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Title:   Author  item
                                            SZEWCZYK, EDYTA - University Of Californiaitem
                                            Kasuga, Takaoitem
                                            FAN, ZHILIANG - University Of California
  Submitted to: Journal of Microbiological Methods
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 2/18/2014
                                    Publication  Date: 5/18/2014
                                    Publication URL: http://www.sciencedirect.com/science/article/pii/S0167701214000475#Citation: Szewczyk, E., Kasuga, T., Fan, Z. 2014. A self-excising beta-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa. Journal of Microbiological Methods. 91(100):17-23.Interpretive Summary: A plant decomposing fungus, Neurospora crassa, has been used for the study of biochemistry and cellular biology.  We have previously reported a molecular biological method, which allows us to sequentially manipulate genes to create desirable genotypes.  In this study, we have further modified and improve efficiency and selectivity of the genetic manipulation strategy. This method is highly valuable for bioengineering as well as functional genomics study, and applicable to genetic study of devastating plant pathogenic fungi.
                                Technical Abstract: In a previous study we developed a cassette employing a bacterial beta-recombinase acting on six recognition sequences (beta-rec/six), which allowed repetitive site-specific gene deletion and marker recycling in Neurospora crassa.  However, only one positive selection marker was used in the cassette.  A subsequent procedure is needed to purify homokaryons due to the lack of a negative selection after eviction. Additionally, the xylanase promoter from Penicillium chrysogenum used in the construct is not strongly regulated in N. crassa, which led to low efficiency in cassette eviction. Herein we report an improved variant of the self-excising beta-recombinase/six cassette for repetitive gene deletion in N. crassa using a native xylanase promoter from N. crassa, plus the introduction of a bidirectional selection marker to facilitate homokaryon selection using a thymidine kinase (tk) gene.


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