of choice [individual adaptation of transcript level by the plasmid origin of replication (ori)]. This approach also keeps the respective plasmid size small to allow a simple experimental workflow.
As selection markers, we chose tetracycline and chloramphenicol, as Vibrio natriegens is not majorly susceptible to beta-lactamase inhibiting antibiotics (e.g. ampicillin, kanamycin, ...), as advised by the iGEM team Marburg 2018.
The mentioned parts were combined, using the iGEM's BioBrick cloning vector pSB3T5as a template, to create our ptRNA vector backbone.
Source
This part was synthesized by Integrated DNA Technologies.