Difference between revisions of "Part:BBa K4879010"

Latest revision as of 16:09, 11 October 2023

Yarrowia lipolytica guaB construct

The transcriptional unit for guaB, designed for expression in Y. lipolytica in order to help select for ScRAD52 transformants.

Characterization

Cloning: The construct, along with homologous flanking sequences corresponding to the A08 gene locus, were synthesized by iGEM’s DNA synthesis partner IDT. The resulting gene fragment was then PCR amplified with the respective primers to attain a higher copy number of the fragment. The amplification of the fragment was verified by running a 0.8% agarose gel, with the fragments expected to show at around the 3000 bp region.

Fig 1: Lane 1 is the DNA ladder, and lanes 7-10 have the successful amplicons.

The amplified fragment was then gel-extracted and assembled together with the ScRAD52 expression construct (BBa_K4879011) with the help of the NEBuilder HiFi DNA Assembly kit, and subsequently transformed into our chassis.

Validation: The transformed yeast culture was then plated onto the selection plates containing about 2000 µg/ml of mycophenolic acid and was incubated at 30°C. The growth of the yeast on the selection plates confirms the functional validity of the expression construct.

Fig 2: The growth of transformed Y. lipolytica on the MPA selection plate, confirming part functionality.

Sequence and Features