% IMPORTANT: The following is UTF-8 encoded.  This means that in the presence
% of non-ASCII characters, it will not work with BibTeX 0.99 or older.
% Instead, you should use an up-to-date BibTeX implementation like “bibtex8” or
% “biber”.

@ARTICLE{Lenz:902912,
      author       = {Lenz, Patrick and Hilgers, Fabienne and Burmeister, Alina
                      and Zimmermann, Leonie and Volkenborn, Kristina and
                      Grünberger, Alexander and Kohlheyer, Dietrich and Drepper,
                      Thomas and Jaeger, Karl-Erich and Knapp, Andreas},
      title        = {{T}he i{S}plit {GFP} assay detects intracellular
                      recombinant proteins in {B}acillus subtilis},
      journal      = {Microbial cell factories},
      volume       = {20},
      number       = {1},
      issn         = {1475-2859},
      address      = {London},
      publisher    = {Biomed Central},
      reportid     = {FZJ-2021-04667},
      pages        = {174},
      year         = {2021},
      abstract     = {BackgroundBacillus subtilis is one of the most important
                      microorganisms for recombinant protein production. It
                      possesses the GRAS (generally recognized as safe) status and
                      a potent protein secretion capacity. Secretory protein
                      production greatly facilitates downstream processing and
                      thus significantly reduces costs. However, not all
                      heterologous proteins are secreted and intracellular
                      production poses difficulties for quantification. To tackle
                      this problem, we have established a so-called intracellular
                      split GFP (iSplit GFP) assay in B. subtilis as a tool for
                      the in vivo protein detection during expression in batch
                      cultures and at a single-cell level. For the iSplit GFP
                      assay, the eleventh β-sheet of sfGFP is fused to a target
                      protein and can complement a detector protein consisting of
                      the respective truncated sfGFP (GFP1-10) to form fluorescent
                      holo-GFP.ResultsAs proof of concept, the GFP11-tag was fused
                      C-terminally to the E. coli β-glucuronidase GUS, resulting
                      in fusion protein GUS11. Variable GUS and GUS11 production
                      levels in B. subtilis were achieved by varying the ribosome
                      binding site via spacers of increasing lengths (4–12
                      nucleotides) for the GUS-encoding gene. Differences in
                      intracellular enzyme accumulation were determined by
                      measuring the GUS11 enzymatic activity and subsequently by
                      adding the detector protein to respective cell extracts.
                      Moreover, the detector protein was co-produced with the
                      GUS11 using a two-plasmid system, which enabled the in vivo
                      detection and online monitoring of glucuronidase production.
                      Using this system in combination with flow cytometry and
                      microfluidics, we were able to monitor protein production at
                      a single-cell level thus yielding information about
                      intracellular protein distribution and culture
                      heterogeneity.ConclusionOur results demonstrate that the
                      iSplit GFP assay is suitable for the detection,
                      quantification and online monitoring of recombinant protein
                      production in B. subtilis during cultivation as well as for
                      analyzing production heterogeneity and intracellular
                      localization at a single-cell level.},
      cin          = {IMET / IBG-1},
      ddc          = {570},
      cid          = {I:(DE-Juel1)IMET-20090612 / I:(DE-Juel1)IBG-1-20101118},
      pnm          = {2172 - Utilization of renewable carbon and energy sources
                      and engineering of ecosystem functions (POF4-217)},
      pid          = {G:(DE-HGF)POF4-2172},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:34488765},
      UT           = {WOS:000693256700003},
      doi          = {10.1186/s12934-021-01663-7},
      url          = {https://juser.fz-juelich.de/record/902912},
}