Activities summary

I. MOLECULAR TOOLS IN BIOTECHNOLOGY

A. Site-specific genome editing based on CRISPR-associated nuclease Cas9. (Theoretical session: 90 minutes). Dra. María Esther Fárez

CONTENTS:

The ability to engineer biological systems and organisms holds enormous potential for applications across basic science, medicine and biotechnology. Programmable sequence-specific endonucleases that facilitate precise editing of endogenous genomic loci are now enabling systematic interrogation of genetics elements and causal genetic variations in a broad range of species, including those that have not previously been genetically tractable.The talk will focus on description of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in eukariotic cells, as well as generation of modified cell lines for downstream functional studies. A description of a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs to minimize off-target cleavage will be discussed. This talk will provide experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity.

Aim of the course:

International Course in Biotechnology of prokaryotic and eukaryotic cells

Antofagasta, Chile. From 22nd to 25th of August 2017

-Description of the Cas9 RNA-guided nuclease for genome editing. Limitations of the Cas9 system. PAM sequence.-Target selection for sgRNA: minimization and detection of off-target cleavage activities.-Approaches for sgRNA construction and delivery: PCR amplicons containing an expression cassette or sgRNA-expressing plasmids.-Design of repair template: conventional double-stranded DNA targeting constructs with homology arms flanking the insertion sequence or single-stranded DNA oligonucleotides (ssODNs)-Clonal isolation-SURVEYOR nuclease assay for functional testing. Homology-directed repair detection. Analysis of nonhomologous end joining mutations.-Discussion on tantalizing opportunities offered by genome editing for tackling a number of diseases that are beyond the reach of previous therapies.

B. Data Base for gene and protein analysis. (Theoretical/Practical session: 90 minutes). Dra. María Esther Fárez

CONTENTS:

The course will focus on available data base to input a target genomic DNA sequence and identify and rank suitable targets sites or search for all members of a given protein family. Aim of the course:-Use of Ensembl and NCBI genomic browsers to access genomic information with a number of visualisation tools. Levels of access and flexibility. Download data. Primer design. BLAST searches. Search for all members of a given protein family. Configure and personalize your page and export data.-We will also discuss the CRISPR design tool. Identify suitable targets and computationally prediction for off-target sites.

C. Discovering the dark side: tools for microbial biodiversity(Theoretical/practical session: 180 minutes). Dr. Manuel Martinez Bueno

CONTENTS:

The microbial world is considered the largest and unexplored reservoir of biodiversity on Earth, and because of this is considered the next frontier in biology. Studies on microbial communities raise questions about their composition, structure and stability and about the activity and function of their individual members. Traditional microbiological techniques and conventional microscopy are insufficient means to answer these questions. Most of the bacteria in natural samples cannot be detected by traditional methods as microscopy or culture techniques. The microbial cells adhere on to particles and therefore remain ‘invisible’. Culture-based approaches, despite their tremendous utility in basic microbiological research, cannot accurately describe naturally occurring prokaryotic assemblages simply because all such methods are incomplete. Most important, organisms that are recovered on standard microbiological media are rarely representative of the naturally occurring community, and for the majority of prokaryotic species no appropriate media

and conditions for growth are available. To deal with this problem, microbiologists have employed culture-independent approaches in studying naturally occurring prokaryotic assemblages. Culture-independent approaches most commonly target the cell’s DNA because of its information content and discriminating capacity. Many of these methods were only developed during the last decade. With recent advances in genomics and sequencing technologies, microbial community analyses using culture-independent molecular techniques have initiated a new era of microbial ecology. Molecular approaches such as genetic fingerprinting, metagenomics, metaproteomics, metatranscriptomics, and proteogenomics are vital for discovering and characterizing the vast microbial diversity and understanding their interactions with biotic and abiotic environmental factors. These techniques try to determine the microbial diversity in the ecosystems, as much in the space as in the time, without isolation of the bacterial strains. Limitations of these procedures are often associated with technical aspects, such as extraction of DNA / RNA from the sample or problems in DNA amplification (lack of primer annealing, wrong annealing, competition between primers, primer preferences) among others. The most commonly used gene in the analysis of microbial communities is the 16S rDNA, which in spite of its many advantages also has some drawbacks, such as intra-specific heterogeneity or low discrimination among nearby species. The use of other genes, such as RNA polymerase B (rpoB), recA gene family, gyrase B (gyrB), Tu (tuf) elongation factor, dnaK gene, in addition to other functional genes, could solve some of these problems. This talk will briefly review methods without using cell culture that are usually used for the study of microbial diversity, from genetic fingerprinting techniques of a community to mass sequencing by different platforms (The “Omics”).

Topics to talk about: Methods to study microbial diversity Culture methods in microbial ecology: applications and limitations Molecular methods for microbial community analysis Partial community analysis approach. Clone library method. Molecular

fingerprinting (RISA, RFLP (T-RFLP), DGGE, TTGE) Next-generation DNA sequencing techniques. Functional microbial ecology. The contribution of “omics” sciences in

microbial ecology.

Theoretical/Practical: Familiarize the student with molecular techniques without using cell

culture, for the study of microbial biodiversity and bacterial identification. DNA extraction. Ribosomal intergenic spacer length polymorphism

analysis (RISA)

Practical:1) DNA extraction and gel analysis 2) Ribosomal Intergenic Spacer Analysis. Ribosomal intergenic spacer

analysis (RISA) involves PCR amplification of a portion of the intergenic spacer region (ISR) present between the small (16S) and large (23S) ribosomal subunits. The ISR contains significant heterogeneity in both

length and nucleotide sequence. By using primers annealing to conserved regions in the 16S and 23S rRNA genes, RISA profiles can be generated from most of the dominant bacteria existing in an environmental sample. RISA provides a community-specific profile, with each band corresponding to at least one organism in the original community. Amplicons will be detected by acrylamide gel electrophoresis

3) The automated version of RISA is known as ARISA and involves use of fluorescence-labeled primers. ISR fragments are detected automatically by a laser detector (i.e. ABI 3100).

D. Identification of species through High resolution melting.(Theoretical/practical session: 180 minutes). Dra. Mariela Rivas

CONTENTS:

A brief introduction will be made on microalgae, focusing on eukaryotic microalgae. It will highlight the properties of the same, the bioactive compounds that synthesize. Also as they are grown and produced on a larger scale worldwide. On the other hand, the key aspects in the taxonomic identification of the same will be mentioned through molecular tools. In this sense we will discuss the development of primers and the techniques used. We will detail the real-time PCR technique linked to High Resolution Melting, which serves as methodology, the basis of the technique and how it can be applied in species identification.

Practical Session

An HRM reaction bound qPCR will be performed using Melt Doctor. To do this the reaction will be prepared using gDNA of 3 different microalgae and the Step-One equipment of Applied Biosystems will be used. Analysis of results in laboratory.

E. Current challenges in the study of the human microbiome(Theoretical/practical session: 180 minutes). Dra. Cristina Dorador

CONTENTS:New sequencing DNA/RNA technologies has provide new knowledge regarding microbial diversity and function in the environment including human. In this topic, we will analyse the history and the impact of the knowledge of human microbiome either in medicine or in ecology. Also, we will understand the ecology of microorganisms in the build environment.

Aims of the session: The idea of the course is to give a general overview of the “new” vision of the human in terms of an ‘ecological community’ of Archaea, Bacteria, Eukaryotic cells and viruses and the consequences in the understanding of disease and immunity.

Methodology of the session: The session will have two parts. The first one is an introductory class about human microbiome and the concept of hologenome, including the main results of initiatives as Human Microbiome Project, Earth Microbiome Project, Microbiology of the build environment, etc. The second part will be the analysis of real data obtained from open databases using Qiime and Mothur. After this, we will analyse critically the impacts of the study of the human microbiome and their perspectives.

Expected results of student´s learning: This session will give the students a general overview of the human microbiome from an ecological perspective.

II. VACCINE DEVELOPMENT AND OTHER CONTROL STRATEGIES

A. Exovesicles release in parasites and their immunological role(Theoretical session: 60 minutes). Dr. Antonio Osuna

CONTENTS:

B. Immunization against parasitic helminths(Theoretical session: 60 minutes). Dr. Antonio Osuna

CONTENTS:

C. Potential chemotherapeutic role of nanoparticles(Theoretical/practical session: 240 minutes). Dr. Jorge González and Dr. Kevin Sepúlveda

CONTENTS:

Parasitic infections of man are considered as neglected diseases. They affect vast sectors of the world's population, especially those associated with underdevelopment and poverty.A good example of these is Chagas disease, whose causative agent is a flagellate protozoan called Trypanosoma cruzi. The disease, affects 7-8 million people worldwide, and although it was described in 1909, after more than one hundred years there are only two drugs available, the nitrofurmimox and the benznidazole. Both drugs are mainly active in the acute phase of the infection, they have long therapeutic schemes and multiple side effects. Therefore, they are so far from being considered ideal drugs and new drugs are required. Likewise, in urban áreas, the main form of transmission of infection, occur by blood transfusion. Although in some countries there are protocols that through surveys and serological screening of the blood donors, may reduces the risk of infection, the chemoprophylaxis of blood bags contaminated with T. cruzi is a real necessity. However, the use of Gentian Violet was proposed more than 60 years ago, its use was restricted and currently there is no compound or molecule that can be used in the chemoprophylaxis of total blood.Nanotechnology is the engineering and manipulation of materials and devices with sizes in the nanometer range. Colloidal gold, iron oxide nanoparticles and quantum dot semiconductor nanocrystals are examples of nanoparticles, with

sizes generally ranging from 1 to 20 nm. These nanotechnologies have been researched tremendously in the last decade and this has led to a new area of "nanomedicine" which is the application of nanotechnology to human health-care for diagnosis, monitoring, treatment, prediction and prevention of diseases. Then, nanomaterials themselves can be used as image agents or therapeutic drugs, and for drug and gene delivery, biological devices, nanoelectronic biosensors or molecular nanotechnology. Then, in the theoretical class, a description will be presented respect to the main and potential applications of nanomedicine and nanotechnology in the treatment of human pathologies such as cancer and infectious diseases. Potential applications in the field of parasitic diseases caused by protozoa will be described. Likewise, results will be presented on the effect of metallic nanoparticles against Trichomonas vaginalis and T. cruzi.Laboratory activities will aim to evaluate the trypanocidal activity of nanoparticles. To do that, tests will be carried out in microplates evaluating the anti-T. cruzi, effect at 24 or 48 hours by means of quantitative assays that will determine the LD50 of each nanoparticle.

III. USES OF ENTOMOPATHOGENS FOR THE CONTROL OF VECTORS AND PESTS A. Uses of entomopathogens for the control of vectors and pest insects (Theoretical: 90 min) Dra. Susana Vilchez

CONTENTS:

The course will be focused in the description of one of the activities in Biotechnology, the use of living organisms for the control of insect populations that represent agricultural or health problems. Topics as the problem with insects, the methods for the control of insect populations, the concept of Biological Control, and the methods and practical examples of tools used in Biological control will be included in this course. In addition, an overview of the most common molecular mechanism used by entomopathogenic bacteria to colonize their insect host will be addressed.

A special emphasis will be made on the use of the entomopathogen Bacillus thuringiensis israeliensis in the control of Aedes aegypti the insect vector of yellow feber, Dengue, Chikungunya and Zika virus.

IV. INDUSTRIAL BIOTECHNOLOGY

A. Enzymatic protein hydrolysates: uses in agriculture, aquaculture and human nutrition. (Theoretical session: 120 minutes) Dra. Emilia Guadix

CONTENTS:

The properties and functionality of proteins can be changed by physical, chemical and enzymatic treatments. Numerous advantages and possibilities

exist for improving the functional, nutritive and bioactive properties of food proteins by partial enzymatic hydrolysis. The peptide bond cleavage leads to change in pH and osmolality affecting properties such as solubility, emulsifying activity, foaming capacity and viscosity. Moreover, the hydrolytic process generates numerous peptides with different peptide-chain-length and different amino acid sequences. A rigorous control of the reaction and an adequate purification process can lead to useful hydrolysates for enteral nutrition, infant formulas and functional foods.This section will focus on the factors affecting enzymatic hydrolysis of proteins, the design and control of the process and the application of protein hydrolysates.The principles of the protein hydrolysis will be discussed. The substrates and commercial enzymes will be mentioned. The production of hydrolysates and control of the reaction in continuous and discontinuous reactors will be studied. Special attention will be given to the continuous membrane reactor. An important part of the section will be to analyze the industrial applications of these hydrolysates. These can be used as foliar fertilizer, food for juvenile fish or as protein content in products for infant or clinical nutrition. On the other hand, in the last decade, an enormous research effort has been made to demonstrate the bioactivity of certain peptides obtained by enzymatic hydrolysis as modulators of blood pressure, cholesterol levels, stress and fatigue, among others. These capacities make them interesting ingredients in the formulation of functional foods

B. Pigments from bacterial origins

(Theoretical/practical session: 180 minutes) Dra. Catherine Lizama. Colaborators: Dr. Pedro Cerezal Mezquita, Dr. Adrián Paredes; Assistans: Juan Morales Espinoza, Carolina Espinoza, Marjorie Jauregui, Waldo Bugueño.

CONTENTS:

Carotenoids are natural pigments synthesized as hydrocarbons (carotenes, lycopene, α-carotene and β-carotene) or their oxygenated derivatives (xanthophylls, lutein, α-cryptoxanthin and β-cryptoxanthin, zeaxanthin, canthaxanthin and astaxanthin) by plants and microorganisms. In recent years, interest in the production of natural carotenoids by microbial fermentation has increased. Carotenogenic microbes such as Dunaliella salina, Xanthophyllomyces dendrorhous, Haematococcus pluvialis, and Blakeslea trispora have been investigated for large-scale production (Jacobson et al., 2000, Mehta et al., 2003, Olaizola 2000 and Raja et al. 2007). Extreme halophilic bacteria and archaea represent an excellent source of research for carotenoid pigments. The reddish pigmentation of these environments is due to the colonization by haloarqueas and halophilic bacteria. A high production of carotenoids including beta-carotene and canthaxanthins has been reported in extreme halophilic bacteria (Asker and Ohta, 1999) and the production of canthaxanthins by Haloferax alexandrinus (Asker and Ohta, 2002). However, the production of these haloarqueal pigments has not been developed at the industrial level.Carotenoids are natural pigments that exhibit many biological functions: as antioxidants (i.e., promote oxidative stress resistance), membrane stabilizers and

precursors for vitamin A. The link between these biological activities and many health benefits: anticancer activity, prevention of chronic diseases, etc. β-Carotene is used in the food industry as a natural food colorant. As a precursor of vitamin A, it is of importance as an additive in cosmetics, multivitamin preparations, and health food products.

Theoretical session (one sesión the 60 minutes) Biological Diversity of extremely halophilic Archaea Pigments of extremely halophilic archaea Intellectual and industrial property the pigments produced by halobacteria. Pilot and industrial scale production of pigments of microbial origin. Antioxidant capacity of carotenoid pigments and phenolic compounds produced

by halophilic Archaea.

Practical session

Visit to Pilot Plant ( 30 minutes)

Demonstrative activities at the Department of Food Microencapsulation laboratory.(2 hours)

Determination of antioxidant capacity Pigment tests in different food matrices Microencapsulation techniques of pigments

V. PLANT PHYSIOLOGY AND BIOTECHNOLOGY

A. Metabolomics and System Biology are powerful tools for the improvement of Crops against biotic and abiotic stressors.

(Theoretical/practical session: 150 minutes) Dr. Cristián Wulff Zottele

CONTENTS:

General overview of the course: Since the beginning of the 21th century, the development of high scale DNA sequencing methods improved the discovery of new genes involved in biochemical and physiological responses of plants against biotic and abiotic factors. However, plant genome sequencing projects did not provide information of the functionality of new genes related to plant physiological functions, and requires of other biochemical methodologies related comprehending the functionality of genes at transcriptional, translational levels in higher plants. Besides, metabolite profiling is an additional “omics” tool that allows the discovery of metabolic pathways involved in plants physiological responses against biotic and abiotic stressors. The information acquired by these “omics” techniques generates new challenges related to comprehend the complexity of the plants physiological responses against environmental

stresses, and it have implicated the development of data mining techniques for the discovery of the functionality of new genes.

Aims of the course: The main task of the present course consist to show to the students the basic concepts and strategies that allows the discovery of primary, and secondary, metabolic pathways, such oxidative stress scavenging mechanisms, that are affected in plants tissues as consequence of environmental stressors, using GC/MS metabolomic techniques and system biology methodologies. Furthermore, the course will guide the student to use open sources software’s that allow the development of system biology methodologies that will be employed in their research, such as Real time PCR analisys tools and data mining techniques of information obtained by GC/MS metabolite profiling.

Methodology of the course: The course will be divided in two sections. The first section will be a theoretical class about transcriptomics using Real time PCR method, metabolomics, and system biology. Then the students will be trained in the basic handle of open access software’s that are related to Real time PCR data analyses and data mining techniques, such as MEV. Results of the GC/MS metabolite profiling obtained of A. thaliana leaves exposed to combined conditions of High light intensities and sulfate starvation will be analyzed by the student with bioinformatics, statistical, and data mining tools, such as principal component (PCA) and hierarchical clustering (HCA) analyses.

Expected results of student´s learning: This course will give to the students a basic overview of the topics of “omics” and system biology tools used for the discovery of biochemical and physiological responses higher plants against environmental factors, and the applications in plant biotechnology.

Bibliography that will be used in the : Wulff-Zottele C, Gatzke N, Kopka J, Orellana A, Hoefgen R, Fisahn J, Hesse H, (2010) “Photosynthesis and metabolism interact during acclimation of Arabidopsis thaliana to high irradiance and sulfur depletion”, Plant Cell Environm, 33: 1974-1988