Investigation Title	Transcription profiling of yeast mutant at fast growth rates	
Comment[Submitted Name]	S. cerevisiae Mutant Competition at Fast Growthrates	
Experimental Design	ex_vivo_design	time_series_design	co-expression_design	transcription profiling by array	
Experimental Design Term Source REF		The MGED Ontology	The MGED Ontology	EFO	
Comment[AEMIAMESCORE]	4	
Comment[ArrayExpressReleaseDate]	2010-12-01	
Comment[SecondaryAccession]		
Comment[ArrayExpressAccession]	E-TABM-892	
Comment[MAGETAB TimeStamp_Version]	2010-10-14 14:48:46 Last Changed Rev: 14677	
Experimental Factor Name	DevelopmentalStage	
Experimental Factor Type	developmental_stage	
Experimental Factor Term Source REF	
Person Last Name	Pir	
Person First Name	Pinar	
Person Mid Initials	
Person Email	pp305@cam.ac.uk	
Person Phone	
Person Fax	
Person Address	Cambridge Systems Biology Department of Biochemistry 80 Tennis Court Road CB2 1GA Cambridge	
Person Affiliation	University of Cambridge	
Person Roles	submitter	
Person Roles Term Source REF	The MGED Ontology	
Quality Control Type	
Quality Control Term Source REF	
Replicate Type	
Replicate Term Source REF	
Normalization Type	
Normalization Term Source REF	
Date of Experiment	
Public Release Date	2010-12-01	
PubMed ID	
Publication DOI	
Publication Author List	Pir et al.	
Publication Title	Controls and Barriers to Rapid Growth in Yeast	
Publication Status	
Publication Status Term Source REF	
Experiment Description	Our approach for the identification and quantification of growth control by the components of yeast cells relies on two complementary screens: In the first, growth rate is changed and the effect on the concentration of cell components is measured (Castrillo et al. 2007, Genome Biology, 6,4). In the second, the concentration of the components is changed (by reducing gene copy number) and the effect on growth rate is measured (Delneri et al., 2008, Nat Gen, 40, 113). The heterozygous deletant (hemizygote) library we use is composed of diploid mutants each lacking one copy of a different gene (Giaever et al., 2002, Nature, 418, 387). When such a pool of mutants is grown in competition, some will grow slower than the others due to their particular mutation. Over time, the proportion of these mutants in the population will decrease – a phenotype termed `haploinsufficient’. Conversely, some mutants will have a growth-rate advantage and so will increase their proportion in the population; we call these strains `haploproficient’. Screening a pool of hemizygotes for their haploinsufficiency (HI)/haploproficiency (HP) phenotypes thus identifies cellular components that have high flux control (HFC) over growth rate. We found that genes that are major controllers of growth rate under nutrient-limitation are not, themselves, subject to growth rate control of their transcription. In this study we have investigated the generality of this rule, first by identifying HFC genes in turbidostat culture, where biomass concentration sets the rate of nutrient addition, growing at the maximum rate (mmax=0.32h-1) permitted by the complex defined medium (FPM) employed. And to study growth-rate effects, competition experiments were performed at higher growth rates (D=0.2h-1 and 0.3h-1) in nitrogen-limited chemostats and, to control for the effect of nitrogen limitation, in FPM using a chemostat at D=0.3h-1 (Pir et al., manuscript in preparation)	
Protocol Name	P-TABM-4632	P-TABM-4630	P-TABM-4629	P-TABM-4631	P-TABM-4628	P-TABM-4633	P-TABM-4635	
Protocol Type	grow	grow	grow	grow	nucleic_acid_extraction	labeling	bioassay_data_transformation	
Protocol Description	1ml aliquot of heterozygous deletion pool was grown in 100ml of YPD for 24 hours in an orbital shaker kept at 30oC and 180rpm. Ten ml of this preculture was used to inoculate 1L of nitrogen-limited F1 medium in the sterilized fermentor vessels. The cultures were first grown in batch in fermentors kept at 30oC with 1L/min aeration and 750rpm stirring for 24 hours. During batch culture, no pH control was made and within 24 hours the pH dropped from 4.5 to approximately 3. After 24 hours, the fermentors were to continuous mode at dilution rate 0.3h-1 which corresponds to 300 ml fresh media input per hour. Turbidity and pH control was also started at this point, target turbidity was set to approximately half of the maximum reading from the batch cultivation, which corresponds to concentration at mid-exponential phase, pH was kept at 4.5 until the end of the fermentation. The maximum growth rate reached in FPM turbidostats was approximately 0.32h-1 and cultures were sampled at 0, 11.0, 22.2, 33.2 generations. Generation count had started at switch to continuous mode. Number of generations were calculated using the doubling time formula (ln(2)/D). FPM: Yeast Nitrogen Base (1.7g/l), ammonium sulfate (5g/l), arginine (0.174g/l), aspartate (0.171g/l), glutamate (0.169g/l), histidine (0.155g/l), leucine (0.131g/l), lysine(0.183g/l), methionine (0.149g/l), serine (0.105g/l), threonine (0.119g/l), tryptophan (0.204g/l), valine (0.117g/l), citrate (0.210g/l), fumarate (0.160g/l), malate (0.134g/l), pyruvate(0.110g/l), succinate (0.270g/l), cytosine (0.111g/l), uracil (0.112g/l), glucose (20g/l)	1ml aliquot of heterozygous deletion pool was grown in 100ml of YPD for 24 hours in an orbital shaker kept at 30oC and 180rpm. Ten ml of this preculture was used to inoculate 1L of nitrogen-limited F1 medium in the sterilized fermentor vessels. The cultures were first grown in batch in fermentors kept at 30oC with 1L/min aeration and 750rpm stirring for 24 hours. During batch culture, no pH control was made and within 24 hours the pH dropped from 4.5 to approximately 3. After 24 hours, the fermentors were to continuous mode at dilution rate 0.2h-1 which corresponds to 300 ml fresh media input per hour. pH control was also started at this point and kept at 4.5 until the end of the fermentation. Nitrogen-limited D=0.3h-1 experiments were sampled at 0, 10.4, 20.8, 31.1 generations, generation count started at switch to continuous mode. Number of generations were calculated using the doubling time formula (ln(2)/D). N-lim F1: KH2PO4 (2g/l), MgSO4·7H20 (0.55g/l), NaCl (0.1g/l), CaCl2·2H2O (0.09g/l), Uracil (0.02g/l), L-Histidine (0.02g/l), L-Leucine (0.1g/l), ZnSO4·7H2O (0.7 × 10-4g/l), CuSO4·5H2O (0.1 × 10-4g/l), H3BO3 (0.1 × 10-4g/l), KI (0.1 × 10-4g/l), FeCl3·6H2O (0.5 × 10-4g/l), myo-inositol (0.12g/l), thiamine/HCl (0.014g/l), pyridoxine (0.004g/l), Ca-pantothenate (0.004g/l), biotin (0.0003g/l), ammonium sulfate (0.46g/l) and glucose (20g/l)	1ml aliquot of heterozygous deletion pool was grown in 100ml of YPD for 24 hours in an orbital shaker kept at 30oC and 180rpm. Ten ml of this preculture was used to inoculate 1L of nitrogen-limited F1 medium in the sterilized fermentor vessels. The cultures were first grown in batch in fermentors kept at 30oC with 1L/min aeration and 750rpm stirring for 24 hours. During batch culture, no pH control was made and within 24 hours the pH dropped from 4.5 to approximately 3. After 24 hours, the fermentors were to continuous mode at dilution rate 0.2h-1 which corresponds to 200 ml fresh media input per hour. pH control was also started at this point and kept at 4.5 until the end of the fermentation. Nitrogen D=0.2h-1 experiments were sampled at 0, 7.6, 20.8 and 29.1 generations, generation count started at switch to continuous mode. Number of generations were calculated using the doubling time formula (ln(2)/D). N-lim F1: KH2PO4 (2g/l), MgSO4·7H20 (0.55g/l), NaCl (0.1g/l), CaCl2·2H2O (0.09g/l), Uracil (0.02g/l), L-Histidine (0.02g/l), L-Leucine (0.1g/l), ZnSO4·7H2O (0.7 × 10-4g/l), CuSO4·5H2O (0.1 × 10-4g/l), H3BO3 (0.1 × 10-4g/l), KI (0.1 × 10-4g/l), FeCl3·6H2O (0.5 × 10-4g/l), myo-inositol (0.12g/l), thiamine/HCl (0.014g/l), pyridoxine (0.004g/l), Ca-pantothenate (0.004g/l), biotin (0.0003g/l), ammonium sulfate (0.46g/l) and glucose (20g/l)	1ml aliquot of heterozygous deletion pool was grown in 100ml of YPD for 24 hours in an orbital shaker kept at 30oC and 180rpm. Ten ml of this preculture was used to inoculate 1L of nitrogen-limited F1 medium in the sterilized fermentor vessels. The cultures were first grown in batch in fermentors kept at 30oC with 1L/min aeration and 750rpm stirring for 24 hours. During batch culture, no pH control was made and within 24 hours the pH dropped from 4.5 to approximately 3. After 24 hours, the fermentors were to continuous mode at dilution rate 0.3h-1 which corresponds to 300 ml fresh media input per hour. pH control was also started at this point and kept at 4.5 until the end of the fermentation. FPM D=0.3h-1 experiments were sampled at 0, 10.4, 20.8, 31.1 generations, generation count had started at switch to continuous mode. Number of generations were calculated using the doubling time formula (ln(2)/D). FPM: Yeast Nitrogen Base (1.7g/l), ammonium sulfate (5g/l), arginine (0.174g/l), aspartate (0.171g/l), glutamate (0.169g/l), histidine (0.155g/l), leucine (0.131g/l), lysine(0.183g/l), methionine (0.149g/l), serine (0.105g/l), threonine (0.119g/l), tryptophan (0.204g/l), valine (0.117g/l), citrate (0.210g/l), fumarate (0.160g/l), malate (0.134g/l), pyruvate(0.110g/l), succinate (0.270g/l), cytosine (0.111g/l), uracil (0.112g/l), glucose (20g/l)	Approximately 1/10 of the cell pellets (from 20-40ml samples) were used for DNA isolation, using Promega Wizard Genomic DNA purification kit, according to the protocol provided together with the kit. Isolated DNA was visualized on 1% agarose gel and quantified via NanoDrop.	200ng of the DNA from each sample was used to prepare the PCR reaction aliquot, by adding 192 ?L of Invitrogen Platinum PCR Supermix and 100ng of up-tag or down-tag primers, and made up-to 200 ?L using water.  PCR reactions for amplifying up-tags and down-tags were carried out separately using the following biotinylated universal primers synthesized by MWG/Operon: B-U1           5'-GATGTCCACGAGGTCTCT-3' B-U2 comp 5'-GTCGACCTGCAGCGTACG-3' B-D1           5'-CGGTGTCGGTCTCGTAG-3' B-D2 comp 5'-CGAGCTCGAATTCATCG-3' In each case the 200 ?L PCR mix was divided into 50 ?L aliquots and amplified using the following PCR program: 95oC 5 min., (94oC 20 sec., 56oC 20 sec., 72oC 30 sec.) x 35 cycles, 72 oC 5min. 5 ?L of the 200 ?L re-combined PCR products were visualized on 2% agarose gel, purified and concentrated to 40 ?L using either Microcon YM (Cat No. 42408) or Amicron Ultracell-10K (Cat No. UFC501096) filters, by loading the product to the filters, adding 300?L of water and centrifuging for 15min at 13000g for three times and then eluting the product to a fresh eppendorf tube for 3min at 1000g. 1 ?L of the cleaned PCR product was visualized and quantified on 2% agarose gels with respect to PCR Marker (New England Biolabs, N3234L). 500ng of the up/down-tag PCR product was used to prepare the hybridization mixes. Hybridization, array washing and scanning was carried out as in Affymetrix protocols for custom DNA arrays. Tag3 microarrays (Affymetrix) were used for Nitrogen-limited D=0.2h-1 and the FPM turbidostat samples and Tag4 (Affymetrix, GenFlex 16K v2) for Nitrogen-limited and FPM D=0.3h-1. Genechip 640 hybridization oven, Genechip 450 fluidics wash-station and Genechip 7G scanner were used.	Linear modeling of the normalized Tag3 data for each mutant with respect to time was made as described in Delneri et al., 2008 (Delneri, D. et al. Identification and characterization of high-flux-control genes of yeast through competition analyses in continuous cultures. Nat Genet 40, 113-117 (2008)), growthrates of individual mutants and their functional analysis are given in Pir et al., 2010 (manuscript in preparation)	
Protocol Parameters	
Protocol Hardware	
Protocol Software	
Protocol Contact	
Protocol Term Source REF							The MGED Ontology	
SDRF File	E-TABM-892.sdrf.txt	
Term Source Name	EFO	The MGED Ontology	ArrayExpress	The MGED Ontology	EFO	
Term Source File	http://www.ebi.ac.uk/efo/	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/	
Term Source Version