Difference between revisions of "Part:pSB6A1:Experience"

iGEM2016 Tokyo_tech

It was found that the registered sequence of pSB6A1 was wrong; our sequencing of the plasmid showed that 272 bases was lost and the lost sequence was located at the downstream of the PstⅠ recognition site.

When the lasR (BBa_C0179) gene fragment was inserted into pSB6A1, we tried to know whether the ligation was correctly performed by cutting with SalⅠ. We expected generation of a fragment of 800 bp, but unexpectedly, a fragment of approximately 500 to 600 bp was observed(Fig.5-1) in electrophoresis. The result of sequencing showed that the recognition sequence of PstⅠ and SalⅠ were adjacent.

Fig. 5-1. Agarose gel electrophoresis of lasR (pSB6A1) cut with SalI. Lane 1 shows that approximately 300 bases are missing in lasR or pSB6A1

iGEM2012 WHU-CHINA

This plasmid backbone works very well. When we tested the function of our promoter PfadR BBa_K861061 in high copy number plasmid pSB1A2, almost all the clones went light red. The result from plate reader also indicted that in high copy number plasmid pSB1A2, the promoter is quite leaky. However, when we transferred the part into pSB6A1, the device becomes less leaky. We therefore highly recommend iGEM teams to use this backbone to test the function of factor-relugated promoter.

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This plasmid has been adapted to the version 5 of BioBrick vector backbones by the ETHZ 2011 iGEM team, resulting in pSB6A5. This means, that the cassettes for the ORI and antibiotic resistance have been minimized (reduction of the size and transcriptional terminators flanking the prefix and the suffix have been added, to trancriptionally insulate the inserted parts from the vector backbone machinery and vice versa. This minimization yielded a reduction in size from 4022 bp to 2743 bp.

For comparison of the copy number of the plasmids, triplicates of OD normalized bacterial cultures containing the respective plasmids were miniprepped. The DNA concentration was then determined by an agarose DNA gel electrophoresis of EcoRI digests of the purified plasmids (Figure 1). This experiment showed a clear reduction in copy number of the minimized version of the original plasmid (Figure 2).

Figure 1: Agarose gel with 2-log-ladder loaded in in lane one, digests of triplicates of the EcoRI digests of pSB6A1 (lane 2-4) and pSB6A5 (lane5-7).

Figure 2: Agarose gel with 2-log-ladder loaded in in lane one, digests of triplicates of the EcoRI digests of pSB6A1 (lane 2-4) and pSB6A5 (lane5-7).