This project led to a number of educational resources and useful DNA parts for the transfer the high molecular weight DNA between synthetic biology chassis - contributing to overcoming a major bottleneck of rational genome engineering.
Outcomes, Outputs and Progress
Bacterial artificial chromosomes (BACs) are frequently used for manipulating high molecular weight DNA fragments in E. coli. To facilitate integration of the high molecular weight DNA into M. polymorpha plastid, we engineered the well-characterized BAC pBeloBAC11 by inserting sequences homologous to the trnG-trnfM intergenic region of the M. polymorpha plastid DNA. Two integration target sequences introduced into pBeloBAC11, the leading (5') integration sequence (mppl1) and the trailing (3') integration sequence (mppl2) are 922 bp and 742 bp long, respectively. Any high molecular weight DNA inserted between the leading and trailing integration sequence will integrate into M. polymorpha by homologous recombination following microprojectile bombardment (biolistic method). Furthermore, the engineered iBAC (iBAC_Mp_mTurq) harbours cyan fluorescent protein-encoding gene (mTurq) and the spectinomycin resistance-encoding gene aadA, located between two flanking mppl integration sequences. The homologous sequences to the trnG-trnfM intergenic region of the M. polymorpha plastid DNA, cyan fluorescent protein-encoding gene (mTurq) and aadA conferring resistance to spectinomycin were obtained from plasmid pCSCL0*b (Boehm et al, Plant Cell Physiol, 2015).
We used Gibson Isothermal Assembly (Gibson et al, Nat Methods, 2009, Merryman and Gibson, Metab Eng, 2012) to assemble iBAC_Mp_mTurq. Briefly, PCR amplified pBeloBAC11 backbone and the gene cassette comprising mTurq and aadA flanked by mppl integration target sites were joined to generate iBAC_Mp_mTurq. E. coli cells harbouring correctly assembled iBAC_Mp_mTurq grew on selective medium containing spectinomycin. The engineering of iBAC_Mp_mTurq was confirmed by diagnostic PCR with the flanking primers and sequencing. The engineered iBAC_Mp_mTurq can accept virtually any high molecular weight DNA for integration into the M. polymorpha plastid.
Follow Up Plans
The iBAC_Mp_mTurq-mediated system for the high molecular weight DNA integration into the M. polymorpha plastid allows rapid selection of transformants by spectinomycin resistance and the cyan fluorescent protein, mTurquoise, expression. In the follow up work we would like to adapt this system for expression of 17 different fluorescent proteins and chromoproteins. These fluorescent proteins and chromoproteins will be engineered for maximal expression in the M. polymorpha plastid.
The additional funding and any remaining funding will be spent within six months of this report as requested.