Yeast BioBrick Assembly (YBA)

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1 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 RFC 1 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

2 Contents I Introduction 2 1 Homologous Recombination System of Yeast Design of DNA Fragments for Gap Repair Cloning Example: Vector Assembly via Gap Repair Cloning for Expression of Three Genes in Yeast 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 Checklist Vector Checklist PCR Checklist Yeast Transformation Promoters and Terminators of Yeast Promoter Terminator Ligated Terminator-Promoter Parts Standardized YBA Primers for Assembly of Yeast Expression Vectors Design of YBA Standard Primers Primer Gene Amplication (with annealing temperature) Primer Promoter Amplication (with annealing temperature) Primer Terminator Amplication (with annealing temperature) Primer Terminator-Promoter Part Amplication (with annealing temperature) Methods General procedure of a transformation via gap repair Polymerase Chain Reaction (PCR) Restriction Digestion for Linearization of a Plasmid Control Restriction Digestion of Plasmid with Insert Yeast Transformation Production of competent cells Transformation E.coli Transformation Production of competent cells Transformation (on ice) IV Assembly of E.coli Vectors 19 V Acknowledgements 20 2

3 Part I. Introduction 1 Homologous Recombination System of Yeast There are many endogenous and exogenous factors (for example reactive oxygenspecies, 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 repair 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 exploited 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 fragments 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 shuttle 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

4 Promoter 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 Terminator Promoter Gene 1 Gene 3 Terminator 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 together 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

5 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 advantages of the gap repair method is to avoid this. Moreover it leaves no scars between the assembled fragments like restriction digest and religation does. Another 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 reasons 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 standard 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 unimportant 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 fragments 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. Any yeast expression plasmid has a similiar ordered insert region. Depending on the number of genes on the nal plasmid the insert region consists of several repeats of the general scheme promoter-gene-terminator. Gap repair suitable DNA can simply be created by choosing promoters and terminators of yeast and the desired genes from the parts registry. Now every gen has to be amplied with YBA standard primers which contain homologue DNA overlaps to the end of a chosen promoter and the beginning of a proper terminator. With such PCR products it is possible to do the homologue recombination in yeast like it is shown in picture two. Since any Biobrick (from the same standard) can be amplied with the same primers it is sucient to construct one standard primer for every promoter, overlapping with the end of the promoter, and one for every terminator, overlapping with the beginning of the terminator. We designed one standard primer compartible to BioBrick RFC 10 for any of the yeast promoters and terminators we used in our project. Of course the annealing sequence of every standard primer can be adapted to all other BioBrick RFCs. 5

6 The connecting terminator-promoter fragments between the BioBrick devices shall not have BioBrick prex or sux at their ends because this could lead to incorrect assembly of targeting vector and reduces the eciency of gap repair cloning. After all there are two ways of connecting the terminator of one gene with the promoter of the following gene. The rst one is to design specic combinations of ligated terminator-promoter parts (shown in picture2). At the vector assembly you just have to transform the linearized plasmid, the amplied genes with homologue overlaps and the terminator-promoter parts to get the targeting vector. The advantage is that you need a lower number of DNA fragments which increases the eciency of gap repair cloning. We designed a few of this terminator-promoter parts for the registry. The disadvantages are rst that you are not able to choose all possible combinations because they are too many. Secondly you have to amplify the parts out of the BioBrick plasmid anyway. Therefore you must use a forward primer annealing behind the prex and a reverse primer anneling in front of the sux. This leads to the second way. The second way allows much more possibilities of combining terminators and promoters (picture3). Here you can choose BioBricks of single terminators and promoters from the parts registry. First you amplify the terminator with two specic primers annealing behind the prex and previous to the sux. The next step is amplication of a suitable promoter which comes after this terminator. There you take one primer annealing at the prex. This primer has an additional homologue overlap to the previous terminator to connect both at gap repair cloning. The second primer is annealing in front of the sux. In the end you transform all amplied genes, promoters and terminators and the linearized plasmid into yeast cells to get the targeting plasmid. Whenever you want to assemble a high number of BioBrick devices in a specic order to a targeting vector you can use standardized YBA methode for an easy, eective and cheap assembly. 6

7 Primer Prefix Promoter Primer Suffix Terminator Promoter Primer Suffix Suffix Homologous T Homologous P Primer Prefix Prefix Suffix Prefix Gene 1 Gene 2 Prefix Prefix Suffix Suffix Homologous P Terminator Homologous T Plasmid* Uracil Gene *not all attributes are shown Picture2: Example of BioBrick Assembly via gap repair cloning with YBA standard. First primers are assembled to the respective gene via PCR. The primer overlap to sux or prex is about 20 bp. Now the assembled genes have homologous overlaps to the respective promoters and terminators of about 40 bp length. In a yeast transformation the shue plasmid, a ligated terminator-promoter part and the assembled genes are put into the yeast cells. They put all parts together via homologous recombination to form the complete vector. Primer 2 Primer 2 Prefix Suffix Terminator Promoter Suffix Prefix Primer 1 Homologue Terminator Primer 1 Prefix Picture3: Variable assembly of terminator-promoter parts via gap repair cloning. 7

8 Part III. Assembly of Yeast Vector 4 Assembly of a Yeast Expression Vector The targeting yeast expression vector consists of a shue plasmid (containing selection markers for yeast and E.coli, one yeast promoter, one yeast terminator) and a specic number of genes, each possessing one promoter and one terminator (example at picture2). To assemble the nal vector we use YBA method. First you have to choose a suitable shue plasmid for your project. After that you need one specic restriction enzyme to linearize the shue plasmid. It has to cut specically at the MCS (Multiple Cloning Site). Now you have to look for BioBrick devices of the genes you want to express in the parts registry and arrange them in the desired order. Afterwards choose an appropriate number of yeast promoters an terminators for every gene. Fit the respective promoters and terminators between the genes. Pay attention that you already have one promoter and terminator on your shue plasmid. At the end the insert region of your vector construct should consist of suitable numbers of the general scheme promoter-gene-terminator. You can decide if you want to have BioBricks of specic combinations of already ligated terminator-promoter parts which you can transform into the yeast cell as one fragment or if you want to amplify your own combination of terminator-promoter parts. Than you have to take single terminator and promoter BioBricks. (cf. Checklist Vector) To use YBA method you have to amplify the genes, promoters and terminators with suitable primers. First you have to look for the right BioBrick standard of your primers. The most common BioBrick assembly standard which is used by most igem teams and most BioBricks in the parts registry is BBF RFC 10. The BioBrick standard of your genes, promoters and terminators has to be suitable to your choosen primer standard. For every gene amplication you have to choose the appendant forward primer with a homologue overlap to the promoter in front of the gene and the suitable reverse primer with a homologue overlap to the terminator at the end of the gene. If you want to amplify your own combination of terminator-promoter parts you also have to look for the suitable primers to the respective promoters or terminators. The terminator has to be ampied with primers annealing behind the prex and in front of the sux of the BioBrick. For promoter ampication you need one primer annealing to the prex and having a homologue overlap to the suitable terminator which is in front of this promoter. The second primer has to anneal in front of the sux of this promoter. If you have choosen BioBricks of already ligated terminator-promoter parts you need to amplify them with primers annealing at the beginning of the terminator and the end of the promoter. This amplication is just to get the terminator-promoter part out of the BioBrick plasmid and to increase the amount of terminator-promoter fragments. After you have choosen the right primers (pay attention that you need two 8

9 primers (forward- and reverse-primer) for every fragment) for your fragments you have to amplify all of them (genes, promoters, terminators) in a PCR reaction. (cf. Checklist PCR) Then you transform all PCR products and the linearized plasmid into yeast cells and select the transformants on LB medium lacking of one specic substance which is your yeast selection marker on the shue plasmid. (cf. Checklist Yeast Transformation) With positive clones you are able to inoculate yeast cultures. After plasmid preparation you are able to transform the isolated targeting vector into E.coli to increase amount of it. You should control if your targeting vector possesses the complete insert via control restriction digestion and agarose gel electrophoresis Checklist Vector 1. Shue plasmid (stable in S.cerevisiae and E.coli) with yeast promoter and terminator 2. (x) Biobrick genes 3. (x-1) Promoters/Terminators (a) Single (x-1) promoter and (x-1) terminator BioBricks or (b) (x-1) BioBricks with already ligated terminator-promoter parts Pay attention on promoter-gene- Assemble all parts into the desired order. terminator scheme. 4.2 Checklist PCR 1. Gene Amplication (a) BioBrick of gene (b) fw-primer annealing at prex with homologue overlap to promoter of gene (fw_pg_aprexx_hpyyy) (c) rev-primer annealing at sux with homologue overlap to terminator of gene (rev_pg_asufxx_htyyy) 2. Promoter/Terminator Amplication (a) Single Promoter/Terminator BioBricks i. Promoter Amplication A. BioBrick of promoter (pyyy) 2 Please take solution of E.coli plasmid preparation because it possesses a higher amount of targeting vector. This is important for identication of the bands at agarose gel electrophoresis. 9

10 B. fw-primer annealing at prex with homologue overlap to terminator in front of promoter (fw_pp_aprexx_htzzz) C. rev-primer annealing at promoter in front of sux (rev_pp_apyyy) ii. Terminator Amplication A. BioBrick of terminator (tzzz) B. fw-primer annealing at terminator behind prex (fw_pt_atzzz) C. rev-primer annealing at terminator in front of sux (rev_pt_atzzz) (b) BioBricks of ligated Terminator-Promoter Parts i. BioBrick of ligated terminator-promoter part (tzzz-pyyy) ii. fw-primer annealing at terminator behind prex (fw_ptp_atzzz) iii. rev-primer annealing at promoter in front of sux (rev_ptp_apyyy) Pay attention that you have choosen the same primer standard as your BioBrick standard! 4.3 Checklist Yeast Transformation 1. Linearized shue plasmid 2. Amplied Genes which now have homologue overlaps to appendent promoters and terminators 3. Amplied Promoters/Terminators (a) Amplied terminators and amplied promoters which contain homologue overlap to appendent termintor (b) Amplied ligated terminator-promoter parts 5 Promoters and Terminators of Yeast 5.1 Promoter phxt7: ppfk1: 5' gag ctc gta gga aca att tcg ggc ccc tgc gtg ttc ttc tga ggt tca tct ttt aca ttt gct tct gct gga taa ttt tca gag gca aca agg aaa aat tag atg gca aaa agt cgt ctt tca agg aaa aat ccc cac cat ctt tcg aga tcc cct gta act tat tgg caa ctg aaa gaa tga aaa gga gga aaa tac aaa ata tac tag aac tga aaa aaa aaa agt ata aat aga gac gat ata tgc caa tac ttc aca atg ttc gaa tct att ctt cat ttg cag cta ttg taa aat aat aaa aca tca aga aca aac aag ctc aac ttg tct ttt cta aga aca aag aat aaa cac aaa aac aaa aag ttt ttt taa ttt taa tca aaa a 3' 5' gaa aaa tat aag gat gag aaa gtg aaa tcg gtt ttt ttt ttc cat tgt cgt cat caa cat gat ttt tta aat aaa taa ata cga ttt ttt att ttt ttt ccc ttc ttt gtt ttt gtt ttg ctt att ccc atc ttc att att aaa ttc ttc cgc tct taa taa agg agt ttt ttt att atc ttc ttg tgt aat cat cct ttt tct tta att ttc 10

11 ttc ctt ttc ttt ttc tct tta ctg gtt ttt tta ctt ctt tat tct caa cca tct aaa gaa tat tat tgc ttt cta cca ata aaa tct gtt aat tct att tgg att gtc gtc tac tca agt ctc gcc tag taa ata aac gat aaa caa att tga agt aag aat aac aat ata ggg aga gaa att ttt cta ttt tta att tcg aaa cag gta cca aaa aat cta agt tca ctt tag cac tat ttg gga aag ctt tta tat aaa aaa tct gaa aca aaa tca tat caa ag 3' ppgk1: 5' tg ttt gca aaa aga aca aaa ctg aaa aaa ccc aga cac gct cga ctt cct gtc ttc cta ttg att gca gct tcc aat ttc gtc aca caa caa ggt cct agc gac ggc tca cag gtt ttg taa caa gca atc gaa ggt tct gga atg gcg gga aag ggt tta gta cca cat gct atg atg ccc act gtg atc tcc aga gca aag ttc gtt cga tcg tac tgt tac tct ctc tct ttc aaa cag aat tgt ccg aat cgt gtg aca aca aca gcc tgt tct cac aca ctc ttt tct tct aac caa ggg ggt ggt tta gtt tag tag aac ctc gtg aaa ctt aca ttt aca tat ata taa act tgc ata aat tgg tca atg caa gaa ata cat att tgg tct ttt cta att cgt agt ttt tca agt tct tag atg ctt tct ttt tct ctt ttt tac aga tca tca agg aag taa tta tct act ttt tac aac aaa tat aaa aca 3' 5.2 Terminator thxt7: 5' ttt gcg aac act ttt att aat tca tga tca cgc tct aat ttg tgc att tga aat gta ctc taa ttc taa ttt tat att ttt aat gat atc ttg aaa agt aaa tac gtt ttt aat ata tac aaa ata ata cag ttt aat ttt caa gtt ttt gat cat ttg ttc tca gaa agt tga gtg gga cgg aga caa aga aac ttt aaa gag aaa tgc aaa gtg gga aga agt cag ttg ttt acc gac cgc act gtt att cac aaa tat tcc aat ttt gcc tgc aga ccc aca tct aca aat ttt ggt 3' tpfk2: tcyc1: 5' aa gaa aat gac ctt tta tta cac ttt cta tta tta atg tca att aat gtt aac cca tgt ttt tct ttt gtg tct ata att ctt ttt ttt tat ctc taa gct ttt gaa caa tga att ttt tgt tcc ttt ctt tta ata ata caa gta cta ccc cat gaa acc aat att atc atg cat ttt tat gaa tgt caa gaa taa aga tac tgt tat ttt ttg tgt ctt att ttt ttt ctc ttt gtt tat tta aac gtt ttc taa aat taa aac tta tgt ata ctg gaa tat gtg ata tag acg att t 3' 5' t cat gta att agt tat gtc acg ctt aca ttc acg ccc tcc ccc cac atc cgc tct aac cga aaa gga agg agt tag aca acc tga agt cta ggt ccc tat tta ttt ttt tat agt tat gtt agt att aag aac gtt att tat att tca aat ttt tct ttt ttt tct gta cag acg cgt gta cgc atg taa cat tat act gaa aac ctt gct tga gaa ggt ttt ggg acg ctc gaa ggc ttt aat ttg c 3' 5.3 Ligated Terminator-Promoter Parts thxt7-ppfk1: 5' ttt gcg aac act ttt att aat tca tga tca cgc tct aat ttg tgc att tga aat gta ctc taa ttc taa ttt tat att ttt aat gat atc ttg aaa agt aaa tac gtt ttt aat ata tac aaa ata ata cag ttt aat ttt caa gtt ttt gat cat ttg ttc tca gaa agt tga gtg gga cgg aga caa aga aac ttt aaa gag aaa tgc aaa gtg gga aga agt cag ttg ttt acc gac cgc act gtt att cac aaa 11

12 tat tcc aat ttt gcc tgc aga ccc aca tct aca aat ttt ggt gaa aaa tat aag gat gag aaa gtg aaa tcg gtt ttt ttt ttc cat tgt cgt cat caa cat gat ttt tta aat aaa taa ata cga ttt ttt att ttt ttt ccc ttc ttt gtt ttt gtt ttg ctt att ccc atc ttc att att aaa ttc ttc cgc tct taa taa agg agt ttt ttt att atc ttc ttg tgt aat cat cct ttt tct tta att ttc ttc ctt ttc ttt ttc tct tta ctg gtt ttt tta ctt ctt tat tct caa cca tct aaa gaa tat tat tgc ttt cta cca ata aaa tct gtt aat tct att tgg att gtc gtc tac tca agt ctc gcc tag taa ata aac gat aaa caa att tga agt aag aat aac aat ata ggg aga gaa att ttt cta ttt tta att tcg aaa cag gta cca aaa aat cta agt tca ctt tag cac tat ttg gga aag ctt tta tat aaa aaa tct gaa aca aaa tca tat caa ag 3' tpfk2-ppgk1: 5' aa gaa aat gac ctt tta tta cac ttt cta tta tta atg tca att aat gtt aac cca tgt ttt tct ttt gtg tct ata att ctt ttt ttt tat ctc taa gct ttt gaa caa tga att ttt tgt tcc ttt ctt tta ata ata caa gta cta ccc cat gaa acc aat att atc atg cat ttt tat gaa tgt caa gaa taa aga tac tgt tat ttt ttg tgt ctt att ttt ttt ctc ttt gtt tat tta aac gtt ttc taa aat taa aac tta tgt ata ctg gaa tat gtg ata tag acg att ttg ttt gca aaa aga aca aaa ctg aaa aaa ccc aga cac gct cga ctt cct gtc ttc cta ttg att gca gct tcc aat ttc gtc aca caa caa ggt cct agc gac ggc tca cag gtt ttg taa caa gca atc gaa ggt tct gga atg gcg gga aag ggt tta gta cca cat gct atg atg ccc act gtg atc tcc aga gca aag ttc gtt cga tcg tac tgt tac tct ctc tct ttc aaa cag aat tgt ccg aat cgt gtg aca aca aca gcc tgt tct cac aca ctc ttt tct tct aac caa ggg ggt ggt tta gtt tag tag aac ctc gtg aaa ctt aca ttt aca tat ata taa act tgc ata aat tgg tca atg caa gaa ata cat att tgg tct ttt cta att cgt agt ttt tca agt tct tag atg ctt tct ttt tct ctt ttt tac aga tca tca agg aag taa tta tct act ttt tac aac aaa tat aaa aca 3' 6 Standardized YBA Primers for Assembly of Yeast Expression Vectors All parts are named by a specic nomenclature to indentify the right parts for the respective amplication. The following legend shows how the parts are named. For every primer is said what kind of amplication is done with it, where it is annealing and what kind of homologue overlap it has. 3 3 Example: fw_pg_apre_hpxxx is the name of a forward primer for gene amplication annealing at the BioBrick prex (BioBrick standard RFC 10) having a homologue overlap to a specic promoter. 12

13 Table1 Legend for primer nomenclature Abbreviation Meaning P Primer PG Primer Gene Amplication PTP Primer Terminator-Promoter Part Amplication PP Primer Promoter Amplication PT Primter Terminator Amplication fw forward Primer rev reverse Primer p Promoter t Terminator Pre10 Prex BioBrick RFC 10 Suf10 Sux BioBrick RFC 10 a annealing at h homologue to 6.1 Design of YBA Standard Primers Of course it is possible to design new YBA standard primers for other promoters/terminators of yeast but also for E.coli promoters/terminators. Note that all of these primers must have a specic annealing temperatur in the eld of 58 ± 1 C to combine dierent primers for every possible PCR amplication. All primers for gene amplication must anneal at prex or sux of the BioBrick device. The annealing sequence must be compartible to at least BBF RFC 10 standard. Additionally it can be also compartible to other BBF RFC standards. The homologue overlap to the new promoters/terminators must be not less than 40 bp. Primers for amplication of singel promoters or terminators as well as for ligated terminator-promoter parts must anneal behind the prex or in front of the sux, except of the forward primer of single promoter parts which is intended to be the only one having an homologue overlap of 40 bp to previous terminators. The homologue overlap of this primer is essential for possibility of combining own terminator-promoter parts. Pay attention that you must design two primers (forward and reverse) for every PCR reaction. Each primer must have a free 3'-ending in direction of the DNA fragment so the DNA-Polymerase can synthesise the new strand in 5' to 3' direction. The consequence of this is that the forward primer must anneal to the 3'-5' singel strand DNA and the reverse primer must anneal to the 5'-3' single strand DNA of the template. The standardization of primers from YBA standard is only depending on the annealing sequence which must be homologue to the respective BioBrick standard prex or sux. The fact that it is insignicant what kind of homologue overlaps the primers have oers the option to choose also other fragments (not only promoters/terminators) incorporating between the BioBrick devices. For 13

14 assembly of such fragments the suitable homologue overlaps have to be designed onto the primers. 6.2 Primer Gene Amplication (with annealing temperature) fw_pg_apre10_hphxt7: 5' aag aat aaa cac aaa aac aaa aag ttt ttt taa ttt taa tca aaa aga att cgc ggc cgc ttc tag 3' (58.6 C) fw_pg_apre10_hppfk1: 5' tgg gaa agc ttt tat ata aaa aat ctg aaa caa aat cat atc aaa gga att cgc ggc cgc ttc tag 3' (58.6 C) fw_pg_apre10_hppgk1: 5' atc atc aag gaa gta att atc tac ttt tta caa caa ata taa aac aga att cgc ggc cgc ttc tag 3' (58.6 C) rev_pg_asuf10_hthxt7: 3' atg atc atc gcc ggc gac gtc aaa cgc ttg tga aaa taa tta agt act agt gcg aga tta aac acg 5' (57.2 C) rev_pg_asuf10_htpfk2: 3' atg atc atc gcc ggc gac gtc ttc ttt tac tgg aaa ata atg tga aag ata ata att aca gtt aat 5' (57.2 C) rev_pg_asuf10_htcyc1: 3' atg atc atc gcc ggc gac gtc agt aca tta atc aat aca gtg cga atg taa gtg cgg gag ggg ggt 5' (57.2 C) 6.3 Primer Promoter Amplication (with annealing temperature) fw_pp_apre10_hthxt7: 5' aa ata ttc caa ttt tgc ctg cag acc cac atc tac aaa ttt tgg tga att cgc ggc cgc ttc tag 3' (58.6 C) fw_pp_apre10_htpfk2: 5' aa aat taa aac tta tgt ata ctg gaa tat gtg ata tag acg att tga att cgc ggc cgc ttc tag 3' (58.6 C) fw_pp_apre10_htcyc1: 5' ac ctt gct tga gaa ggt ttt ggg acg ctc gaa ggc ttt aat ttg cga att cgc ggc cgc ttc tag 3' (58.6 C) rev_pp_aphxt7: 3' gtt ttt gtt ttt caa aaa aat taa aat tag ttt tt 5' (58.7 C) rev_pp_appfk1: 3' gaa aat ata ttt ttt aga ctt tgt ttt agt ata gtt tc 5' (58.7 C) rev_pp_appgk1: 3' gt tcc ttc att aat aga tga aaa atg ttg ttt ata ttt tgt 5' (59.9 C) 14

15 6.4 Primer Terminator Amplication (with annealing temperature) fw_pt_athxt7: 5' ttt gcg aac act ttt att aat tca tga tca c 3' (59.9 C) fw_pt_atpfk2: 5' aa gaa aat gac ctt tta tta cac ttt cta tta tta atg 3' (57.9 C) fw_pt_atcyc1: 5' tca tgt aat tag tta tgt cac gct tac att cac 3' (59.3 C) rev_pt_athxt7: 3' cg tct ggg tgt aga tgt tta aaa cca 5' (58.4 C) rev_pt_atpfk2: 3' gaa tac ata tga cct tat aca cta tat ctg cta aa 5' (56.5 C) rev_pt_atcyc1: 3' ctg cga gct tcc gaa att aaa cg 5' (58.3 C) 6.5 Primer Terminator-Promoter Part Amplication (with annealing temperature) fw_ptp_athxt7: 5' ttt gcg aac act ttt att aat tca tga tca c 3' (59.9 C) fw_ptp_atpfk2: 5' aa gaa aat gac ctt tta tta cac ttt cta tta tta atg 3' (57.9 C) fw_ptp_atcyc1: 5' tca tgt aat tag tta tgt cac gct tac att cac 3' (59.3 C) rev_ptp_aphxt7: 3' gtt ttt gtt ttt caa aaa aat taa aat tag ttt tt 5' (58.7 C) rev_ptp_appfk1: 3' gaa aat ata ttt ttt aga ctt tgt ttt agt ata gtt tc 5' (58.7 C) rev_ptp_appgk1: 3' gt tcc ttc att aat aga tga aaa atg ttg ttt ata ttt tgt 5' (59.9 C) 7 Methods 7.1 General procedure of a transformation via gap repair 1. Amplication of the required DNA fragment with homologue ends to the gene/promoter/terminator/plasmid beside this DNA fragment via PCR and an agarose gel to review the correct size 2. Linearization of the shue plasmid with one specic restriction enzyme and an agarose gel for review 3. Yeast transformation with the PCR products and the linear plasmid (homologue recombination). Every time negative control with water and maybe positive control with circular plasmid. 4. Selection on an agar plate without the metabolite, which is on the plasmid for yeast selection (e.g. uracil or histidin) 15

16 5. Inoculation of clones and isolation of the plasmids from yeast (plasmid preparation) 6. Transformation of a plasmid in E.coli and selection on LB medium with antibiotic (the shue plasmids also have antibiotic resistance) 7. Isolation of the plasmids from E.coli (plasmid preparation) 8. Diagnostic restriction of the plasmid in order to nd the correct plasmid with all inserts 9. Retransformation of the correct plasmid in E.coli to make a permanent culture 10. Transformation of the correct plasmid in yeast to do further experiments 7.2 Polymerase Chain Reaction (PCR) Table2 PCR protocol Components Final Concentration Volume of Example PCR Polymerase-buer (e.g. for 1x 10 µl (5x) Phusion Polymerase) dntps 200 µm 5 µl (2 mm) Primer fw/rev respectively 0.2 µm respectively 1 µl (10 µm) Template 0.2 ng/µl 1 µl (10 ng/µl) DNA-Polymerase (e.g. Phusion 0.02 U/µl (mostly µl) 0.5 µl (2 U/µl) Polymerase) H 2 O add to 50 µl solution 31 µl MgCl 2 10 mm 0.5 µl (1 M) Total Volume 50 µl 50 µl Add all components with the right concentrations to a PCR tube. Pay attention to defrost phusion-polymerase on ice and add it at the very end. Put the PCR tube, containing a 50 µl reaction mixture, into a PCR cycler. Make the following settings for running the PCR programm: Table3 PCR programm for one PCR run Steps Temperature Time First Denaturation 98 C 30 sec Denaturation 98 C 10 sec Annealing Depending on Primer sec Polymerization 72 C 1 min/kb for Taq-Polymerase; 20 sec/kbp for Phusion Polymerase Final Polymerization 72 C 5 min Let the PCR-Programm run for about 35 cycles. 16

17 7.3 Restriction Digestion for Linearization of a Plasmid The choosen shue plasmid has to be digested with one suitable restriction enzym at the multiple cloning site (MCS). Table4 Example for digestion reaction Component Concentration Shue Plasmid 1 µl (3-5 µg/µl) Restriction Enzyme respectively 1 µl Suitable Buer 5 µl (10x) Water add to 50 µl Total Volume 50 µl Incubate the digestion mix 2 hours at 37 C. After that purify the solution with e.g. with a PCR purication kit. 7.4 Control Restriction Digestion of Plasmid with Insert Table5 Example for control digestion reaction Component Concentration Plasmid with Insert ng Restriction Enzyme respectively 1 µl Suitable Buer 1 µl (10x) H 2 O add to 50 µl Total Volume 50 µl Incubate the digestion mix 2 hours at 37 C. After that seperate the dierent fragments in an agarose gel electrophoresis and look for the right bands. 7.5 Yeast Transformation Production of competent cells 1. Inoculate the synthetic complete medium (SC) with the yeast strain 2. Incubate with shaking overnight at 30 C 3. Harvest the cells at a OD600 0,5-0,6 by centrifugation (3000x g, 5 min, RT) 4. Wash the cells with 0,5 vol of sterile water (resuspend by shaking, centrifugate with 3000x g, 5 min, RT) 5. Resuspend the cells in 0,01 vol of sterile water, transfer the suspension to a reaction tube and pellet the cells (3000x g, 5 min, RT) 6. Resuspend the pellet in 0,01 vol of sterile ltrated FCC (frozen competent cell) solution 17

18 7. Aliquot 50 µl of the solution into the reaction tubes 8. Store the cells at -80 C for at least one night (up to one year) Transformation 1. Mastermix for the FCC transformation mixture: Table6 FCC mastermix Substance Volume PEG 3350 (50% (w/v)) 260 LiAcetat 1.0 M 36 single-stranded carrier DNA (10 mg/ml) 10 Total Volume Prepare DNA aliquots: Solute enough DNA (e.g. determine 500 ng plasmid (5000 pb) and add equimolar amounts of every fragment) in 54 µl of water 3. Unfreeze the cells in a 37 C block for sec 4. Centrifuge the solution at 13000x g for 2 minutes 5. Remove the supernatant 6. Add 306 µl of FCC transformation mixture to the cells 7. Add 54 µl of the DNA to the solution and vortex shortly 8. When all the reaction tubes are prepared, vortex the samples well until all pellets are completely resoluted 9. Incubate the samples for 40 minutes at 42 C in a heating block 10. Centrifuge the cells at 13000x g for 30 sec and pour o the supernatant 11. Resuspend the cells in sterile water by vortexing 12. Spread onto the appropriate selection medium 13. Let the cells grow at 30 C 7.6 E.coli Transformation Production of competent cells 1. Inoculate 400 ml LB medium (pre-warmed to 37 C) in a 1 l ask with 100 µl of a fresh over night culture 2. Harvest the cells at a OD600 0,60-0,65 (in a room with 37 C, sterile) 18

19 3. Aliquot the solution in 8 x 50 ml falcons (sterile, pre-cooled on ice) 4. Incubate for 30 min on ice 5. Centrifuge for 12,5 min with 4000x g, 4 C 6. Resuspend the pellets in each 10 ml sterile water (4 C) 7. Centrifuge for 10 min with 4000x g, 4 C 8. Repeat steps 6 and 7 two times 9. Resuspend the cells in 10 ml 10% glycerine (+millipor, sterile, 4 C) 10. Centrifuge for 15 min with 4000x g, 4 C 11. Add 800 µl of 10% glycerine (sterile, 4 C) to the cells 12. Aliquot 40 µl of the solution in each reaction tube (pre-cooled) and freeze them in liquid nitrogen 13. Store them up to 6 months at -70 C Transformation (on ice) 1. Add DNA (e.g. 1 µl of extracted yeast plasmids) to the frozen cells 2. Unfreeze the cells on ice 3. Fill the cells into cuvettes for electroporation 4. Electroporation (for 2 mm cuvettes: voltage I = 2,5 kv; resistance R = 2000 ohm; electrical current A = 25 µf; for 1 mm cuvettes: I = 2,2 kv; resistance R = 2000 ohm; electrical current A = 25 µf) 5. Fill the cells with 1 ml of SOC medium back in a reaction tube immediately 6. Shake them for 1 hour at 37 C in a heating block 7. Centrifuge for 1 min 8000x g 8. Decant the supernatant 9. Spread onto the appropriate selection medium Part IV. Assembly of E.coli Vectors YBA method can also be used for assembly of E.coli vectors. Therefore you need a special shue plasmid which has an E.coli promoter and terminator next to the MCS (multiple cloning site) and is also stable in yeast. After that you can design specic primers annealing at the prex and sux of your BioBrick device 19

20 to amplify homologue overlaps to promoters and terminators like it is shown above for yeast expression vectors. Even though E.coli as a bacteria possesses polycistronic mrna 4 you have the possibility to regulate every gene by it's own if you assemble additional promoters and terminators between the genes. But you are also able to incorporate other parts, for example other regulatory fragments, between the genes by designing suitable homologue overlaps to these fragments at the primers. Another possibility is to create primers for one BioBrick device with pre- x/sux annealing sequence and an additional homologue overlap to the coding sequence of a successive BioBrick device. It is important that every second BioBrick device of the insert region must be amplied with specic primers in such a way that only the coding sequence remains. So you can assemble a vector with only one promoter and terminator and a few aligned BioBricks as insert. 5 After amplifying all fragments in an PCR reaction you transform them into yeast cells. Then you just have to isolate the complete targeting plasmid from yeast, transform it into E.coli and check after another plasmid preparation and a control restriction digestion if it possesses the complete insert. Up to this point all mentioned primers are designed as standard parts of YBA standard annealing at prex or sux of a BioBrick device. More interesting for E.coli vector assembly are the possiblities you have if you are independent of any standard. Then gap repair cloning and homologue recombination system of yeast gets a usefull tool for creating new biobrick devices by combining parts. For example you have the possibility to amplify a RBS (ribosom binding site) to a BioBrick device containing a pure gene. Therefore you have to design a suitable primer paar of respectively app. 20 bp length which must anneale behind of the prex and in front of the sux to amplify the pure gene without any BioBrick endings. After that you creat a second primer pair. The forward primer has to anneal at the beginning of the gene having an overlap containing the Shine-Dalgarno-Sequenz/RBS and maybe additionally an homologue overlap to a previous part (e.g. another gene). It is important that you have the pure gene because RBS must be app. 6-7 bp in front of the beginning of the gene. The reverse primer must anneal at the end of the gene and could also have a homologue overlap to a following part. With homologue recombination you can assemble all of your amplied fragments to a completely new BioBrick device by only one yeast transformation. Moreover at all times you want to assemble a higher number of fragments ecient, fast and with the lowest possible costs homologue recombination in yeast is the better methode compared to restriction digestion and ligation. 4 E.coli only needs one promoter and one terminator to express more than one gene. 5 Compare to picture2: terminator-promoter part has to be replaced by coding sequence of a BioBrick device. 20

21 Part V. Acknowledgements We gratefully acknowledge our instructor Prof. Eckhard Boles who inducted us in the methode of gap repair cloning and also provides the sequences of our mentioned yeast promoters and terminators. 21