A Quick Guide to my current work
This article is based on a poster I presented on “Forschungsforum HTW Berlin” in October 2021. The poster is available in German language on DOI: 10.13140/RG.2.2.26595.43048.
What’s so cool about Telomeres?
Telomeres are relevant to both aging and cancer. Drugs elongating or stabilizing them could delay aging. Drugs that shorten them could be used in cancer therapy.
Drug development that affects Telomere length and dynamics requires systems to screen for these parameters. In such a system the influence of thousands of substances on Telomeres has to be examined in parallel. It has to be cheap, quick and easy.
Idea: A high-throughput system that uses optical signals to estimate the telomere lengths of living yeast cells. For this purpose, proteins that naturally bind to Telomeres will be labeled fluorescently and/or luminescently.
Problem: Yeast use Telomerase to keep their Telomeres constant. In addition to the marking the Telomers, I need to integrate a switch on the Telomerase of the yeast to activate and deactivate it.
Markers for Telomer Length
Methods for Marking
The marking is done in the genomic code of the target proteins (RAP1, RIF1 and RIF2) by fusion with reporter proteins. The genetic code of the reporter (a fluorescent or luminescent protein) is attached to the code for the target protein in the genome by means of transformation and recombination.
Methods to turn Telomerase on/off
In order to make the Telomerase switchable the promoter of one of the genes that together form the Telomerase is exchanged. A promoter is a regulatory sequence in front of the actual gene (the blueprint of a protein). It controls how and when the encoded protein is produced.
The natural promoter of the EST2 gene, which is one of the components of yeast Telomerase, is replaced with the promoter of the DDI2 gene. While Telomerase is always active in yeast, the DDI2 gene is only activated under certain chemical stimuli. If the promoter is successfully exchanged the Telomerase is inactive but can be activated by adding small amounts of the chemical cyanamide.
One way to achieve this is to exchange the promoter directly in the yeast genome via recombination.
Another slightly simpler method would be to destroy (knock-out) the gene in the yeast genome and introduce a functional version with the exchanged promoter on an artificial chromosome (plasmid) into the cell.
Status of work after year 1 of 4
Libraries of yeast strains with GFP-tagged proteins are available. These were used to carry out the first preliminary tests to establish the measurement methods.
A DNA construct was designed and produced that makes it possible to replace the promoter of the EST2 gene. The construct was transformed into strains with different GFP tags.
Transformed colonies are currently being examined to determine whether and where the construct is integrated in the genome. It is also checked at the protein level whether the control works. This has just been confirmed by a Western blot which I will explain in one of my next articles.
Next the new strain will be tested in the fluorescence assay.