University of Ottawa iGEM team

Figure 2. Schematic of PCR-mediated homologous recombination using the ADE4 targeting vector.

References•Guido, N. et al. A bottom-up approach to gene regulation. Nature 439, 856-860, doi:10.1038/nature04473 (2006). •Hua, S.-B., Qiu, M., Chan, E., Zhu, L. & Luo, Y. Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast. Plasmid 38, 91-96 (1997). •Gray, M. & Honigberg, S. M. Effect of chromosomal locus, GC content and length of homology on PCR-mediated targeted gene replacement in Saccharomyces. Nucleic Acids Research 29, 5156-5162 (2001). •Sikorski, R. S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27 (1989). •Ugolini, S. & Bruschi, C. V. The red/white colony color assay in the yeast Saccharomyces cerevisiae: Epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells. Curr Genet 30, 485-492 (1996). •Phillips, I. & Silver, P. A new biobrick assembly strategy designed for facile protein engineering. dspace.mit.edu (2006). •Ellis et al. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nat Biotechnol (2009) vol. 27 (5) pp. 465-71 •Ruohonen et al. Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins. Journal of Biotechnology (1995) vol. 39 (3) pp. 193-203

Materials and Methods• YPH500 yeast strain containing a deletion in the ADE2 gene was used

 

A B C D

Figure 4. YPH500 yeast transformed with ADE4 targeting vectors. A) ADE4 targeting vector containing the natMX6 resistance marker. The plate

contains YPD media with 100 ug/mL Nourseothricin. The cells transformed with a Nourseothricin-resistant ADE4 targeting vector gave only white colonies.

B) Wild type YPH500 cells plated on YPD media with 100 ug/mL Nourseothricin. Wild type cells in media with Nourseothricin gave no colonies.

C) ADE4 targeting vector containing the kanMX resistance marker. The plate contains YPD media with 0.2 mg/mL G418. Areas of high cell density appear red presumably due to cells growing on top of each other. Areas of lower cell density are white

D) Wild type YPH500 cells plated on YPD media with 0.2 mg/mL G418. Wild type cells plated on media with kanamycin gave no colonies.

Project I New Yeast Selection Markers and

ADE4 Targeting Vector

Project IICharacterization of the ADH1 Promoter

Project IIIProximal-Distal Promoter Distinction

Project IIICharacterization of ADH1 Promoter

Introduction

• Auxotrophic selection markers use deletions and mutations in genes in a pathway that generate an essential metabolite.There are two major drawbacks :

1) Small background colonies without the integrated construct often appear

2) The selection marker might recombine with the wild type open reading frame due to their substantial homology, and cause a yeast colony without the construct to survive on the selection media

• Drug selection markers are much stronger and have less background. We BioBricked natMX6 and kanMX, which give resistance to nourseothricin sulfate and G418 (kanamycin) respectively. They are completely heterologous and do not share any homology with the yeast genome, and therefore are less likely to recombine into an incorrect locus

• Existing yeast integrating vectors do not comply by the BioBrick standards. Moreover, they have short homologous sequences and require special yeast deletion strains to function. Also, most of them do not provide colorimetric validation of successful integration.

• The yeast ADE4 targeting vector we constructed (BBa_K319043) is able to integrate into the ADE4 locus by homologous recombination using the flanking ATM1 and DYN3 sequences and knocking out the ADE4 open reading frame with high specificity

• In an ADE2-mutant strain, 5-amino imidazole ribotide (AIR) gets converted into a red pigment. When successfully integrated, the ADE4 targeting vector knocks out the ADE4 gene, undercuts the adenine biosynthesis pathway and therefore restores the white colony phenotype (Ugolini, 1996). The colour indication eliminates the need for PCR validation

Introduction

• Although there are several versions of the alcohol dehydrogenase (ADH1) promoter already available in the registry, they are longer than needed for optimal expression from the promoter. Furthermore, they are inhibited by ethanol, which is used as a source of energy during part of the yeast life cycle (Ruohonen et al.)

• We made an improved version of the ADH promoter in yeast with a more manageable length for the construction of larger networks, and also without ethanol inhibition

Material and Methods

• ADH1 promoter was cloned in front of the yeast enhanced green fluorescence protein (yEGFP) sequence with an ADH1 terminator. This construct was put into a pRS403 vector and integrated into the YPH500 genome

• The resulting strain was inoculated in yeast peptone dextrose (YPD) medium with 2% glucose and 2% adenine overnight. The overnight culture was reinoculated into the same media at an optical density of 0.02

• After 3 hours, the strain was analyzed on a Cyan ADP flow cytometer using a 488 laser for excitation. Emission was captured with a standard FITCH filter set

Results

Figure 6. Left: Fluorescence of Wild Type YPH500. Right: Fluorescence of ADH1 promoter driving yEGFP expression in a YPH500 background.

• An 100 fold increase in GFP expression was observed in YPH500 yeast transformed with the improved promoter expression than in the wild YPH500

Discussion

•Figure 6 shows that the improved ADH1 promoter is able to increase the yEGFP gene expression by 100 fold. The improved promoter is able to overcome the problem of ethanol inhibition, and the length is also more manageable

Future Directions• The proximal-distal shuffling: Testing combination's of the 24 proximal promoters we submitted to the registry with the distal promoter elements we have submitted and plan on submitting

• Characterizing the TEF1, TEF2 and MYO2 full-length promoters submitted

• Double (two parts at once) and triple recombination (three parts at once) with the ADE4 Targeting Vector - so far we have only managed to integrate one part at a time using the Ade4 Targeting Vector successfully (the Kan and Nat integrations)

•Characterizing the LacI and TetR repressors

Figure 3. Vector Maps of the Kanamycin-resistant and Nourseothricin-resistantADE4 targeting vectors

Results

Figure 1. Adenine biosynthesis pathway in yeast

Figure 5. A transformation of a construct of interest with URA3 selection into the ADE2 locus of BY4742 using 40 bp overhangs. There are 21 red colonies and 64 white colonies.

Discussion

• The ADE4 targeting vector has an 100% specificity indicated by 100% white colonies in Figure 4, in contrast with a 25% specificity for a typical transformation of an ADE2-targeting vector as shown by the percentage of red colonies in Figure 5. The ADE4 targeting vector has a much higher specificity and avoids the need for PCR-screening, which is usually used for loci that do not allow for colour selections

Project II Proximal-distal Promoter Shuffling

• Many interesting wild type eukaryotic promoters in nature respond to a particular signal, and they are often used in biosensors. Although they respond correctly to a signal, they may not produce the desired output• We hypothesize that it would be possible to shuffle around distal and proximal components from various promoters. By adding the correct proximal promoter, it should be possible to tune the desired output• We proceeded to BioBrick Act 1 distal promoter and combine it with the proximal elements from a library of 40 variants of the inducible Gal1 promoter that contain operator sites for the binding of TetR and LacI repressors (generously donated by Ellis et al.)• We were not able to finish this project; however we made all the BioBricks available to the registry