Bee gut bacterial isolation and genome sequencing (Orbaceae)

Knowledge graph centered on Bee gut bacterial isolation and genome sequencing (Orbaceae) with 28 nodes and 103 connections. Top connected: Bombus, Bombus terrestris, Bombus appositus, Bombus impatiens, B. appositus.

Method synopsis

Isolation of pure bacterial cultures from bee gut homogenates followed by whole genome sequencing using Illumina MiSeq and PacBio platforms with subsequent assembly and annotation.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Metabolism of toxic sugars by the bee gut symbiont <i>Gilliamella apicola</i> (2017), mBio. Implementations in other papers (listed below) may differ.

1. Bee gut sample collection
   Collected honey bees from 4 species (Apis mellifera, A. cerana, A. dorsata, Meliponula andreniformis) and bumble bees from 10 species. Bees were identified based on morphology. Dissected guts were either homogenized in 10 mM MgSO4 for immediate bacterial isolation or crushed in 19% glycerol and frozen directly after sampling.
   Quantities: 15 strains from honey bees, 27 strains from bumble bees, total 42 G. apicola strains isolatedDuration: Not specifiedConditions: Field collection from various locations
   Equipment: 10 mM MgSO4, 19% glycerol
2. Bacterial isolation and cultivation
   Pure cultures of G. apicola strains were isolated from bee guts using previously described methods. Fresh gut homogenates or glycerol stocks were plated on heart infusion agar (HIA) supplemented with defibrinated sheep's blood (5% vol/vol) at final concentration using Hardy Diagnostics materials. After 2 days incubation at 37°C under CO2-enriched atmosphere (5%), visible colonies were identified by sequencing of 16S rRNA gene.
   Quantities: 42 pure culture strains establishedDuration: 2 days incubationConditions: 37°C, 5% CO2 atmosphere
   Equipment: heart infusion agar, defibrinated sheep's blood, Hardy Diagnostics materials
3. Genome sequencing and assembly
   Genomic DNA was extracted using phenol-chloroform method and purified on DNAeasy spin columns (Qiagen). Paired-end libraries were constructed and sequenced on Illumina MiSeq platform. Strain wkB7 received complete genome sequencing on PacBio RS II platform and assembly with HGAP method. Other genomes were assembled with MaSuRCA version 3.1.0 and closed/verified by mapping Illumina MiSeq reads onto HGAP-generated contigs.

Typical Equipment

• 10 mM MgSO4
• heart infusion agar
• Illumina MiSeq
• PacBio RS II

Output Measurements

• bacterial isolate cultures
• genome sequences
• phylogenetic relationships

Papers Using This Protocol (1)

Species Studied With This Protocol