fingerprinting of yeast with interdelta primers

References:

Legras and Karst, Optimisation of interdelta analysis for Saccharomyces cerevisiae strain characterisation, FEMS Microbiology Letters, Volume 221, Issue 2, April 2003, Pages 249–255

https://academic.oup.com/femsle/article/221/2/249/630262

Ness

 et al.,

1993. 

Identification of yeast strains using the polymerase chain reaction. 

J. Sci. Food Agric.

62,89-94.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/jsfa.2740620113

Primer ordered:

delta2 5'-GTGGATTTTTATTCCAACA-3'

delta12 5′-TCAACAATGGAATCCCAAC-3′ 

delta21 5′-CATCTTAACACCGTATATGA-3′

Fingerprints 20231221

 

Fingerprints 20231221

The results are quite good. However, we will need to improve the quality of the DNA extracted from the yeast extract for nanopore sequencing. What are the sizes of the markers?

We should try whole genome sequencing first and if not satisfactory, we may try AP-PCR-nanopore sequencing

Workflows for 2023 HT Yeast Extract Project Phase 1

 Phase 1--December 2023 to May 2024

1A. December 2023 to 12 January 2024

• Isolate and identify the yeast strain that is nearly identical to that of the Reference Yeast Extract
• Streak from source for single colonies
• YPD plates
• Pick single colony
• Observe under microscope
• Grow test-tube cultures in YPD medium aerobically
• Extract DNA from yeast cells
• Extract DNA from Reference Yeast Extract
• Interdelta Fingerprint comparison
• Observe the patterns of lower bands
• Nanopore sequencing for WGS
• DNA sequence comparison-- target >90% similarity

1B. 15 January 2024 to 26 January 2024 

• Lab-scale fermentor cultivation 
• Medium follows the previous optimized recipe 
• 4x500ml fermentors for as many times as needed for >20g of Yeast extract--Need about 10L of culture--5 times
• Collect yeast cells by centrifugation
• Pool enough yeast cells
• Autolysis of yeast cells to make yeast extracts, following the previous optimized treatment conditions
• Freeze-dry/oven-dry the yeast extract--check with Eliz

1C. 29  Janurary 2024 to 16 February 2024

• Large lab-scale fermenter cultivation
• HKPolyU fermenter familiarization
• HKPolyU fermenter test-runs
• >50g Yeast extracts production