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Improvement of an Existing Part

Modifying a Shuttle Vector by iGEM Team MarBurg 2019

Design of panS-loxP-MCS

We needed a shuttle vector to clone AK, DavB and DavA, and CF3BD into so that high yield of the constructs could be obtained in E.coli DH5α for transformation and expression in S.elongatus UTEX 2973. Work done by iGEM Team Marburg 2019 included a shuttle vector pMC_0_7+8_panS_KanResLVL2 BBa_K3776010 based on Golden Gate Assembly. This shuttle vector contains both the ColE1 origin of replication for replication in E.coli and the pANS origin replication for replication in S.elongatus. We modified this shuttle vector and registered it as BBa_K3776010 called panS-loxP-MCS. This first thing we included was a multiple cloning site (MCS) to provide flexibility for other cloning systems. We modified the pUC19 MCS by switching the placements of some restriction enzyme cut sites to fit the restriction enzymes we need to use in cloning in our project but we did not omit any so that future users can still use cut sites they necessary. We then flanked the ColE1 bacterial origin of replication with loxP sequences facing the same direction so that ColE1 can be removed with Cre recombinase. We included this design to reduce the metabolic burden of S.elongatus UTEX 2973 after this shuttle vector is transformed into it. This is because after we clone our genes to be expressed in S.elongatus UTEX 2973 at the MCS, we will transform the shuttle vector in E.coli DH5α for high yield plasmid miniprep before transforming it into S.elongatus UTEX 2973. Since the ColE1 has no more use after the shuttle vector is transformed into S.elongatus UTEX 2973, we hypothesized that removing it would minimize energy use in S.elongatus UTEX 2973 for replication.


Build

We transformed E.coli DH5α cells with BBa_K3776010 for high yield plasmid miniprep and have transformed them into S.elongatus UTEX 2973 cells by electroporation. The cells have shown resistance to kanamycin, expected due to the kanamycin cassette in the shuttle vector. We will clone our genes of interest into this shuttle vector and assay their expression S.elongatus UTEX 2973 in the second phase of our project.