Riding to work

This is a hard job, and it’s taking a toll on my brain. So I’m committing here and now to commuting to the lab by bike at least three days a week, as long as it’s not pouring rain, and temp is 40<t<100 degrees. And you can…


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Hold me accountable! Call me on my excuses! I need it!

More hyphal growth time lapse imaging

More capture of sample video time-lapses of some random bread mold (Rhizopus? Penicilium?) hyphae to use as sample images for…well, some stuff we want to do.  It’s surprising that software and imaging platforms to do what we want to do don’t seem to exist.  So I’m writing it myself…or trying, at least.

Training the new summer help…

The students just keep looking younger and younger the older I get. 

Measuring hyphal growth

Since my lab is shifting the focus of some of our fungal work to Candida albicans, one of the things we’d like to be able to do (in a cheap, reproducible, and automated fashion) is to measure the adherence and growth of fungal biofilms, and specifically to measure the linear rate of growth at the hyphal tip.  So to start, here is a time lapse I recorded of some bread-mold spores growing on the surface of a YPD agar plate.  Seems like a good enough mimic of germ-tube formation and hyphal growth of C. albicans

Yeast wild types…

There are a bunch of them, and here are the ones we use.  Just putting this up here so I have something to which I can link to keep this info handy:

 

SEY6210/SEY6211

Genotype: MATa/MATα leu2-3,112/leu2-3,112 ura3-52/ura3-52 his3-Δ200/his3-Δ200 trp1-Δ901/trp1-Δ901 ade2/ADE2 suc2-Δ9/suc2-Δ9 GAL/GAL LYS2/lys2-801

Notes: SEY6210/SEY6211, also known as SEY6210.5, was constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers, good growth properties and good sporulation.

References: Robinson et al. (1988) Mol Cell Biol 8(11):4936-48

Sources: ATCC:201392

SEY6210

Genotype: MATα leu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 suc2-Δ9 lys2-801; GAL

Notes: SEY6210 is a MATalpha haploid constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers and good growth properties.

References: Robinson et al. (1988) Mol Cell Biol 8(11):4936-48

Sources: ATCC:96099

Additional note: SEY6210 is the strain in which all or most of the lipids transport mutants from Voelker lab have been constructed or selected. This is the preferred strain for mitochondrial studies and for protein and lipid transport work

SEY6211

Genotype: MATleu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 ade2-101 suc2-Δ9; GAL

Notes: SEY6211 is a MATa haploid constructed by Scott Emr and has been used in studies of autophagy, protein sorting etc. It is the product of crossing with strains from 5 different labs (Gerry Fink, Ron Davis, David Botstein, Fred Sherman, Randy Schekman). It has several selectable markers and good growth properties.

References: Robinson et al. (1988) Mol Cell Biol 8(11):4936-48

Sources: ATCC:96100

W303

Genotype: MATa/MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} [phi+]

allele locus mutation (1) 
ade2-1 YOR128C nonsense, glu64STOP
trp1-1 YDR007W nonsense, glu83STOP
can1-100  YEL063C frameshift, lys47
leu2-3,112  YCL018W frameshift, gly83
his3-11,15  YOR202W 2x frameshifts, ala70 and glu106

Notes: The W303 genome is to 85.4% derived from S288c, part of the other regions are similar to non-S288c regions of Sigma1278b. In total, some 800 CDS differ between W303 and S288c, but in most cases only one or two residues differ [2]. These include a bud4 mutation that causes haploids to bud with a mixture of axial and bipolar budding patterns. In addition, the original W303 strain contains the rad5-535 allele. As S288c, W303 has an allelic variant of MIP1 which increases petite frequency.

The W303 genome was sequenced at the Sanger Institute and by Ralser M. et al. (2012) Open Biol 2: 120093. 1 (DDBJ/EMBL/GenBank ALAV00000000).
References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).
bud4 info: Original mutant description Voth et al. (2005) Eukaryotic Cell, 4:1018-28. Mutation: deletion of one of four Gs at positions 2456-2459 of BUD4 ORF. Seq data from: Ralser et al above
rad5-535 info: see detailed notes

Sources: ATCC:200060

W303-1A

Genotype: MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15}

Notes: W303-1A possesses a ybp1-1 mutation (I7L, F328V, K343E, N571D) which abolishes Ybp1p function, increasing sensitivity to oxidative stress.

References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).
ybp1-1 info: Veal et al. (2003) J. Biol. Chem. 278:30896-904.

Sources: ATCC:208352

W303-1B

Genotype: MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15}

References: W303 constructed by Rodney Rothstein (see detailed notes from RR and Stephan Bartsch).

Sources: ATCC:201238

BY4743

Genotype: MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0

Notes: Strain used in the systematic deletion project, generated from a cross between BY4741 and BY4742, which are derived from S288C. As in S288c, this strain as well as haploid derivatives BY4741, and BY4742 have allelic variants of MIP1SAL1 and CAT5 and these polymorphisms, described in the respective locus history notes for these genes (MIP1SAL1 and CAT5) all contribute to the high observed petite frequency. Details regarding the contributions of these variants to petite formation are referenced in Dimitrov et al. (2009) Genetics 183(1):365-83. See the Brachmann et al., 1998 reference for details of strain construction.

References: Brachmann et al. (1998) Yeast 14:115-32.

Sources: ATCC:201390

BY4742

Genotype: MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0

Note: This strain is gal-

BY4741

Genotype: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0

Note: This strain is gal-

Notes: S288C-derivative laboratory strains. BY4741 and BY4742 are part of a set of deletion strains derived from S288C in which commonly used selectable marker genes were deleted by design in order to minimize or eliminate homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. The yeast strains were all directly descended from FY2, which is itself a direct descendant of S288C. Variation between BY4741 and 4742 and S288C is miniscule. BY4741 and BY 4742 were used as a parent strains for the international systematic Saccharomyces cerevisiae gene disruption project.

 

References: Brachmann CB, et al. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14(2):115-32 PMID:9483801

Winston F, et al. (1995) Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11(1):53-5

 

 

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