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Education

Novel cloning technologies for future biotechnology

Traditional cloning methods are not always suitable for nowadays scientific research. Therefore novel cloning technologies not only need to be developed but also teached to students as they are future scientists. Therefore we used the University environment to develop and offer a full 11cts module on novel cloning technologies

Aim and scope of the modul

Main area of interest for this modul is the generation and integration of artificial biochemical pathways into organisms like bacteria and plants. As the construction of highly complex pathways is nearly impossible with standard cloning methods, the modul will introduce the students to novel cloning technologies for future construction of genes and genomes.

The lecture will give main basic aspects of novel cloning technologies and biological pathways. Seminar will introduce examples and show the usage of appropriate software tools. Practical training deals with design of artificial pathways and planning of needed cloning strategies.

Entry test

Students coming from different backgrounds might have different knowledge about cloning of genes. Therefore, first lecture was started with several questions to gain some insight into what the students already know.

Lecture and Seminar

Main basic information on the topic of cloning genes and genomes is provided by the lecturer Katrin Messerschmidt. Sequences of theoretical input are accompanied by practical exercises, discussions and question-and-answer schemes. Later on, students present peer reviewed publications to each other to introduce different cloning methods. Besides cloning methods also additional information as signal-peptides, protein-tags, protein purification and artificial transcription factors are introduced to the audience. Seminar times are used for discussion of cloning strategies on different given projects and problems. Furthermore, a laboratory handbook for lecture topics is designed and produced.

Cloning technologies:

  • “Rescue” of PCR products

  • Basics of Gateway cloning

  • Gateway destinations

  • Cloning with BioBricks

  • Cloning with Bgl Bricks

  • Golden Gate cloning

  • ligation independent cloning (LIC and SLIC)

  • PCR-based cloning (OE-PCR and CPEC)

  • Cloning technologies IV

  • Gibson assembly

  • SLiCE

  • TAR

Usage of peptide and protein tags:

  • tags for protein detection and purification

  • SplitGFP tag for protein quantification

Signal sequences for prokaryotes:

  • Sec-pathway of E. coli

  • Tat-pathway of E. coli

Signal sequences for eukaryotes:

  • targeting to membrane

  • nuclear import

  • nuclear export

Artificial transcription factors:

  • TALE-based artificial TF

  • dCas9-based artificial TF

  • Tetracyclin repressor-based artificial TF

Artificial operons:

  • 2A peptides

  • IRES elements

  • Protein depletion with degrons

Internship

As part of the modul the internship should introduce the students to different cloning methods within a scientific research project. Different cloning projects of Cell2Fab and different cooperation projects were used to prepare the internships. Project descriptions offered the material available as well as a description of the desired constructs. Some hints on work flow were given too. Within the projects students would work self-organized. Daily work included experimental setups, design of cloning strategies, analysis of cloning results and documentation of work done.