1. YEAST MOLECULAR GENETICS (B)

2. Homologous recombination is quite a frequent event!

3. Gene replacement one step

4. Deleting a yeast gene
There are a number of different ways to generate the piece of DNA for yeast transformation, i.e. the marker flanked by fragments with DNA from YFG1
It can be done using restriction enzymes and DNA ligation
It can be done by PCR/restriction/ligation; the entire plasmid is amplified by PCR with the exception of the ORF; restriction sites in the PCR primers generate a site where the marker can be cloned in
It can be done by PCR without any cloning step; in two separate PCR reactions the flanking regions of YFG1 are amplified and used in a second round as primers to amplify the marker gene; this requires the primers to be designed accordingly (see below)
It can also be done with long PCR primers, in which only the marker is amplified and recombination is mediated by the primer sequences; as little as 30bp can be enough to mediate recombination; in such cases the use of a heterologous marker is recommended to make integration in the right place more reliable
The latter two approaches do not even require the gene to be cloned!! A gene deletion project hence may take only a couple of days
First PCR to amplify the flanking
parts of your favourite gene
YFG1
URA3
Second PCR to amplify the marker
URA3
Final PCR product ready
for transformation

5. Smart gene deletion
There are very smart ways to make most out of a gene deletion/disruption approach, depending on the marker cassette used
For instance, if the marker cassette contains in addition the lacZ reporter gene a precise fusion can be generated that places the lacZ gene under control of the yeast promoter of YFG1
If such a construct is used for gene deletion in a diploid, it can be used to study the expression of the gene by monitoring b-galactosidase activity in that diploid and after sporulation of the diploid the mutant phenotype can be studied in the haploid progeny
In a similar way, a gene can be tagged. For instance, if the casette is inserted in frame to the end of the ORF it will generate a fusion protein, with lacZ, GFP or an immuno-tag for protein detection
URA3
lacZ
YFG1
Diploid cell

6. Smart gene deletion
In a similar way, a gene can be tagged. For instance, if the cassette is inserted in frame to the end of the ORF it will generate a fusion protein, with lacZ, GFP or an immuno-tag for protein detection and purification
For instance, there are now sets of strains available in which each yeast protein has been tagged with GFP or TAP-tag
YFG1
URA3
GFP

7. Smart gene deletion
There are some ways to delete a yeast gene without leaving any trace behind, i.e. no marker gene
This is very important if one wants to re-use the marker in order to make many deletions in one and the same strain (there are strains with more than 20 deletions!)
It is also important for industrial yeast strains; when one wants to engineer those at the end no foreign DNA should be left behind (but for hardliners on genetic engineering the intermediate presence of foreign DNA in a yeast is already ”dangerous”)
All these methods use homologous recombination a second time, i.e. to pop-out the integrated DNA again
An example for this are the loxP-kanR-loxP cassettes; recombination between the two loxP cassettes is stimulated by the Cre-recombinase (transformed on a separate plasmid); recombination just leaves behind a single loxP site

8. Gene replacement two steps
Questa tecnica può essere utilizzata solo con marcatori selezionabili e controselezionabili
Facciamo un esempio….

9. Smart gene deletion
A very useful marker to work with is URA3 because one can select for and against its presence
Selection for URA3 is of course done on medium lacking uracil
Selection against URA3 uses the drug 5-flouro-orotic acid, which is toxic to URA3 cells
An example is shown below
URA3
plasmid
YFG1
genome
YFG1
URA3
Integration of the plasmid, which only contains YFG1 flanking regions, creates a duplication; recombination between
the blue sequences leads to a pop-out of the entire plasmid plus the YFG1 coding region

10. From gene disruption to transposon mutagenesis
The gene deletion/disruption technique has been taken a step further to be used in random mutagenesis
For this a gene library is first constructed as discussed before such that the inserted yeast DNA can be cut out with NotI, an enzyme that only cuts a very few times in the yeast genome
Then this library is mutagenised with a transposon in E. coli, where the Tn randomly integrates into the yeast DNA
Subsequently, the entire mix of NotI fragments is transformed into yeast where it is expected to replace genes; with about 30,000 yeast clones a more then 90% coverage of the genome is achieved
The Tn used is a quite sophisticated example of such a transposon, that can be partially cut out again through the lox-sites. This creates a tag, which allows immunolocalisation of the gene product
TR: Tn3 terminal inverted repeats
Xa: Factor Xa cleavage recognition site
loxR: lox site, target for Cre recombinase
lacZ: 5'-truncated lacZ gene encoding b- galactosidase
URA3 gene from S. cerevisiae
tet: tetracycline resistance gene
res: Tn3 site for resolution of transposition intermediate
loxP: lox site, target for Cre recombinase
3xHA: Hemagglutinin (HA) triple epitope tag

11. From gene disruption to transposon mutagenesis
The reason why transposon mutagenesis is so powerful lies in the fact that the gene affected by the insertion can be determined very easily
For this, the entire genomic DNA of the mutant is isolated and cut with an enzyme that does not cut within the transposon
In this way of course many fragments are generated but only one will contain the transposon plus some flanking yeast DNA
Ligation generates a circular plasmid that can be transformed into E. coli and further analysed
Sequencing using a primer binding to the transposon but directing into the yeast DNA will reveal exactly where the transposon was integrated when the sequence is compared to that of the yeast genome
This method works so well that it has been used for a comprehensive genome analysis
For instance, we have recently screened 25,000 Tn-mutants for a number of properties and could allocate functions to a number of uncharacterised genes with relevance to stress tolerance
Derivative of the transposon with antibiotic markers are very useful tools to mutagenise and study industrial strains

12. Analisi genetica delle tetradi
Consente di attribuire una funzione a geni mutati (analisi fenotipo dell’aploide mutato)

13. Visualizza le interazioni proteina-proteina
Two-hybrid system
Visualizza le interazioni proteina-proteina

14. Getting further: two-hybrid system
The yeast two hybrid system is a method to detect the interaction of two proteins in the yeast cell and it can be used to select for an interacting partner of a known protein
The original version uses a transcriptional read-out to monitor interaction, nowadays there are also other methods
The method is so powerful since it is not restricted to yeast proteins; the interacting partners can origin from any organism; in fact some versions do not use any yeast sequences
Basis for the system is the modular nature of transcription activators that consist of exchangeable DNA binding and transcriptional activation domains
The gene of interest, the bait, is cloned in fusion with a DNA binding domain, such as that of the E. coli lexA protein
The potential binding partner, the target or prey, which may be a library, is cloned in fusion to a transcriptional activation domain, such as that from VP16, a viral protein
Only when bait and target interact, a reporter gene whose only promoter is a lexA binding site will be activated
lexA
site
reporter

15. Application of the yeast two-hybrid system
The possible applications to the two-hybrid system are absolutely tremendous
The system can be used to detect interaction between two proteins
The system can be used to characterise the domains and residues in the two proteins that mediate interaction; this can be done by mutagenesis and the use of a counterselectable reporter, such as URA3
The system can of course be used to find interaction partners
The system can be used to find proteins that regulate the interaction between two proteins
The system can be used to screen for drugs that inhibit the interaction between two proteins
The system is actually used to construct an genome-wide map of protein interactions in yeast; using laboratory robots 6000 bait strains are crossed to 6000 prey strains to study all possible protein intercations
etc

16. Use of recombinant yeast in industrial biotechnology

17. Yeast as cell factory for recombinant protein production (RPP)

19. Looking for unconventional yeasts

20. Promoters to be used in RPP

21. Promoters to be used in RPP

22. RPP in S. cerevisiae
Usata nella terapia riperfusiva post-infarto per minimizzare il danno ossidativo!
Farmaco salvavita

24. RPP in S. cerevisiae

25. RPP in Pichia pastoris

26. Which fermentation process for your strain?