The goal of this course is to give the basic theoretical notions and practical skills of the most popular DNA cloning techniques used to investigate the structure and function of genes (from DNA to protein). Lectures will provide an overview of the different cloning vectors, host cells and of the principal screening strategies usually employed in the recombinant DNA technology. During the laboratory experiences students will perform an experimental strategy aimed to clone a specific gene and to express the corresponding recombinant protein in E. coli host cells.

Laboratory experiences

1. Defining the goal of the cloning experiment and design of the cloning strategy.

1.1 Selection of gene to be cloned and cloning vector.

1.2 Selection of restriction enzymes to be used and design of degenerate primers.

2. Isolation of total RNA from the selected source.

2.1 Spectrophotometric quantification and electrophoresis of purified RNA.

3. cDNA synthesis from total RNA, by reverse transcription (RT).

4. PCR with degenerate primers specific for the insert to be cloned.

4.1 Analysis of the amplified product by agarose gel electrophoresis.

4.2 Purification of the PCR product.

5. Ligase of the PCR product with a plasmid vector, by the TA cloning strategy.

6. Transformation of competent E. coli cells with the ligase reaction.

7. Screening and identification of recombinant clones.

7.1 PCR from bacterial colonies.

7.2. Extraction of one positive recombinant clone (plasmid miniprep).

8. Transfer of the target gene into a prokaryotic expression vector, by directional cloning, with restriction enzymes.

8.1 Restriction enzyme digestion of both insert and vector.

8.2 Ligase reaction.

9. Transformation of the expression construct into a host strain, suitable for recombinant protein expression.

9.1 Transformation of E. coli BL21(DE3) competent cells.

9.2 Screening of recombinants.

9.3 Propagation and storage of recombinant clones.

10. Basic Bioinformatics Toolkit for cloning.

Lectures (supporting practical experiences)

1. DNA cloning: overview of enzymes, vectors, host cells.

1.1 Enzymes for DNA cloning: nucleases, ligases, polymerases, phosphatases.

1.2 Cloning vectors for E. coli and eukaryotic cells: plasmids, phages, cosmids, BAC, YAC, shuttle vectors.

1.3 Other common cloning strategies besides the traditional restriction-based method: Golden Gate Cloning; Topo Cloning; Sequence and Ligation-Independent Cloning (SLIC); Gateway Cloning; Gibson Assembly.

1.4 Biological systems of molecular biotechnology: bacteria, yeast; higher eukaryotes.

2.DNA introduction into live cells.

2.1 Genetic transformation of prokaryotes.

3. Construction and screening of DNA libraries.

3.1 Preparation of genomic DNA and cDNA for library construction.

3.2 Library screening strategies: colony and plaque hybridization.

3.3. Production of labeled probes.

3.4 Isolation of recombinant vectors.

3.5 Restriction mapping.

3.6 DNA sequencing.

4. Polymerase chain reaction (PCR) as an alternative to cell-based DNA cloning.

5. Basic techniques of recombinant DNA technology.

5.1 Isolation of nucleic acids.

5.2 Quantification of nucleic acids: standard spectrophotometric assay and NanoDrop assay (with exercises).

5.3 Agarose gel electrophoresis and sizing.

5.4 Making LB agar plates and streaking of bacteria.

5.5 Inoculation of bacterial cultures and preparation of glycerol stocks for long term storage.

6. Introduction to expression vectors.

6.1 Essential features of an expression vector (cis-elements).

6.2 Promoters: constitutive, inducible.

6.3 Orientation and Reading Frame.

6.4 Codon usage.

None.

D1 - Knowledge and ability of comprehension.  After completing the course, students will have acquired the theoretical-practical knowledge of the most popular laboratory techniques employed for nucleic acid purification and analysis, of the different types of vectors and host cells used in gene cloning and of the main strategies employed for the screening and characterization of recombinant products.

D2 - Ability to apply knowledge and comprehension. Students will have to demonstrate to be able to understand and apply a laboratory protocol and to have developed skills to design and perform a cloning experiment autonomously.

D3 - Autonomy of judgement. Students will be able to critically analyze results and to publicly discuss them.

D4 - Communication skills. Students must acquire adequate scientific language skills by taking a participatory and critical attitude to the topics of the classroom lectures and laboratory experiences.

D5 - Learning skills. Students will demonstrate to possess the ability to independently increase basic knowledge and practical skills of newly emerging molecular biotechnologies.

The teaching material prepared by the lecturer in addition to recommended textbooks (such as for instance slides, lecture notes, exercises, bibliography) and communications from the lecturer specific to the course can be found inside the Moodle platform › blended.uniurb.it

20-hour supplementary contract.

Teaching

Learning activity will mainly consist of lab experiences. Lectures will be intended to supply the theoretical knowledge of the leading techniques employed in molecular biology.

The schedule will be introduced at the beginning of the course.

Attendance

Students must attend at least 2/3 of lab hours and few compulsory lectures, to be admitted to oral examination.

To attend the laboratory lessons, students must before attend the General Safety training and the Chemical and Biological Safety Course and pass the final examination.

A good knowledge of basic Molecular biology is a prerequisite to understand the contents of the course.

Course books

·  S. Carson, H. B. Miller, D. S. Witherow, M. C. Srougi. MOLECULAR BIOLOGY TECHNIQUES. A Classroom Laboratory Manual. 4th Edition. Elsevier Science & Technology, 2019.

·  F. Amaldi, P. Benedetti, G. Pesole, P. Plevani. TECNICHE E METODI PER LA BIOLOGIA MOLECOLARE. Casa Editrice Ambrosiana, 2020.

·  T. A. Brown. BIOTECNOLOGIE MOLECOLARI, Principi e tecniche, Zanichelli, 2017.

Supplementary books:

·  D. P. Clark, N. J. Pazdernik. BIOTECHNOLOGY, Academic Cell, Elsevier Inc, 2015.

·  K. Wilson, J. Walker. BIOCHIMICA E BIOLOGIA MOLECOLARE, Principi e tecniche, Raffaello Cortina Editore, 2019.

Additional educational material or scientific reviews will be made available during the course.

Assessment

Oral examination.

Evaluation of the digital Laboratory notebook.

Specific indications for the preparation and presentation of the Lab notebook will be established during the lessons. In any case, students will have to provide the report to the teacher by the end of the course.

The final exam consists of four questions on different topics of the program, two of which are related to the laboratory experience (with specific references to the report provided), and is aimed at determining the knowledge acquired, the degree of depth and the ability of the student to connect different themes to each other and to solve practical problems. The final grade will also take into consideration the ability to present the topics with an appropriate scientific language and to critically discuss the results of laboratory experiences reported in the Laboratory notebook.

Disability and Specific Learning Disorders (SLD)

Students who have registered their disability certification or SLD certification with the Inclusion and Right to Study Office can request to use conceptual maps (for keywords) during exams.

To this end, it is necessary to send the maps, two weeks before the exam date, to the course instructor, who will verify their compliance with the university guidelines and may request modifications.

Teaching

NOT APPLICABLE - course with mandatory attendance.

Attendance

NOT APPLICABLE - course with mandatory attendance.

Course books

NOT APPLICABLE - course with mandatory attendance.

Assessment

NOT APPLICABLE - course with mandatory attendance.

Disability and Specific Learning Disorders (SLD)

Students who have registered their disability certification or SLD certification with the Inclusion and Right to Study Office can request to use conceptual maps (for keywords) during exams.

To this end, it is necessary to send the maps, two weeks before the exam date, to the course instructor, who will verify their compliance with the university guidelines and may request modifications.

Part of the course will be taught in English on student request.
The student can request to sit the final exam in English with an alternative bibliography.