Nel mio curriculum, alla voce “expertise” ci sono le tecniche di biotecnologia, dal clonaggio, alla biologia molecolare, alla produzione di proteine ricombinanti, ai saggi di attività, poi vengono le metodiche di medio o high-throughput, la PCR su micropiastre, il sequenziamento di DNA in colonnine microcapillari (fino a 96 campioni al giorno), i vetrini per la lettura di fluorescenza allo scanner laser, per vedere l’interazione proteine/inibitore o l’attività proteasica, o per ibridare campioni di DNA con sonde oligonucleotidiche.
Sono stato fortunato a fare parte di progetti e sovvenzionamenti (NATO-Russia, EU framework programmes, progetti ministeriali), per cui sono stato coinvolto in attività in cui queste expertise, skills, abilità, sono state utilizzate. Ogni pubblicazione relativa porta il riferimento al progetto, al finanziamento.
Quello di cui mi pregio però, sono i lavori extra-curriculari, le attività che non hanno visto finalità di documentazione, report istituzionali, ma che sono serviti a studenti per svolgere ricerca (progetti regionali per la collaborazione pubblico-privato).
All’inizio della mia attività, uno studio su 20 varietà di uva da vinificazione, raccolte nel campo sperimentale di Valenzano, Bari, spremute e fermentate in laboratorio (hanno sviluppato meno alcool perchè coperte da un ostrao di olio, a bassa ensione di ossigeno): oltre all’analisi dei lieviti di ogni campione, come output ci sono stati i campioncini del fermentato alcolico, che per ossidazione ha originato 20 campioni di aceto monovarietale leggero, mamolto aromatico, l’ho consumato per condire le insalate (ogni varietà ha prodotto aromi unici, dal Malbek, all'aglianico, al montepulciano, alla malvasia, ai bianchi). Un giorno arriveranno sulle nostre tavole, se gli imprenditori capiranno questa opportunità.
Gli insaccati senza nitrati/nitriti. All’incirca verso il 2009, insieme all’IRTA in Catalogna, sperimentammo un salume a base carnea in cui al posto dei conservanti fu aggiunto un concentrato di melograno acido, prodotto da me privatamente, melagrane selvatiche incluse. Il laboratorio di Agraria di Portici fece lo studio antimicrobico su piastra del concentrato, che risulto antimicrobico perchè ricco in acido citrico. I colleghi spagnoli fecero il panel test per la valutazione organolettica del salume, che risultò idoneo al consumo con un voto elevato. Due anni dopo, al convegno sull’innovazione negli alimenti a Bari, un relatore dell’università di Napoli presentò il loro progetto finanziato su un insaccato senza nitrati, con altri prodotti vegetali antiossidanti. L'importante è se poi lo si produce. A Monteroni c'è l'azienda Mocavero, che produce il capocollo ubriaco conservato nel vino negramaro, ed un salume alla melagrana, simile a quello che abbiamo prodotto. A Matino c'è l'azienda Offishina, insaccati di pesce, che per il trattamento del salume di pesce usa il mosto cotto.
Quello che ho cercato di fare nel mio lavoro è l’aspetto biotecnologico. Abbiamo depositato 398 sequenze nucleotidiche in GenBank (per differenziare, funghi, batteri, e Solanaceae, e per snoRNAs e noncoding RNAs di topo, e per 60 sequenze amminoacidiche di proteine funzionali
progetto NATO-Russia, con il Bakh Institute of Biochemistry di Mosca. Geni di Solanaceae: inibitori di proteasi e di poligalatturonasi
I/le colleghi/e russi hanno amplificato 26 nuove sequenze da geni per inibitori di proteasi di tipo Kunitz, da tubero di patata, le proteine ricombinanti sono state prodotte al CNR, ed abbiamo fatto i saggi di inibizione di varie proteasi. I protein chips sono stati testati anche su estratti di vari acari. Nei risultati non pubblicati ci sono altri inibitori non da patata ma da specie non commestibili ma filogeneticamente vicine (Solanum brevidens, S. stoloniferum, S. palustre, S. nigrum).
AA Krinitsina, AS Speransky, P Poltronieri, A Santino, AM Bogacheva, NL Buza, MA Protsenko, AB Shevelev. Cloning of polygalacturonase inhibitor protein genes from Solanum brevidens Fill. Geneika, 2006, 42:376-84.
Speransky AS, Cimaglia F, Krinitsina AA, Poltronieri P, Fasano P, Bogacheva AM, Valueva TA, Halterman D, Shevelev AB, Santino A. Kunitz-type protease inhibitors group B from Solanum palustre. Biotechnol J. 2007 Nov; 2(11):1417-24.
Poltronieri P, Cimaglia F, Santino A, De Blasi MD, Krizkova-Kudlikova I, Liu S, Wang Y, Wang Y. Protein chips for detection of mite allergens using Kunitz-type protease inhibitors. Biotechnol J. 2010 Jun; 5(6):582-7.
Poltronieri P; Liu S; Cimaglia F; Santino A; Wang Y. Characterization of Kunitz-type inhibitor B1 performance using protein chips and AFM. Sensors and actuators. B, Chemical (Print) 168 (6), pp. 231–237, 2012.
Speranskaya AS, Krinitsina AA, Kudryavtseva AV, Poltronieri P, Santino A, Oparina NY, Dmitriev AA, Belenikin MS, Guseva MA, Shevelev AB. Impact of recombination on polymorphism of genes encoding Kunitz-type protease inhibitors in the genus Solanum. Biochimie. 2012 Aug;94(8):1687-96
Krinitsina, A.A., Melnikova, N.V., Belenikin, M.S. et al. Polymorphism of the KPI-A gene sequence in the potato subgenera Potatoe (Sect. Petota, Esolonifera, and Lycopersicum) and Solanum. Mol Biol 47, 358–363 (2013)
COST853 Agricultural biomarkers for array technology. Editors: Frey J.E, and Pasquer F. Berti Druck AG, Rapperswil, Switzerland.
progetto EU FP6 TRANS-BIO
Clonaggio della sequenza codificante per bioplastiche, la poliidrossialcanoato sintasi PhaC, da Ralstonia, suo trasferimento mediante plasmide ricombinante in E. coli, e produzione di PHB in fermentatore
Mezzolla V, D'Urso OF, Poltronieri P. Optimization of Polyhydroxyalkanoate Production by Recombinant E. coli Supplemented with Different Plant By-Products. Biotechnol Ind J. 2017;13(3):138.
Poltronieri P. , Mezzolla V, D’Urso O.F. PHB production in biofermentors assisted through biosensor applications. The 3rd International Electronic Conference on Sensors and Applications (ECSA 2016), 15–30 November 2016; Sciforum Electronic Conference Series, Vol. 3, 2016.
Cupriavidus necator ATCC-17699 (LGC Standards), deposited as Ralstonia eutropha, was used to amplify phaCAB operon. Escherichia coli TOP10 chemically competent [F−mcrA ∆(mrr.hsdRMS-mcrBC) φ80lacZ∆M15 ∆lacΧ74 recA1 araD139 ∆(ara-leu) 7697 galU galK rpsL (StrR) endA1 nupG λ–] and Escherichia coli BL21(DE3) chemically competent [F−ompT hsdISB(rB−mB−) gal dcm(DE3)] (Invitrogen, Life Technologies) were used for cloning and gene expression, respectively. Linear cloning pUC19 vector (Invitrogen, Life Technologies) was used for gene cloning by homologous recombination. Expression vector pET24b characterized by strong hybrid T7/LacO promoter (Novagen, Inc.) was used for inducible phaCAB genes expression.
Figure 1: Molecular detection of recombinant plasmid pET24/phaCAB by Nested PCR and Digestion. Nested PCR of phbC gene (550bp) to confirm insert in presumed positive colonies (1a) and digestion of positive pET24/phaCAB plasmid with BglII restriction enzyme to linearize it (9400bp) (1b)
when bacteria reached a high density, phaCAB operon was induced and expressed, under the strong hybrid T7/LacO promoter of pET system, by addiction of lactose or galactose. The pET vector is a powerful system for expressing recombinant proteins as it possesses a strong and inducible hybrid T7/LacO promoter, but the costs of the chemical inducing agent, IPTG, are very high. Therefore, several authors used lactose as inducer. In fact, in the absence of lactose, the lac repressor (LacI) binds to the operator sequence, blocking access of T7 RNA polymerase to the promoter site; conversely, when lactose binds to LacI, T7 RNA polymerase can bind to the promoter site and genes are expressed. The bacterial β-Galactosidase (lacZ) hydrolyzes lactose to galactose and glucose; glucose is used as carbon source, while galactose is used only as inducer as E. coli BL21 is a gal(-) strain and it is not able to metabolize it.
Recombinant E. coli BL21(DE3) harbouring heterologous phaCAB operon from Cupriavidus necator ATCC-17699 was cultivated at 30 °C and 150 rpm in fed-batch conditions, both in 500 mL shake flasks with a starting volume of 150 mL and in 20 L bioreactor (BIOSTAT®, Sartorius) with a starting volume of 15 L, maintained at 40% oxygen saturation with a constant flow of compressed air (2 vvm) and with a cascade control speed. The pH was controlled automatically at 6.9 ± 0.1 by the addition of 15% v/v H2SO4 and NH4OH (20% v/v). The induction phase was conducted adding lactose 30 mM at 25 °C, pH 6.9 and air flow 3 L/min after 24h, when the bacteria reached their stationary phase. Feeding solution was added at 4 mL/min for 48 h. The fed-batch hydrolysate media containing Sweet Corn Enzymatic Hydrolysate 10%, Banana juice 5% or Potato Skin Enzymatic hydrolysate 25% were supplied from TRANS-BIO Consortium. The sugar content in Sweet Corn Enzymatic Hydrolysate, Potato Skin waste and Banana juice hydrolysate was reported in Table 1.
Na2HPO4/Na2PO4 x H2O buffer 10% (v/v) (stock 1M, pH 7) was used; to maintain plasmid stability, kanamycin antibiotic (50μg/mL) was added to the medium. A Trace Element Solution (TES) stock was prepared as follows, (g/L): 10 FeSO4x7H2O, 2 CaCl2x2H2O, 2,2 ZnSO4x7H2O, 0,5 MnSO4x4H2O, 1 CuSO4x5H2O, 0,02 Na2B4O7x10H2O and 1% of TES solution was added to hydrolysate medium.
The reducing carbohydrates content in the bacterial medium was daily measured by Sucrose, D-Fructose and D-Glucose kit (Megazyme, Wicklow, Ireland), through which NADH is quantified by its absorbance. This was made using microwell plates and spectrofluorometer reads at 340 nm in an Infinite 200 Pro instrument (Tecan). Glucose and fructose have been quantified every 12 h. The enzymatic reactions allowed to determine the levels of consumed sugars.
Fermentatore
Recombinant E. coli BL21(DE3) harbouring heterologous phaCAB operon was cultivated at 30°C and 150 rpm in fed-batch conditions, both in 500 mL shake flasks with a starting volume of 150 mL and in 20 L Biostat bioreactor (Sartorius, Goettingen, Germany) with a starting volume of 15 L. Optical density (OD) was used to monitor the bacteria biomass, achieved by maintaining a 40% oxygen saturation with a constant flow of compressed air (2 vvm) and with a cascade control speed. pH was monitored automatically at 6.9 ± 0.1 through addition of a stock 15% v/v H2SO4 and NH4OH (20% v/v). The induction phase was conducted adding galactose 10 mM or lactose 30 mM at 25°C, pH 6.9 and air flow 3 L/min after 24 h, when the bacteria reached their stationary phase. Feeding solution was added at 4 mL/min for 48 h.
Progetto europeo RIBOREG: novel non-coding RNA in development and disease. Nove sequenze uniche registrate in GenBank per ncRNAs espressi durante lo sviluppo embrionale di topo.
Collaborazione con 6 partner europei e 3 aziende private, concluso con un workshop europeo
ESF Workshop: MOLECULAR MECHANISMS INVOLVING NON-PROTEIN CODING RNAsAt: Carry le Rouet. Marzo 2007. Poster: DNA array analysis of gene expression of npcRNAs in cancer cell lines and in retinoic acid-differentiated NB4 cells.Rossi A, D'Urso OF, Gatto G, Poltronieri P, Ferracin M, Remondelli P, Negrini M, Caporaso MG, Bonatti S, Mallardo M. Non-coding RNAs change their expression profile after Retinoid induced differentiation of the promyelocytic cell line NB4. BMC Res Notes. 2010 Jan 27;3:24.
Mallardo M, Poltronieri P, D'Urso OF. Non-protein coding RNA biomarkers and differential expression in cancers: a review. J Exp Clin Cancer Res. 2008 Jul 16;27(1):19.
Progetto abbinato al finanziamento SiMiSa (Ministero per le attività economiche e l'industria).
Engineering Resistance receptors in plant immunity by modifying LRR specificity
The chimera FLS2-EFR, carrying the LRR ectodomain of FLS2 and the kinase domain of EFR, recognizes flg22 and induces an EFR-like response resulting in a higher ethylene production than the response triggered by FLS2 upon perception of flg22. A recent report on the chimera EFR/Cf9 transferred into tobacco plants showed the effectiveness in conferring resistance to biotrophic pathogens (Wu et al., 2019).
Wu J, Reca IB, Spinelli F, Lironi D, De Lorenzo G, Poltronieri P, Cervone F, Joosten MHAJ, Ferrari S, Brutus A. An EFR-Cf-9 chimera confers enhanced resistance to bacterial pathogens by SOBIR1- and BAK1-dependent recognition of elf18. Mol Plant Pathol. 2019 Jun;20(6):751-764.
DNA fragments representing the coding sequence (CDS) of the ectodomain of EFR (Lys649EFR) and FLS2 (Arg806FLS2) were fused to the CDS of the TM and cytoplasmic domain of Cf-9 by splicing overlapping extension polymerase chain reaction (SOE-PCR) (Higuchi et al., 1988) to generate DNA fragments encoding EFR-Cf-9 and FLS2-Cf-9, respectively, and cloned into the binary vector pBI121 under the control of the 35S promoter, or, by Gateway cloning, the fragments were inserted into pK7FWG2.0 to generate 35S:EFR-Cf-9-eGFP and 35S:FLS2-Cf-9-eGFP, and inserted into pGWB20 to generate 35S:EFR-Cf-9-10xMyc and 35S:FLS2-Cf-9-10xMyc.
Ringraziamenti: Cyril Zipfel (The Sainsbury Laboratory, Cambridge, UK) for providing elf18 peptides, Pierre J.G.M. de Wit (Wageningen University and Research, Netherlands) for the plasmids carrying 35S:Cf-9 and 35S:Avr9, and Jonathan Jones (The Sainsbury Laboratory, Cambridge, UK) for providing the tobacco line expressing Avr9. This work was supported by the Institute Pasteur-Fondazione Cenci Bolognetti, by the Ministero dell’Istruzione, dell’Università e della Ricerca, by ERA-Net (ERA-CAPS project “SIPIS”) and by Sapienza Università di Roma (Progetti di Ricerca 2016 and 2017).
the Leucine-Rich-Repeat (LRR) sequence recognising mouse interleukin-6 (found by Alexandre Brutus during the screening of a library of LRR solenoids, at Michigan State University) was swapped to the transmembrane and intracellular domain of Cf9 receptor, through two rounds of PCR, using target-sequence tagged primers followed by long PCR for the whole swapped receptor, using the Splicing by overlapping extension (SOE) PCR method.
The LRR domain sequence that recognises IL-6 is well known, and the use of IL-6 protein is easy to handle and cheap. The stimulation of tobacco leaves with an IL-6 solution induced the appearance of necrotic lesions after 24 hours, confirming the suitability of use of specific LRR sequences to recognise unequivocally their new protein partner, IL-6 instead of Avr9 (unpublished results, Reca B., Francocci F., Brutus A.) (figure 2).