Synthetic Biology:Vectors/Single copy plasmid
Goal
Design and fabricate a single copy vector in which BioBricks devices can be characterized. To date most characterization work has been done in low or high copy vectors which have several issues including
- Copy number is uncertain or variable making it difficult to infer PoPS per DNA copy.
- At high copy, devices place a high metabolic load on the cell thereby altering host physiology and observed device behavior.
The proposed solution to these two problems is to characterize devices at single copy in the cell. Obviously, such a vector will vary between 1 and 2 copies per cell over the cell cycle but nevertheless will hopefully present an improvement over the current situation. The advantage of using a single copy plasmid rather than simply integrating the device into the genome is that a separate plasmid offers some isolation from the host and makes moving the device between different host strains slightly easier.
Design
The F plasmid origin needs to be designed. The complete F plasmid with partitioning genes in ~10kb in length. It contains several BioBricks restriction sites in both coding and noncoding regions.
Once designed, the F plasmid origin can be assembled with an antibiotic resistance marker and cloned into the vector scaffold to generate a new single copy BioBricks plasmid.
Chris Anderson suggested inclusion of the R6K origin in these plasmids (rather than inclusion of a pUC19 origin in the multiple cloning site). The R6K origin is a conditional origin. It only works in the presence of the trans-acting protein Π (encoded by pir) for replication. R6K replicates at a medium copy (15 per cell) in pir+ strains and high copy (250 per cell) in pir-116 (high-copy-number mutant) E. coli hosts.
Fabrication
- Reshma 15:27, 27 February 2007 (EST): Tom suggested that mutations of the BioBricks sites could be done via Pete Carr, Farren Isaacs and George Church's single stranded mutation method.
Notes
Drawbacks
- Can only be used in F- strains
- Should likely be used in recA- strains to avoid integration onto the genome and ensure plasmid stability.
- It is unclear whether this vector would truly be operating at single copy. If it is not, perhaps it is easier to stick with the pSB2* plasmids.
Relevant pages
See the list of parts for plasmid engineering.
See notes on bacterial artificial chromosomes.
See Synthetic Biology:Vectors for information on vector nomenclature, existing vectors and vectors that we would like constructed.
Vectors has a lot of general information on vectors.
References
- Jones KL and Keasling JD. Construction and characterization of F plasmid-based expression vectors. Biotechnol Bioeng. 1998 Sep 20;59(6):659-65.
- Metcalf WW, Jiang W, and Wanner BL. Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers. Gene. 1994 Jan 28;138(1-2):1-7. DOI:10.1016/0378-1119(94)90776-5 |
