Version :
Integrated Structural Biology Grenoble

Contact person(s) related to this article / VERNET Thierry / VILLARD Anne-Marie

RoBioMol

Molecular Biology and Protein Expression Automated Platform


Summary

The RoBioMol platform, hosted by the Pneumococcus Group of the Institut de Biologie Structurale, and integrated in ISBG, offers high throughput molecular biology processes using automates.

Current services include:

  • gene cloning,
  • site-directed mutagenesis,
  • expression and purification test of proteins expressed in E. coli
  • plasmid preparation
  • screening of detergents for membrane protein solubilization and affinity purification
  • detergent exchange on solubilised membrane proteins.

These services are available for a minimal number of cloning / Expression tests / Minipreparation / Detergent screening of 24 samples.
Data are processed and managed via a Laboratory Information Management System (RoBioLIMS) specifically developed for the platform by the CEA (GIPSE).

The RoBioMol platform also provides its facilities: the Microlab Star from Hamilton and the CFX real time PCR from BioRad.

Key words

Gene cloning, site-directed mutagenesis, small-scale recombinant protein expression/purification, plasmid preparation / affinity chromatography, membrane proteins, detergents, molecular biology, liquid handling automates, .

Dedicated staff

Contact and localization

IBS-PG 419

Equipements

  • Microlab Star Hamilton for versatile molecular biology processes
  • Nanovue GE Healthcare for matrix quality control
  • CFX Connect Biorad for real time PCR and thermal shift assay processes

All these equipments are maintained and serviced rigorously.

Accessibility

RoBioMol is accessible to PSB members and academics for services or collaborations following successful shared grant applications, and industrials.

How to make a request ?

Contact the RoBioMol team to submit your request.

After discussion and acceptance of your application, you will:

  • for gene cloning, site directed mutagenesis and protein expression: enter your order on RoBioNET. Discussion with RoBioMol team is necessary before login and order on RoBioNET.
    If you already have a password then click RoBioNET
    Request login/password to RoBioMol
  • for plasmid minipreparation, screening of detergent and detergent exchange, CFX or microlab star providing, please contact RoBioMol staff.

Acceptance of the quote (signed by both parties) will be needed prior to the beginning of the work.

Samples provide by the requesting scientist

Sample input depends on the type of requested service:

  • for gene cloning and site-directed mutagenesis, the gene to be mutated or cloned should be provided either cloned into a plasmid (10 ng minimum/cloning) or as a chromosomal template (ie: bacterial DNA, 100 ng/cloning).
    The quality of the DNA template will be controlled by the RoBioMol team (concentration and 260nm/280nm ratio determination, analysis by agarose gel electrophoresis).
  • for plasmid minipreparation, bacterial colonies containing the plasmids to be prepared should be provided in petri dishes at RoBioMol place.
  • for detergent screening for membrane protein solubilization and detergent exchange, membrane preparation, which has to be previously analysed to ascertain the presence of overexpressed His-tagged protein, has to be put down at RoBioMol place.

Samples must have security level L1 and conform to the corresponding GMO statement (GMO agreement number is required).
Should the quantity or quality of the matrix departs from the RoBioMol standards, the service will be delayed and the requesting scientist will be contacted.

Template will be renamed for internal use, stored frozzen for plasmid or genomic DNA and membrane preparation, or between 0°C and 10°C for bacterial petri dishes. Template will be discarded at the end of the work unless specified otherwise on the order form.

Results

Depending on the type of requested service:

  • Gene cloning and site directed mutagenesis deliverables: the plasmid DNA containing the cloned or mutated gene is delivered in solution (a few nanogram, enough to perform a bacterial transformation for amplification of the plasmid) when appropriate. Validation of cloning and mutagenesis is determined by full-length sequencing. The given sequence is 100% garanteed. A report (PDF / doc) containing a review of cloning / mutagenesis performed, sequencing results is provided.
  • Expression and purification test of proteins deliverables: a detailed technical report containing an estimated expression/purification level is provided.
    An analysis of expression level of recombinant protein is made by sub-cellular fractionation followed by SDS-PAGE gel migration, to estimate the expression level and integrity of the proteins.
  • Automated plasmid preparation deliverables: for low copy number plasmid, RoBioMol will deliver 30-40µL at 20ng/µL minimum. Concentration trueness is measured by nanovue and garanteed at 3,1%.
  • Detergent screening deliverables: a detailed technical report containing solubilization and purification results analyzed by SDS PAGE and/or Western blot. Detergent concentration trueness is garanteed at 2,5 %.

Products and generated reports are not kept after delivery.

Cost and duration

A quotation will be issued for each application and must be approved by the requesting scientist before the beginning of the work (please, contact RoBioMol team for details).

The start and duration of the work (4 to 6 weeks for gene cloning/mutagenesis, one week for detergents screening) will be specified for each order.

Follow-up/Acknowledgements

The platform remains available for additional information and/ or support for publication (by email or telephone exchanges, meeting/publication for the collaborations and developments).

RoBioMol users will acknowledge the contribution of the platform as follows:

  • Activity Service: Acknowledgement in publication and oral presentation
  • Collaboration: Co-authors in publication and oral presentation
  • Development: Co-authors in publication and oral presentation.

Acknowledgements in publication:
“This work used the platforms of the Grenoble Instruct centre (ISBG ; UMS3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INBS-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB).
We thank Anne Marie Villard and Dr. Marjolaine Noirclerc-Savoye, for the “gene cloning, site-directed mutagenesis, protein purification or Thermal Shift Assay experiments ; and Céline Charavay and Stéphane Segard (GIPSE, CEA, Grenoble) for the RoBioLIMS database development”.

References

  • "Rapid automated detergent screening for the solubilization and purification of Membrane proteins and complexes"
    _V. Lantez, I Nikolaidis, M. Rechenmann, T. Vernet, M. Noirclerc-Savoye. Eng. Life Sci. (2015) 15 : 39–50
  • "A cost-effective protocol for the parallel production of libraries of 13CH3-specifically labeled mutants for NMR studies of high molecular weight proteins"
    E. Crublet, R. Kerfah, G. Mas, M. Noirclerc-Savoye, V. Lantez, T. Vernet, J. Boisbouvier. Methods Mol Biol. (2014) (1091): 229-44
  • "Reconstitution of membrane protein complexes involved in pneumococcal septal cell wall assembly".
    M. Noirclerc-Savoye, V. Lantez, L. Signor, J. Philippe, T. Vernet, A. Zapun. PLOS One (2013) 8(9) : e75522
  • "Large scale purification of linear plasmid DNA for efficient high throughput cloning".
    M. Noirclerc-Savoye, B. Gallet, F. Bernaudat, T. Vernet. Biotechnology Journal (2010) 5(9):978-985
  • "Parallel screening and optimization of protein constructs for structural studies".
    R. Rasia, M. Noirclerc-Savoye, N. Bologna, B. Gallet, M. J. Plevin, L. Blanchard, J. Palatnik, B. Brutscher, T. Vernet and J. Boisbouvier Protein Science (2009) 18, 434-439
  • "Automation of high-throughput expression screening on His MultiTrap HP with vacuum elution using a Hamilton Liquid Handling Workstation".
    J. Lundqvist, B. Gallet, M. Noirclerc-Savoye and T. Vernet. Discovery Matters (2008), 14-15
  • "High throughput automated affinity purification of His-Tagged proteins with Ni Sepharose matrices on the MICROLAB® STAR".
    B. Gallet, T. Vernet and M. Noirclerc-Savoye. Life Science Robotics (2007)
  • "Automated high-throughput process for site-directed mutagenesis, production, purification, and kinetic characterization of enzymes".
    R. Carapito, B. Gallet, A. Zapun, T. Vernet. Analytical Biochemistry (2006) 355:110-6