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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
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.
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.
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.
“Rescue” of PCR products
Basics of Gateway cloning
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
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
Artificial transcription factors:
TALE-based artificial TF
dCas9-based artificial TF
Tetracyclin repressor-based artificial TF
Protein depletion with degrons
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.