Part:BBa_K1033207:Design
Shuttle vector pSBLbE for E. coli and Lactobacillus
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 3286
Illegal suffix found in sequence at 1 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3286
Illegal NheI site found at 1981
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 3292 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3286
Illegal BamHI site found at 1960 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 3286
Illegal suffix found in sequence at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 3286
Illegal XbaI site found at 3301
Illegal SpeI site found at 2
Illegal PstI site found at 16 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
This is a shuttle vector compliant with BioBrick standard 10 and 25 most prominently (but not exclusively). As such it has standard cloning sites, and no illegal restriction sites belonging to any of the most commonly used enzymes, with the possible exception of the less used NheI.
It has the pSH71 replicon commonly used in vectors for lactic acid bacteria (LAB), and can in theory replicate in E. coli and Lactobacillus, as well as many other LAB.[1]
The insert is a standard RFP (BBa_J04450), but it may have to be replaced to use for screening in Lactobacillus.
The replicon and resistance cassette are surrounded by restriction sites selected by Erik Lundin for their usefulness, non-complementarity and not conflicting with BioBrick standards (excepting 12). The replicon is flanked by MluI and NheI, and the resistance cassette uses SacI and SalI
Construction
The basic design is an iGEM backbone where we have changed the replicon and resistance cassette through subcloning and mutagenesis PCR.
The starting backbone was pSB4C15 made by Erik Lundin at Uppsala University by modifying pSB1C3. It is modular, meaning it was made with unique restriction sites around all key parts and we simply had to sublcone. We amplified the broad range replicon from pJP059 with the corresponding restriction sites (MluI and NheI), and did the same with the backbone to remove the old replicon, then cut and ligated them.
We changed the resistance cassette to one conferring erythromycin by amplifying the ermb gene, RBS and promotor of the L. reuteri plasmid pLUL631 with SacI and SalI -overhangs on one end each. Then we digested the product with the old plasmid pSBLbC and ligated them, followed by selection on erythromycin LB-agar plates.
Source
- The backbone comes from pSB4C15.
- The replicon comes from the plasmid pJP059, originally from pSH71.
- The new promotor in the resistance cassette (CP29: BBa_K1033222) comes from the promotor library we synthesized to BioBrick standard based on the article "The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters." by Peter Ruhdal Jensen and Karin Hammer, shown below.[2]