Difference between revisions of "Part:BBa K624004:Experience"

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Review Information: Team Penn_14

In our attempts to be the first iGEM team to successfully transform Magnetospirllum magneticum AMB-1, we attempted to use PYMB essentials as a shuttle vector, as it contains parts for replication in both E. coli and AMB-1. However, we religiously followed the most characterized transformation protocols from research teams in Japan. Despite all our efforts, we were unsuccessful at transforming AMB-1 with PYMB essentials. We carried out a series of careful experiments and concluded that this biobrick is not functional.

In our attempts to transform AMB-1, we attempted to design an experiment that would address why AMB-1 was not growing on plates with antibiotic, despite our very scrupulous adherence to the protocol. We plated the bacteria at every step of transformation in order to address the success of each step. (Table 2.1)

Table 1-Step-by-step Transformation Results with AMB-1 Link: https://static.igem.org/mediawiki/2014/7/72/Pymb_data.pdf

In order to verify our transformation protocol and our electroporation equipment, we used the exact same transformation protocol on E. coli strain W3110. This experiment verified that our electroporator and buffers were working correctly. Similarly, it showed that we could perform the protocol properly and without error.

Table 2-Step-by-step Transformation Results with E.coli Link: https://static.igem.org/mediawiki/2014/7/72/Pymb_data.pdf

We also investigated the paper that isolated a native plasmid in Magnetospirillum Magneticum MGT-1 and created a shuttle vector of AMB-1. This paper concluded that the shortest region required for replication in AMB-1 was more than 2 kB and included the AMB-1 origin of replication and rep gene. This region also included a long sequence of nucleotides that was essential for replication for the replication of the plasmid. In addition to this, the exact sequence of the origin of replication has not been isolated. The PYMB essentials biobrick only contains 80 nucleotides of what could be the origin of replication. This gives us more reason to believe that this part is not functional.

Reference: Okamura, Y., H. Takeyama, T. Sekine, T. Sakaguchi, A. T. Wahyudi, R. Sato, S. Kamiya, and T. Matsunaga. "Design and Application of a New Cryptic-Plasmid-Based Shuttle Vector for Magnetospirillum Magneticum." Applied and Environmental Microbiology 69.7 (2003): 4274-277.