Session 10

Jensen et al., (2014) EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae

Key findings:

In their study Jensen et al. 2014 present a method to simultaneously integrate several genes into the genome of Saccharomyces cerevisiae by the use of different integrative plasmids, corresponding to integration sites on chromosome X, XI and XII. These plasmids were constructed by the USER cloning method (Nour‐Eldin et al. 2006) based on a previously designed vector set by Mikkelsen et al. 2012. The vectors constructed by Mikkelsen et al. 2012 were modified in that way that the selection marker URA, which was flanked by directed repeats, was replaced by a set of 5 different LoxP‐flanked selection markers. These markers can be recycled, enabling repeated cycles of genetic engineering without the loss of the integrated genes. For a proof of concept, Jensen et al. 2014 selected three genes, coding for different fluorescence proteins (CFP, RFP, YFP) and inserted them into three episomal plasmids and three integrative plasmids. The strain CEN.PK102‐5B was either transformed with the three episomal or the three integrative plasmids simultaneously, followed by flow cytometric analysis to test for the presence of the three fluorescent proteins. They could show that the expression of the three proteins is much more uniform in cells containing the three genes integrated in the genome compared to those containing the three episomal plasmids. The standard deviation for cells expressing the fluorescent proteins from additional plasmids was 4‐5 times larger compared to cells containing the integrated genes. In case of the integrated genes, 95% of the cells showed a fluorescence intensity for all three fluorescent proteins within a range of the mean value ± 15%. In contrast, this was only valid for 6% of the cells containing the three genes on episomal plasmids.

Relevance of the article:

In their study Jensen et al. 2014 are able to show the superiority of integrative plasmids compared to episomal plasmids concerning the stability and uniformity of gene expression. Moreover, they are providing a set of 12 different integrative plasmids which can facilitate the simultaneous integration of several genes into the genome of S. cerevisiae. I would rate the article as very good, especially for people who are working in the field of molecular biology, since the article shows a powerful tool for the controllable expression of (foreign) genes in S. cerevisiae.


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