Services

As a collaborative effort within the UPO community, we put our experience in the fields of Recombinant DNA and Cell Biotechnologies available to all interested research labs. In particular, we can provide the following services:

Chemically-competent and electrocompetent bacteria

We offer services to prepare and distribute E.coli competent cells using the chemically-induced method (CCMB80) and also prepare and distribute electrocompetent cells. Before distribution, E.coli competent cells are tested for transformation efficiency and absence of contaminants, and then provided in small, ready-to-use aliquots. We also collaborate with the interested labs to the development and validation of protocols for the competization of non-E.coli bacterial strains.

TA cloning service

We provide a custom TA cloning system for direct one-step cloning of PCR products with 3’-dA overhangs. Our high-quality TA cloning vector is based on the pBluescript II phagemid vector and is provided ready-to-use for efficient ligation with PCR products, thus ensuring high cloning yields and low background (the vector allows a blue/white colony screening). To increase the speed, convenience and efficiency of cloning, the PCR Cloning tools can be combined with our competent E. coli DH5a cells. The DNA insert can be readily excised from the versatile polylinker of pBluescript, sequenced using standard M13/T3/T7 primers or in vitro transcribed with T3/T7 RNA polymerase.

Production of Recombinant proteins into E. coli

We have years of experience in the expression of recombinant proteins into E. coli. Our service include the complete support in designing the strategy to amplify/isolate the gene of interest (GOI) and subcloning it into an expression vector harbouring the desired purification tag (6xHis, or GST or other tags of interest). The correct expression of the target protein (including folding/solubility tests) will be assessed on a pilot scale (typically 50 ml) and then scaled-up to 1 lt culture.

1: Deantonio C, et al. Microb Cell Fact. 2014 Sep 14;13:132. doi:10.1186/s12934-014-0132-1.

Production of Recombinant proteins into mammalian cells (CHO, HEK)

We have also a relevant experience in Mammalian Cell Factories. As for the prokaryotic systems, we can provide a complete service inclusive of 1) strategy design; 2) subcloning the GOI into an expression vector (commercial or customized); 3) cell transfection, clone selection and expansion, biobanking; and 4) culture scale-up to 1 L (benchtop bioreactor).

1: Patrucco L, et alGene. 2015 Sep 15;569(2):287-93. doi: 10.1016/j.gene.2015.05.070.

2: Deantonio C, et al . Methods Mol Biol. 2014;1060:277-95. doi: 10.1007/978-1-62703-586-6_14.

3: Boscolo S, et al . N Biotechnol. 2012 May 15;29(4):477-84. doi: 10.1016/j.nbt.2011.12.005.

Construction of "Domainome" libraries for the high-throughput annotation of protein-coding genes, structural/functional studies and interactomic studies

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. Prof. Daniele sblattero (a former Professor at UPO and leader of the laboratory of Applied Biology) developed a strategy where, by using β-lactamase on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called "domainome", will be well expressed and soluble, making them suitable for structural/functional studies. By cloning and displaying the "domainome" directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification. The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

  1. Soluri MF, et al. J Vis Exp. 2018 Oct 3;(140). doi: 10.3791/56981.
  2. Gourlay LJ, et al. Acta Crystallogr. 2015 Nov;71(Pt 11):2227-35. doi: 10.1107/S1399004715015680.
  3. D'Angelo S, et al. BMC Genomics. 2011 Jun 15;12 Suppl 1:S5. doi: 10.1186/1471-2164-12-S1-S5.
  4. Di Niro R, et al. Nucleic Acids Res. 2010 May;38(9):e110. doi: 10.1093/nar/gkq052.