A user-friendly and streamlined protocol for CRISPR/Cas9 genome editing in budding yeast

Novarina, Daniele; Koutsoumpa, Andriana; Milias-Argeitis, Andreas

doi:10.1016/j.xpro.2022.101358

Cited by 1 publication

(3 citation statements)

References 18 publications

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“…Following this reasoning, we constructed a hxk -null strain, in which the three isozymes of hexokinases (Hxk1, Hxk2, and Glk1) were deleted using CRISPR-Cas9 19 , 20 . To estimate the glucose carryover per surface area ( ) hxk-null , we grew the hxk -null strain in a minimal medium supplemented with galactose until the mid-exponential phase.…”

Section: Resultsmentioning

confidence: 99%

“…The hexokinase-null ( hxk -null) mutant was constructed on the S288C-derived prototrophic YSBN6 strain ( MATa ho::HphMX4 ) 23 via CRISPR-Cas9 using a MoClo Yeast Toolkit 19 , 20 . Target sequences and repair fragments used for CRISPR-Cas9 deletions can be found in Table S2 .…”

Section: Methodsmentioning

confidence: 99%

“…Assuming equilibrium, the amount of intracellular glucose can then be estimated using the total cell volume (V tot ) and the extracellular glucose concentration (c glc, ex ). By subtracting the calculated intracellular amount of glucose from the total glucose determined in the pellet (n glc, pellet ), one can then estimate the amount of glucose carryover (n glc, co ) and normalize it to the total cell surface area ( S tot,hxk−null ), with the following equation: Following this reasoning, we constructed a hxk-null strain, in which the three isozymes of hexokinases (Hxk1, Hxk2, and Glk1) were deleted using CRISPR-Cas9 19,20 . To estimate the glucose carryover per surface area (…”

Section: Evaluation Of the Contribution Of The Sticking Effect To The...mentioning

confidence: 99%

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Quantifying intracellular glucose levels when yeast is grown in glucose media

Li,

Heinemann

2023

Sci Rep

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In Saccharomyces cerevisiae, intracellular glucose levels impact glucose transport and regulate carbon metabolism via various glucose sensors. To investigate mechanisms of glucose sensing, it is essential to know the intracellular glucose concentrations. Measuring intracellular glucose concentrations, however, is challenging when cells are grown on glucose, as glucose in the water phase around cells or stuck to the cell surface can be carried over during cell sampling and in the following attributed to intracellular glucose, resulting in an overestimation of intracellular glucose concentrations. Using lactose as a carryover marker in the growth medium, we found that glucose carryover originates from both the water phase and from sticking to the cell surface. Using a hexokinase null strain to estimate the glucose carryover from the cell surface, we found that glucose stuck on the cell surface only contributes a minor fraction of the carryover. To correct the glucose carryover, we revisited l-glucose as a carryover marker. Here, we found that l-glucose slowly enters cells. Thus, we added l-glucose to yeast cultures growing on uniformly 13C-labeled d-glucose only shortly before sampling. Using GC–MS to distinguish between the two differently labeled sugars and subtracting the carryover effect, we determined the intracellular glucose concentrations among two yeast strains with distinct kinetics of glucose transport to be at 0.89 mM in the wild-type strain and around 0.24 mM in a mutant with compromised glucose uptake. Together, our study provides insight into the origin of the glucose carryover effect and suggests that l-glucose added to the culture shortly before sampling is a possible method that yet has limitations with regard to measurement accuracy.

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Section: Resultsmentioning

confidence: 99%