Plasmid_Backbone
pSB1C3

Part:pSB1C3:Experience

Designed by: Austin Che   Group: iGEM07_Example   (2008-09-08)
Revision as of 22:53, 21 October 2016 by Dsanderson (Talk | contribs) (→‎User Reviews)

This experience page is provided so that any user may enter their experience using this part.
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Applications of pSB1C3

User Reviews

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No review score entered. Genspace 2016

Plasmid Copy Number at Stationary Phase: 24

The Genspace 2016 iGEM team measured the copy number for pSB1C3 in Top10 both by qPCR and gel electrophoresis of lysate generated from cells harboring K909006-pSB1C3 and found the copy number to be much lower than indicated on the main page. qPCR suggested copy number was around 24 copies per cell and lysate electrophoresis indicated copy number was close to that detected by qPCR.

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Enter the review inofrmation here.

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Waterloo iGEM 2014

The pSB1C3 backbone has been made more versatile by turning it into a shuttle vector. By cloning a Staphylococcal selective marker (erythromycin resistance gene) and origin of replication, this part has been improved to be used in both E. coli and Staphylococcal organisms. The Staphylococcal parts have been incorporated into the backbone between the VR and the pMB1 replication origin in pSB1C3. The newly integrated parts have been sequence confirmed and a restriction digest of this part isolated from E. coli shows expected bands:

Shuttle vector restriction digest.png

The shuttle vector was successfully electroporated and maintained in Staphylococcus epidermidis (ATCC 12228) and the newly transformed cell displayed erythromycin resistance. Whether or not the shuttle plasmid confers chloramphenicol resistance due to the E. coli gene is still inconclusive due to the fact that S. epidermidis was shown to have some resistance to chloramphenicol already.

Shuttle Vector Resistance.png

For more information, view the part page for this shuttle vector: BBa_K1323017

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UNIPV-Pavia iGEM 2011

This cloning vector has been improved, cloning in it the strong pTet promoter, between E and X restriction sites; the mRFP coding sequence from BBa_J61002, has been placed between S and P restirction sites, in order to facilitate the cloning of coding sequences downstream pTet promoter in this high copy number plasmid.
For more details see the BBa_K516999 experience page.

The 2011 Nevada iGEM Team attempted to use pSB1C3 as a template to amplify the chloramphenicol resistance cassette for use in a new part. When they attempted to design primers for this purpose, we were unable to identify a start codon. We subsequently contacted Dr. Knight and Austin Che about the problem. They re-examined the sequence and found that the sequence in the registry was missing two nucleotides which resulted in an apparent frameshift. Upon fixing the sequence annotation, we were able to design our amplification primers for the chloramphenicol cassette.

iGEM JHU Wetware 2012 The pSB1C3 backbone was utilized to create a new plasmid backbone specifically engineered for use with the Golden Gate assembly method. Flanking regions were added within the prefix and suffix that facilitate assembly through the use of Bsa1 overhangs. This makes for a scarless ligation into the vector. The resulting sequence is a standard pSB1C3 with a part of choice, as the overhangs are removed during the Bsa1 cleavage. Three varieties of the backbone were made.

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