DNA Manipulation

Code:

BIOL4001

Acronym:

BIOL4001

Keywords
Classification	Keyword
OFICIAL	Biology

Instance: 2021/2022 - 1S

Active?	Yes
Responsible unit:	Department of Biology
Course/CS Responsible:	Master in Functional Biology and Biotechnology of Plants

Cycles of Study/Courses

Acronym	No. of Students	Study Plan	Curricular Years	Credits UCN	Credits ECTS	Contact hours	Total Time
M:BFBP	14	Plano de Estudos M:BFBP_2015_2016.	1	-	6	42	162

Teaching language

Suitable for English-speaking students

Objectives

Aims of the course unit include are i) to provide advanced concepts in DNA manipulation, recombinant DNA technology, cloning and their applications in genetic transformation of plants and PCR, with special emphasis on the molecular techniques involved in the production of recombinant proteins; ii) to provide training in the use of basic bioinformatic tools for a protein expression project, primer design and gene optimization; iii) to provide laboratory “hands-on” training in gene cloning in expression vectors containing different tags and fusion proteins and transformation into appropriate bacterial strains.

Learning outcomes and competences

Students should understand the rationale for recombinant protein production and understand the underlying molecular processes of gene expression and regulation. Describe and understand the relevant molecular techniques and DNA manipulation involved in recombinant protein production. Use basic bioinformatics tools in analysis and planning of a protein expression project. Understand gene optimization and perform primer design.
Obtain laboratory skills in gene cloning using various vector types and bacterial cell strains, preparation of plasmid DNA, agarose gel electrophoresis, restriction analysis, primer design and PCR. Analyze original scientific articles, plan experimental design and analyze data. Communicate orally projects and results and write lab reports.

Working method

Presencial

Program

Structure of genes and regulatory sequences. Gene expression and regulation. DNA sequence analysis. Gene optimization. DNA cloning. PCR and PCR based cloning, restriction enzymes. Cloning vectors. Gateway technology. Expression vectors. Vectors containing different tags and fusion proteins. The pET system and the T7 expression system. Regulators of expression (derived from the pLac promoter, expression of the repressor protein in strains with allele lacIq). Bacterial strains with genetic markers for recombinant protein expression.  Basic bioinformatics tools: database search; BLAST similarity search, restriction analysis, primer design.

Laboratory - Cloning and expression of a gene in E. coli. Minipreparation of plasmid DNA and its analysis by agarose gel electrophoresis. Isolation of genes by PCR. DNA restriction analysis. DNA cloning and transformation into competent E. coli. Screening of recombinants by PCR. Preliminary study of bacterial extracts by western blot.

Mandatory literature

Videira Arnaldo 340; Engenharia genética. ISBN: 9789727577439

Complementary Bibliography

Sambrook Joseph; Molecular cloning. ISBN: 978-087969-577-4 (Vol. 1)
Watson James D. 1928- 070; Recombinant DNA. ISBN: 9781429203128
Greenfield Edward A., 1957- 340; Antibodies. ISBN: 9781936113811

Teaching methods and learning activities

Students are encouraged to engage in projects, both in working groups or individually, and to take the initiative to investigate specific topics. The use of real examples of research and "case studies", provide the context of reality and allows the acquisition of the notion that science is a continuous search for answers to questions that are not ever completely resolved. Activities include lectures, discussion groups, tutorials, problem solving, debates, etc. Emphasis also will be placed in wet lab training.

 Assessment tools include presentation and discussion of original scientific articles, a written laboratory report on the project developed and a final exam.

 

keywords

Natural sciences > Biological sciences > Biological engineering > Genetic engineering
Natural sciences > Biological sciences > Biology > Molecular biology
Natural sciences > Biological sciences > Biology > Functional biology

Evaluation Type

Distributed evaluation with final exam

Assessment Components

designation	Weight (%)
Exame	85,00
Trabalho escrito	15,00
Total:	100,00

Amount of time allocated to each course unit

designation	Time (hours)
Estudo autónomo	105,00
Frequência das aulas	42,00
Trabalho escrito	15,00
Total:	162,00

Eligibility for exams

Compulsory class attendance (minimum 3/4)

Calculation formula of final grade

Evaluation Formula : Final Exam (17 points) + Test exercises (3 points).

Classification improvement

Grade improvement can only be obtained through final exam.