Team:TU-Delft/part3

Content

Introduction
Parts
Results
Conclusions
References

Introduction

Receptor+reporter

By combining the olfactory receptor and the FUS1pr-EGFP reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the FAR1 promoter is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. If the olfactory system will be implemented as a diagnostics tool in developing countries, the EGFP reporter should be changed by a visible reporter.

Growth arrest

Besides the induction of the FUS1 promoter the cells also go in growth arrest mediated by the FAR1 promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefor it is needed to knock out the FAR1 promoter.

Increasing sensitivity

To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. For this, making a knockout of the native GPA1 gene is needed in order to let the subunit work as substitute. In the results we describe how we generated a knockout in yeast. In future work the alpha subunit should be expressed and characterized.

Parts

The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:

BBa_K775005
BBa_K775006
BBa_K775007
BBa_K775008

Results

Knock out GPA1

Setup

• Yeast Transformation
  
• PCR
  
• DNA gel

Outcome

For making a functional knockout, yeast strains BY4741; Mat a; his3D1; leu2D0; met15D0; ura3D0; YJL157c::kanMX4 and BY4741; Mat a; his3D1; leu2D0; met15D0; ura3D0; YHR005c::kanMX4 were used (Euroscarf). Also knockout cassette pUG72 is used (Euroscarf). The LoxP-Ura-LoxP is elongated using the pFx polymerase protocol. The PCR program and primer sequences in table 1 yielded a product which is put on a gel shown in figure 1. Here a band can be recognized between 2000 and 1500 nucleotides, which corresponds to the 1669 nucleotide PCR product.

Table 1 PCR program for elongation of knockout cassette for knocking GPA1 and primers used for elongation.

Repeats	Temperature		Duration
5x	95	Melting	2:00
	51	Annealing	1:00
	68	Elongation	2:00
25x	95	Melting	2:00
	61	Annealing	1:00
	68	Elongation	2:00

GPA ko fw TTAGCATCACATCAATAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC

GPA ko rv TGCATCTTCGGAAACAGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG

GPA ko fw short TAATCCAGAGGTGTATAAATTGATATATTAAGGTAGGAAATAATGCAGCTGAAGCTTCGTACGC

GPA ko rv short AGAATTTACGTATCTAAACACTACTTTAATTATACAGTTCCTTCAGCATAGGCCACTAGTGGATCTG

Figure 1: 1% agarose in TAE ~45 run on 80 Volts. In the picture can be seen: 1 SmartLadder, 2 FAR1 short PCR product, 3 FAR1 long PCR product, 4 GPA1 short PCR product, 5 GPA1 long PCR product.

Yeast strain BY4741; Mat a; his3D1; leu2D0; met15D0; ura3D0; YJL157c::kanMX4 is transformed with the GPA1 ko PCR product, according to the yeast transformation protocol. 100 ng of DNA is used for transformation. After 3 days 3 colonies could be seen on the plate. There where zero colonies on the control plate.
To check whether the knock-out reaction is successful primers A and D bind to 5’ upstream and 3’ downstream regions of the GPA1 gene, the B and C primers anneal in the URA gene which is knocked in.
Cultures of colony 2 and wildtype yeast where grown in YPG (YP+2% glucose) overnight for chromosomal extraction using the MO BIO chromosomal extraction kit (protocol). A PCR reaction was performed on extracted genomic DNA of the grown strains, using an annealing gradient of 45, 55 and 65 C and 15 ng or 30 ng template DNA. Reactions AD, AB and CD where performed according to the conditions summarized in table 11. PCR product is put on gel and shown in figure 5. Here can be seen that colony 2 (B) shows both expected band length in the AB and CD reactions, but no bands in the AD reaction.

Table 11 Taq PCR conditions for KO check.

Repeats	Temperature		Duration
	94	Melting	4:00
35x	94	Melting	0:30
	45/55/65	Annealing	0:40
	72	Elongation	2:15
	72	Elongation	10:00

GPA1 A conf CGTCCTTCTGCGTATTCTTCC

GPA1 D conf CCGAGTATTTACCAGGGAGAAG

KO B CGCCAAGGGTAGAGATCCTAAG

KO C CTTCACGCAGGATGACAGTTC

Figure 5: 1% agarose gel in TAE, 40 min run on 100 V. PCR products of Genomic extracted DNA. Colony 1 (A), Colony 2 (B), and Colony 3 (C) are shown. In the picture can be seen: 1 DNA Smarladder, 2 AD reaction with 15 ng template and 45 C, 3 AD reaction with 15 ng template and 55 C, 4 AD reaction with 15 ng template and 65 C, 5 AD reaction with 30 ng template and 45 C, 6 AD reaction with 30 ng template and 55 C, 7 AD reaction with 30 ng template and 65 C, 8 AB reaction with 15 ng template and 45 C, 9 AB reaction with 15 ng template and 55 C, 10 AB reaction with 15 ng template and 65 C, 11 AB reaction with 30 ng template and 45 C, 12 AB reaction with 30 ng template and 55 C, 13 AB reaction with 30 ng template and 65 C, 14 CD reaction with 15 ng template and 45 C, 15 CD reaction with 15 ng template and 55 C, 16 CD reaction with 15 ng template and 65 C, 17 CD reaction with 30 ng template and 45 C, 18 CD reaction with 30 ng template and 55 C, 19 CD reaction with 30 ng template and 65 C, 20 smartladder.

We concluded that in colony 2 GPA1 has successfully been knocked out. The following test is to transform FUS1-GFP into this double knockout deltafar1 deltagpa1 yeast and to see difference in fluorescence output when compared to deltafar1 yeast strain when induced by pheromone alpha. Expected is that due to lack of alpha subunit of the pheromone receptor, no or less light signal is detected. A full description of the process can be seen here.

Fluorometer experiment

Setup

With yeast strains transformed with the GPR109A receptor and output BBa_K775005 and the R17-ODR10 receptor and output BBa_K775008 a fluorometer experiment was performed. After addition of the ligands (+ control alpha pheromone) OD600 and fluorescence were measured in time.

Outcome

Until the Wiki-freeze no observation of response to both constructs has been observed. This experiment is to be optimized to be able to draw a definite conclusion.

Conclusions

Four olfactory system biobricks were added to the registry. Two of the biobricks were successfully cloned in yeast. To draw conclusive conclusions about the systems further experiments are needed.

References

[1] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)