Advanced techniques in molecular biotechnology

biology
biotechnology

elective courses

Genetics and Genetic Engineering 1400-114GEN
Molecular Biology 1400-215BM

Bacterial and Archaeal Genetics 1400-216GENBA
Genomics and Transcricptomics 1400-216GTR

The prerequisite is the grade obtained in one of two subjects declared by the student.

- molecular biology -exam

- molecular genetics – exam

Or another equivalent subject (in case of Bachelor graduates from outside the University of Warsaw)

The student has basic knowledge in the field of molecular biology, biochemistry, genetics, and microbiology.

Classroom

The course focuses on new methods in molecular biology with potential applications in biotechnology. The lecture will introduce the students to the experimental part. The classes are divided into thematic blocks:

1. CRISPR

2. Protein as products

3. Intellectual Property in Biotechnology

The course covers the following list of tasks and topics:

- preparation of constructs for heterologous expression, alternative cloning methods (SLIC, GATEWAY), computer analysis of genes and genomes,

expression and purification of proteins with biotechnological applications,

- The CRISPR genome editing method, the construction of mutants using this method, and discussion of its applicative variants

- Elements of patent law with particular emphasis on biotechnology patents (so-called 'case study').

During the course, special attention will be paid to the student's work (or work in pairs), both in terms of practical and in silico experiments and terms of theoretical knowledge.

The course focuses on current issues in molecular biotechnology. It is composed of 3 thematic blocks:

1. Genome editing by CRISPR

Basics of genome editing, CRISPR elements, history of CRISPR editing invention, application of CRISPR as molecular biology tool. Molecular aspects of CRISPR. Aplicative variants of different research models (plants, animals, microorganisms, and tissue culture) Students will design their own CRISPR/Cas experiment and perform it during the first 5 classes to finally verify the obtained clones. Students will perform the following practical tasks: primer design, guide RNA cloning, analysis of obtained clones and sequencing results, yeast cell transformation, and phenotyping by diagnostic PCR.

2. Protein as products

-Students will learn the basic principles of direct sequencing of nucleic acids by Oxford Nanopore Technology (4th generation of sequencing). They will prepare RNA or cDNA-based libraries for sequencing, set up runs and finally analyze the collected data. Part of the class will be devoted to the analysis of data obtained during Nanopore sequencing and the latest trends in direct sequencing software development. The use of nanopore sequencing as a method for quality control and the study of intracellular metabolism of therapeutic nucleic acids such as mRNA vaccines.

-High-throughput gene cloning using the no-ligation SLIC method as an example. Design of custom constructs and primers for cloning.

-Metagenomes as a source of new enzyme activities. Sequencing of metagenomes and laccases (oxidases) as examples of enzymes with biotechnological potential.

-Basics of protein purification and activity assessment. Students will purify recombinant proteins produced in bacteria (various new laccases found in bacterial metagenomes and other nucleic acid-binding proteins) and then assess the quality and efficiency of the purification by an SDS-PAGE analysis. They then check the activity of the proteins using a colorimetric method and determine the optimum pH of the reaction. The activity of nucleic acid-binding proteins will be asses by immunostaining of fixed cells followed by state-of-the-art microscopy and analysis of the images collected.

3. Intellectual property in biotechnology.

Students will know different forms of intellectual property, especially this protected in biotechnology,(as well as agriculture and medicine). Students will have the skills to use patent databases as a source of intellectual property knowledge and also as an alternative to scientific papers. Students will analyse the patent application, how it is proceeded, and what is the patent protection. Students will gain knowledge about biological material repositories. As a summary, the student will prepare a short presentation on the patent of choice and present it to the other participants in the course.

The exercises will take place in the classroom, interchangeably, the lectures and the experimental parts. During the course, special attention will be given to the student's individual work or to working in small teams. Communication and individual performance according to a given protocol is essential during classes (simple mathematical calculations, manual skills).

The course is designed for students who plan to work in the research and development of biotechnological companies and for those who plan a scientific career.

The ATMB Handbook (2019) theoretical introduction and practical protocols prepared by the teachers of the class. A PDF file or printed version will be provided for each participant of the classes.

Genome Editing ed. Kursad Turksen, 2016

In terms of knowledge, students should:

-Possess advanced knowledge in the field of molecular biology and biotechnology (K_W01 Bt2).

-Understand the impact of molecular biology on the development of biotechnological solutions and know how to use it in practice(K_W03 Bt2).

Describe the mechanisms of defence of prokaryotic organisms, which became the prototype of CRISPR editing. Knows the CRISPR elements function during an editing event (K_W03 Bt2).

-Be able to use basic vocabulary related to biotechnology and molecular biology in English (K_W12 Bt2).

-Understand the importance of experimental work in biotechnology and the role of molecular analysis in biological and medical research (K_W05 Bt2).

In terms of skills, students should:

- be able to use advanced research techniques appropriate for biotechnology (K_U01Bt2)

- Be able to use basic methods of nucleic acid in silico analysis, apply the theoretical knowledge in this area (searching databases and professional literature) and analyse selected data (K_U17 Bt2).

-be able to plan experiments of heterologous protein expression, understand the applicability of such activities, and assess the chances of success of such attempts. Students are able to choose the cloning technique proposed by the lecturer (SLIC, GATEWAY, shotgun cloning, classic cloning using restriction enzymes), isolate nucleic acids, or analyse the sequencing results (K_U04 Bt2)..

- be able to plan experiments using CRISPR, design RNA guide and repair templates, select the method of introducing the DNA, repair template, and Cas9 nuclease into the cell. Students should be able to carry out a CRISPR experiment and verify the results of this experiment under supervision (K_U13 Bt2).

- be able to isolate and purify proteins as well as obtain biologically active preparations that have potential biotechnological applications. Perform tests of the activity of purified proteins to verify their usefulness (e.g. recombinant antibodies, restriction enzymes, protein-modifying enzymes) (K_U14 Bt2).

- be able to assess the risk of using CRISPR/Cas9 editing for genome modification and successfully counteract this phenomenon (K_U015 Bt2).

-be able to perform correct results interpretation and conclusion. Apply mathematical and statistical methods to describe phenomena and data analysis (K_U05 Bt2).

-be able to present scientific results and professional communication verbally (K_U08 Bt2).

-be able to work in a team and manage the teamwork of a small group (K_U10 Bt2)

Has the ability to use English to read scientific literature and communicate with foreigners (K_U02 Bt2).

In terms of social competences, students should:

-Understand the physical and chemical phenomena that occur in the environment (K_K01Bt2).

- Be responsible for their own work and equipment; demonstrate respect for others work (K_K03 Bt2).

- use objective sources of scientific information and draw critical conclusions while analysing and solving practical problems (K_K04 Bt2).

The final grade depends exclusively on the result of the exam. Other activities are prerequisites for being approved for participating in the exam.

Pass criteria:

- attendance (two absences per semester are allowed)

- preparation of a short presentation of the results obtained during the course

- homeworks prepared individually or in teams (mainly design of the primers used during the course for cloning and genome editing)

- a written exam consisting of mainly closed questions and/or 3 open questions (final score of 51% is required to pass)