Difference between revisions of "Team:NAIT Edmonton/Results"

pSB1C3 is a high copy number plasmid (RFC [10]) carrying chloramphenicol resistance.

The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell).

pSB1C3 has terminators bracketing its MCS which are designed to prevent transcription from *inside* the MCS from reading out into the vector. The efficiency of these terminators is known to be < 100%. Ideally we would construct a future set of terminators for bracketing a MCS that were 100% efficient in terminating both into and out of the MCS region.

Original Sequence

In Spring 2011, pSB1C3 was sequenced using several primers. The current sequence reflects the most recent sequencing of pSB1C3 (w/ BBa_J04450, located in SP 4000 Well 2A). However for posterity, we have included the old sequence that was originally specified in the Registry (PSB1C3-Text-Archival-5-4-11.txt) as well as images of the original sequence with markers pointing out the changes made:

Design Notes

Eliminated ampicillin resistance.

Subversions

Freiburg's pSB1C3 varient of 2010

The iGEM team Freiburg created a modified version of the pSB1C3 in which the two restriction sites SspI and PvuII were removed via site directed mutagenesis. These restriction sites and two others that are not present in the pSB1C3 (BamHI and SalI) were used as single cutting restriction sites to replace the loop coding sequences of the Adeno-associated Virus. For this purpose scar-less cloning is necessary, because of the unpredictable consequences on the viral vector performance arising from mutations or insertions. The final capsid gene adheres fully to the RFC_10 standard.

The sequence and a more detailled description can be found under the BioBrick ID BBa_K404200.

Source

This part was derived from pSB1AC3-P1010.

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:

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.