Project name: Lachancea kluyveri strain:CBS 3082

Description: A single colony from L. kluyveri (CBS 3082) was evolved by a long-term modified serial dilution transfer method (batch cultures) method in which bacteria were sequentially introduced to compete with yeasts under aerobic conditions. Both evolved and the ancestral strains were grown in duplicates in batch cultivation with synthetic minimal media supplied with 20 g/L of glucose. Batch cultures of evolved and ancestor were sampled for RNA extraction between 10 – 11 g/L of residual glucose under both aerobic and anaerobic conditions in fermeters. In addition, we also extracted samples after the diauxic shift (approximately 0.018 g/L of residual glucose and 1.37 ± 0.01 – 1.55 ± 0.03 g/L of residual ethanol) to determine changes in expression of genes responsible for carbon utilization in the evolved strain. RNA was extracted using an RNAeasy kit (Qiagen) according to the manufacturer´s instructions. DNase treatment was performed using a Qiagen on-column kit. RNA quantity and quality was determined using a Nanodrop ND 1000 spectrophotometer (Thermo Scientific) and a Bioanalyzer 2100 (Agilent Technologies) respectively. The cDNA libraries were constructed using the TruSeq™ RNA Sample Preparation Kit (Illumina) according to the manufacturer's instructions. Poly-A containing mRNA was purified from 2 μg of total RNA using oligo(dT) magnetic beads and fragmented into 200 – 500 bp pieces using divalent cations at 94°C for 5 min. The cleaved RNA fragments were copied into first strand cDNA using SuperScript II reverse transcriptase (Life Technologies) and random primers. After second strand cDNA synthesis, fragments were end repaired, A-tailed and indexed adapters were ligated. The products were purified and enriched with PCR to create the final cDNA library. The 6 tagged cDNA libraries were pooled in equal ratios and used for 2 × 100bp paired-end sequencing on a single lane of the Illumina HiSeq2000 II.

Data type: transcriptome

Sample scope: Monoisolate

Relevance: Evolution

Last updated: 2015-01-20