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BBa_K3228069

BBa_K3228069 iGEM_Freiburg_2023

To modify S. elongatus PCC 7942, we, iGEM Freiburg 2023, decided to use this cyanobacteria-specific shuttle vector developed by iGEM Marburg 2019 which they kindly shared with us. This shuttle vector comes with 2 origins of replication (Ori): an Ori from PCC 7942, more specifically, from one of its endogenous pANS plasmids (which makes the vector compatible with our strain), and a high copy number Ori from E. coli, ColE1 (for more information, visit iGEM Marburg 2019 page.)

First, we cloned the aforementioned genes for B12 production (bluB and ssuE) into the shuttle vector, creating a new plasmid, piG_CBM. Next, we attempted to modify PCC 7942 with the piG_CBM via electroporation, conjugation (tri-parental mating), and natural transformation- none of which were successful (we observed no colonies on the plates containing antibiotic resistance). The reason for conjugation not succeeding was eventually found: the shuttle vector does not encompass an OriT (basis of mobility region/bom site) which would need to be added for the conjugation. Furthermore, according to Encinas et al. 2014 [1], the conjugative plasmid used as a helper and the shuttle vector should have the same OriT to improve the efficiency of conjugation.

The electroporation should not be affected by the lack of OriT and yet yielded no colonies after several attempts (with the exception of a faint colony for S. sp. PCC 6803. We did not find an explanation for why it is not working since the protocol we used from Prof Hess group, leader of the CyanoLab [2] at the University of Freiburg, was said to have a high success rate (at least for PCC 6803).

Possibly, a different, PCC 7942-specific electroporation protocol could be tried to further validate the shuttle vector. Also, the natural transformation did not succeed, however, we only tried it once (due to the time limitations) and this method of transformation has a lower efficiency, as a cyanobacteria-focused research group leader Prof. Wilde, also from the University of Freiburg [3], mentioned to us.

Therefore, several repetitions and/or a different transformation protocol might be needed to validate the shuttle vector’s applicability for natural transformation.

References

[1]Encinas D, Garcillán-Barcia MP, Santos-Merino M, Delaye L, Moya A, De La Cruz F. Plasmid Conjugation from Proteobacteria as Evidence for the Origin of Xenologous Genes in Cyanobacteria. Journal of Bacteriology [Internet]. 2014 Apr 15;196(8):1551–9. Available from: https://doi.org/10.1128/jb.01464-13

[2]http://www.cyanolab.de/

[3]https://www.bio.uni-freiburg.de/ag/wilde

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 <I>iGEM_Freiburg_2023</I>
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-To modify <i>S. elongatus<i/> PCC 7942, we, iGEM Freiburg 2023, decided to use this cyanobacteria-specific shuttle vector developed by iGEM Marburg 2019 which they kindly shared with us. This shuttle vector comes with 2 origins of replication (Ori): an Ori from PCC 7942, more specifically, from one of its endogenous pANS plasmids (which makes the vector compatible with our strain), and a high copy number Ori from <i>E. coli</i>, ColE1 (for more information, visit iGEM Marburg 2019 page.)
+To modify <i>S. elongatus</i> PCC 7942, we, iGEM Freiburg 2023, decided to use this cyanobacteria-specific shuttle vector developed by iGEM Marburg 2019 which they kindly shared with us. This shuttle vector comes with 2 origins of replication (Ori): an Ori from PCC 7942, more specifically, from one of its endogenous pANS plasmids (which makes the vector compatible with our strain), and a high copy number Ori from <i>E. coli</i>, ColE1 (for more information, visit iGEM Marburg 2019 page.)
 First, we cloned the aforementioned genes for B12 production (bluB and ssuE) into the shuttle vector, creating a new plasmid, piG_CBM. Next, we attempted to modify PCC 7942 with the piG_CBM via electroporation, conjugation (tri-parental mating), and natural transformation- none of which were successful (we observed no colonies on the plates containing antibiotic resistance). The reason for conjugation not succeeding was eventually found: the shuttle vector does not encompass an OriT (basis of mobility region/bom site) which would need to be added for the conjugation. Furthermore, according to Encinas et al. 2014 [1], the conjugative plasmid used as a helper and the shuttle vector should have the same OriT to improve the efficiency of conjugation.