EDUCATION AND TRAINING
Sep 2013–Oct 2016 Bachelor's Degree in Biological Sciences
Università di Roma "La Sapienza", Roma (Italy)
I have acquired general knowledge about various branches of biology, including cellular biology, histology, biological chemistry, zoology, botanic, physics, molecular biology, genetics, bioinformatics, human physiology.
During the 5 months of internship in the laboratory I was able to deepen the following techniques:
· Protein purification with affinity chromatography
· SDS-PAGE gels preparation and electrophoresis
· Preparation of various buffers
· Western blot
· Bacterial transformation and selection of colonies that have acquired the plasmid
The title of the Bachelor’s thesis is: “Development of new expression and purification strategy of recombinant Glutamyl-tRNA reductase from Acinetobacter baumannii”
Tetrapyrroles, a pigment class that includes heme, chlorophylls and vitamin B12, are required for important processes of life, such as respiration and photosynthesis. 5-aminolevulinic acid (ALA) is the universal precursor of all tetrapyrroles. There are two natural pathways for the production of ALA. In the Shemin pathway, used by animals, mushrooms and some bacteria, ALA is formed by condensation of succinyl-CoA and glycine with the elimination of CO2, catalyzed by ALA-synthase, a PLP-dependent enzyme. The second pathway, used by plants, Archea and most bacteria, is called C5 pathway and involves two enzymes in two sequential steps: first, glutamyl-tRNA-reductase (GluTR), a NADPH-dependent enzyme, reduces glutamyl-tRNA to form glutamate-1-semialdehyde (GSA); second, GSA is isomerised by glutamate-1-semialdehyde aminomutase (GSAM), a PLP-dependent enzyme, with formation of ALA. Due to the importance of ALA for plants and bacteria, any inhibitor of the C5 biosynthetic pathway could potentially be used as herbicide or antibiotic. Our attention focused on the enzyme catalyzing the limiting step in the production of ALA in plants and prokaryotes, GluTR, that is very unstable and difficult to purify. In the reaction catalyzed by GluTR, a cysteine residue in the active site nucleophilically attacks the aminoacyl bond of glutamyl-tRNA, leading to a highly reactive thioester intermediate. In the next step the thioester is reduced via hydride transfer from NADPH to form GSA. GluTR is a planar V-shaped dimer. The current hypothesis is that a complex is formed between GluTR and GSAM, in which the large void in the GluTR structure is occupied by GSAM to allow the efficient synthesis of ALA. This also suggests that a complex between GluTR and GSAM may stabilize both enzymes and make the purification process of GluTR possible. After an initial work of coexpression and purification of GluTR and GSAM, which was successful, we cloned the HemA gene from Acinetobacter baumannii, coding for GluTR, into an expression vector (pET22) to allow GluTR expression with an histidine tag. The histidine tag is essential for the purification of GluTR by affinity chromatography. In this way we could purify this protein without GSAM, missing the histidine tag. Subsequently, we explored several different expression conditions (aerobic and anaerobic conditions, different temperatures and IPTG concentrations) to optimize the expression yield. SDS-page and Western Blot assays were used to estimate the expression yield of our protein. Finally, after purification of GluTR, we developed a new and easy activity assay for this enzyme, using glutamyl-tRNA analogs.
Bachelor's degree score: 110/110 e lode
Sep 2016–Jul 2018 Master Degree in Genetics and Molecular Biology
Università di Roma "La Sapienza", Roma (Italy)
I have deepened my studies in the field of molecular biology and biochemistry.
During the year and a half of internship in the laboratory I was able to deep the following techniques:
· Assays of enzymatic activity
· Native PAGE gels preparation and electrophoresis
· Molecular exclusion chromatography (gel filtration)
· Silver staining
· Plasmid extraction
· Agarose gels preparation and electrophoresis
· Protein concentration
· Bradford assay
· Adsorption spectroscopy
· Use of GraphPad Prism 7
The title of Master thesis is: "Purification of the complex between HemA and HemL, essential enzymes in the biosynthesis of tetrapyrrolic compounds".
Tetrapyrroles (also called porphyrins) are a class of pigments that include heme, chlorophyll and vitamin B12. They are required for different processes that are fundamental to life, such as breathing and photosynthesis. 5-aminolevulinic acid (ALA) is the universal precursor of all tetrapyrroles. In nature there are two distinct pathways of ALA biosynthesis. The first is the Shemin pathway used by animals, mushrooms and some bacteria, in which the synthesis of ALA occurs thanks to the condensation of succinyl-CoA and glycine with the elimination of a CO2 molecule, reaction catalyzed by the ALA synthase. The second way, used by plants, archea and most bacteria is called C5 pathway in which the glutamyl-tRNA reductase (GluTR or HemA) reduces glutamyl-tRNA to form glutamate-1-semialdehyde (GSA) with oxidation of NADPH in a first enzymatic reaction ; in the second reaction, the GSA is isomerized by glutamate-1-semyaldehyde amino-aminase (GSAM or HemL), a pyridoxal 5'-phosphate-dependent enzyme (PLP), with the formation of ALA. GSA can spontaneously be converted into ALA in a physiological pH solution, but it can also give rise to a series of toxic intermediates for the cell. For this reason it was supposed that there is a complex between the two enzymes, thanks to which the GSA is channeled directly from the active site of HemA to the active site of HemL. This hypothesis was supported by experimental evidence and bioinformatic studies conducted on different organisms. Because of the importance of ALA for plants and bacteria, any inhibitor of the C5 biosynthetic pathway could be used as herbicide and antibiotic. The purpose of our laboratory is to characterize the two key enzymes of the C5 pathway of Acinetobacter baumannii, Gram negative bacterium, responsible for infections in humans in the hospital and multi-resistant to various classes of antibiotics, with the aim of purifying the complex and identify specific inhibitors that would not interfere with the human pathway of ALA. HemL is a stable and successfully purified enzyme in our laboratory. Instead HemA is unstable, so its expression was particularly difficult, despite the use of various strategies. Thus, considering that the formation of the complex could stabilize the reductase, we cloned the hemL and hemA genes into a pET28 expression vector, which allows the co-expression of proteins with His-Tag in order to be able to purify them together by affinity chromatography, or in the expression vector pET22 which allows the expression of only HemA with a histidine tag so as to be able to purify it from HemL which is not provided with it. In this way we managed to have a good expression of HemA. The results obtained from the gel filtration, from the enzymatic activity assays performed on HemL and from cross-linking experiments indicate that in the strains in which HemA and HemL are co-expressed, the formation of the complex occurs in vivo. Also the content of ALA in the protein extracts of the bacterial strains in which HemA and HemL are co-expressed is greater than those in which they are not expressed together, indicating that there is an overproduction of the porphyrins, also confirmed by the reddish color of the pellets bacterial. We have tried to recreate the conditions that in the cell favor the interaction between proteins in vitro, adding different HemA ligands, with poor results. Our future goal will be to find the right conditions to recreate the complex in vitro, in order to purify it in such quantities as to be able to solve its crystallographic structure.
Master degree score: 110/110 e lode
Mother tongue: Italian
Foreign language: UNDERSTANDING SPEAKING WRITING
Listening Reading Spoken interaction Spoken production
English B1 B2 B1 B1 B1
Communication skills: Very good communication skills acquired during my studies. I am able to communicate concepts in a simple and clear way. I had the opportunity to be a guide in a museum of comparative anatomy in the university and this activity helped me to improve my comunication skils.
Organisational / managerial skills: Excellent organizational skills. I can organize my work in order to do more activities simultaneously. I never waste my time. I can work well in groups and focus on my role. I can take important decisions about my work.
Job-related skills: Ability to work in a precise, orderly, safe and cooperative way in the laboratory.
Digital skills: Excellent mastery of the programs of the Office package (Access, Excel, Outlook, Power Point, Publisher, Word) and Windows and iOS operating systems. Good mastery in the use of GraphPad Prism7.
Caterina Nardella, Dalila Boi, Martino L. di Salvo, Anna Barile, Jörg Stetefeld, Angela Tramonti and Roberto Contestabile. Isolation of a Complex Formed Between Acinetobacter baumannii HemA and HemL, Key Enzymes of Tetrapyrroles Biosynthesis; Front. Mol. Biosci., 26 February 2019