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Yeast Biobrick Assembly (YBA) Standardized Method for Vector Assembly of Biobrick Devices Via Homologous Recombination in Saccharomyces Cerevisiae

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Yeast Biobrick Assembly (YBA) Standardized Method for Vector Assembly of Biobrick Devices Via Homologous Recombination in Saccharomyces Cerevisiae Yeast BioBrick Assembly (YBA) Standardized method for vector assembly of BioBrick devices via homologous recombination in Saccharomyces cerevisiae Martin Schneider, Leonard Fresenborg, Virginia Schadeweg iGEM Team Frankfurt 2012, Johann Wolfgang Goethe University Frankfurt am Main, 22 September 2012 Keywords: Yeast BioBrick Assembly (YBA), Yeast, Saccharomyces cerevisiae, iGEM, Parts Registry, BioBrick, Homologous recombination, Gap repair cloning Abstract Gap repair cloning is a more and more established methode for ecient, fast and error-free construction of plasmids based on the homologous recombination system of Saccharomyces cerevisiae (common yeast). Naturally yeast uses this process to repair DNA double strand breaks which are one of the most dangerous and life-threatening damages of the DNA for a cell. Therefor this eucaryotic microorganism has developed a few enzymes which have the ability to repair a broken DNA double strand by pairing it with a very similiar DNA region (typically on the homologous chromosome). Using gap repair cloning a series of linear, successive DNA fragments with homologous overlaps to the respectively following fragment can be transformed in only one step into a yeast cell. After that the micoorganism recombines all fragments in the predetermined, specic order to the nal targeting vector. The advantage is that up to eighteen and more successive DNA fragments can be assembled in a single transformation. YBA is a standardized methode that describes a new way of assembling BioBrick devices in a desired order to a targeting plasmid using gap repair cloning. Therefore only one restriction enzyme for linearization of the plasmid and a standardized selection of primers and promoters/termintors is needed. YBA standard is a continuation of the BioBrick standard based on homologue recombination. It is compartible with all BBF RFC1 10 parts. Additionally it can be adapted by specic primer design to all other BioBrick standards. In the following we focuse on assembly of yeast expression vectors by using YBA methode. However it also can be used for E.coli vector design or assembly. 1 BioBrick Foundation Request for Comment 1 Contents I Introduction 2 1 Homologous Recombination System of Yeast . 3 2 Design of DNA Fragments for Gap Repair Cloning . 3 2.1 Example: Vector Assembly via Gap Repair Cloning for Expression of Three Genes in Yeast. 3 II Yeast BioBrick Assembly (YBA) 5 3 YBA: Standardized Gap Repair Cloning for BioBricks Assembly . 5 III Assembly of Yeast Vector 8 4 Assembly of a Yeast Expression Vector . 8 4.1 Checklist Vector . 9 4.2 Checklist PCR . 9 4.3 Checklist Yeast Transformation . 10 5 Promoters and Terminators of Yeast . 10 5.1 Promoter . 10 5.2 Terminator . 11 5.3 Ligated Terminator-Promoter Parts . 11 6 Standardized YBA Primers for Assembly of Yeast Expression Vectors 12 6.1 Design of YBA Standard Primers . 13 6.2 Primer Gene Amplication (with annealing temperature) . 14 6.3 Primer Promoter Amplication (with annealing temperature) . 14 6.4 Primer Terminator Amplication (with annealing temperature) . 15 6.5 Primer Terminator-Promoter Part Amplication (with annealing tem- perature) . 15 7 Methods . 15 7.1 General procedure of a transformation via gap repair . 15 7.2 Polymerase Chain Reaction (PCR) . 16 7.3 Restriction Digestion for Linearization of a Plasmid . 17 7.4 Control Restriction Digestion of Plasmid with Insert . 17 7.5 Yeast Transformation . 17 7.5.1 Production of competent cells . 17 7.5.2 Transformation . 18 7.6 E.coli Transformation . 18 7.6.1 Production of competent cells . 18 7.6.2 Transformation (on ice) . 19 IV Assembly of E.coli Vectors 19 V Acknowledgements 20 2 Part I. Introduction 1 Homologous Recombination System of Yeast There are many endogenous and exogenous factors (for example reactive oxygen- species, ionizing radiation, chemicals and failing of DNA binding enzymes (e.g. collapsed replication forks)) which causes DNA double strand breaks. For the cell this is the most dangerous DNA damage because even if it occurs in rather unimportant regions the cell will not survive the next cell cycle. That's the reason why yeast possesses highly active enzymes which have the ability to re- pair a broken DNA double strand by pairing it with a very similiar DNA region (typically on the homologous chromosome). This process is called homologous recombination. Using the gap repair method this natural process can be ex- ploited for the construction of large cloning vectors in yeast. 2 Design of DNA Fragments for Gap Repair Cloning The idea of the method is to transform a series of linear, successive DNA frag- ments into one yeast cell. The linear fragments have open blunt ends like they occur after a double strand break. If a homologous sequence is available it will be treated like a genomic double strand break and homologous recombination takes place. When the successive DNA fragments are designed in a specic way which includes large sequence overlaps (overall app. 40 bp) to the respectively following fragment yeast will recombinate them together. For the formation of a cloning vector the rst fragment is a yeast-E.coli shuttle plasmid which is linearized by an appropriate restriction digest. A shut- tle plasmid is a plasmid which is stable both in yeast and in Escherichia coli. The rst fragment of the insert has to possess an homologous overlap to both the wished insertion site on the plasmid and to the beginning of the second fragment. The end of the second fragment has to possess an overlap to the beginning of the third one and so on. At least the end of the last fragment of the insert again has to possess an overlap homologous to the second insertion site on the plasmid. At least up to eighteen and more single fragments can be assembled to a targeting plasmid in a single transformation. Another advantage of this method is that no scars are left between the inserted fragments. Assembly of fragments to joint genes is possible. Restriction enzymes only have to be used once for linearization of the shuttle plasmid. 2.1 Example: Vector Assembly via Gap Repair Cloning for Expression of Three Genes in Yeast. In the project of iGEM Team Frankfurt 2012 a plasmid for overexpression of three genes of the Mevalonate pathway was constructed. Therefore synthesized fragments of the mentioned three genes, two promoters, two terminators (both 3 from yeast) and a yeast expression plasmid which already contains one promoter, one terminator and a gene for uracil synthesis were used. Via PCR appropriate homologous overlaps to all fragments were assembled using primers containing these overlaps. Thereby the fragments were arranged in the specic order shown at picture one. Gene 2 Promoter Terminator Promoter Terminator Gene 3 Gene 1 Terminator Promoter Plasmid* Uracil Gene *not all attributes are shown Picture1: Plasmid for expression of three genes in yeast. The overlap sequences at the beginning and the end of each fragment are 20 bp long and homologous to the respectively previous and subsequent fragment. The overlaps to promoter and terminator on the plasmid are longer (40 bp) because the plasmid does not contain any overlaps to the rst and last insert fragment. Thus the homologous region between every fragmentpair is always 40 pb long. To assemble the targeting vector all DNA fragments were transformed to- gether into competent yeast cells. Therefor a mutant yeast strain with a deleted uracil gene is chosen. Only positive clones which contain the complete vector have the abbility to produce uracil because of the intact uracil gene on the plasmid. These transformants are able to grow on selective synthetic medium lacking from uracil. After preparing the plasmid from the positive transfomed yeast clones it can be transformed into Escherichia coli and gained from there in large amounts for further use. 4 Part II. Yeast BioBrick Assembly (YBA) 3 YBA: Standardized Gap Repair Cloning for BioBricks Assembly The BioBrick cloning standards used in the Parts Registry and in the iGEM competition are based on restriction digest and ligation. One of the main ad- vantages of the gap repair method is to avoid this. Moreover it leaves no scars between the assembled fragments like restriction digest and religation does. An- other advantage of gap repair cloning is it´s heightened time eency when a large amount of fragments shall be assembled. Furthermore the expensive use of restriction and ligation enzymes can be reduced signicantly. For these rea- sons gap repair cloning promises to be a useful tool for future iGEM teams. The problem is that the common Biobrick standards are useless by now with regard to gap repair cloning. The idea of YBA is now to design a new stan- dard for assembly of yeast vectors based on standardized PCR primers. YBA is compartible to common BioBrick standards and allows gap repair cloning. Although the restriction sites of the Biobrick pre- and sux are unimpor- tant in our context, the biobricking of genes leads to possiblity to amplify all Biobricks with the same prex and sux type (in our case we focus on BBF RFC 10) with suitable PCR primers. There are already some PCR primers in the Parts Registry which anneal at the pre- or sux sequence. By adding an specic non annealing sequence to the primers a desired overlap to other frag- ments can be produced on every Biobrick device. These primers which consist of an annealing sequenz to either prex or sux and an specic overlap are the stardardized PCR primers for YBA method. The further idea would be to create DNA fragments suitable for gap repair cloning. It would be great if the assembly and cloning of genes in yeast become simpler than the cloning by restriction and ligation. In the following we want to outline the basic idea.
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