10. Complementation I procedure and worksheet - Tatum Kroon

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University of Louisville *

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331

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Biology

Date

Apr 3, 2024

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1

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Lab 10 Complementation Mapping of Mutations in the Lactose ( lac ) Operon Materials 1) Petri plates: 1 X-gal and 1 X-gal + IPTG plate per group 2) Disposable sterile loops – 2 per group 3) DH5α and Lu53 plates (Ask TA) Protocol Day 1 1) Clean your working area by spraying the disinfect solution and wipe with paper towel. 2) Divide the plates in half by drawing a straight line down the middle at the bottom of both plates with a sharpie. Label plates on the corner with group name, section number and date 3) Also label one side of the plate as Lu53 and the other side as DH5α. 4) Use a sterile loop to pick up a colony from LB agar plates containing DH5α and Lu53 (Ask TAs) (DO NOT TAKE TO MANY CELLS) and streak Lu53 colonies on to one side of each plate and DH5α colonies on to the second side of each plate. 5) keep the plates upside down in the 37°C incubator and let cultures grow overnight. 6) See the results on Day 2 complementation lab. Questions (NOTE: MUST BE COMPLETED INDIVIDUALLY and submit the worksheet before leaving the lab for participation points) 1. Name the different structural genes that are a part of the Lac operon. What roles in lactose metabolism do they play? The different structural genes part of the Lac operon are lac z (beta-galactosidase), lac y (permease), and lac a (transacetylase). Beta-galactosidase degrades lactose, permease transports lactose into the cell, and the function of transacetylase is not that well known. 2. What would be the state of the lac operon and the phenotype of DH5α and Lu53 strains of E. coli would you expect in each type of plate (Xgal and Xgal + IPTG), assuming there were no mutations? Xga: DH5α lac repressor binds to the operator; genes would not be transcribed. White color. Lu53 lac repressor binds to the operator; genes would not be transcribed. White color. Xgal + IPTG DH5α transcription of genes. Blue color. Lu53 transcription of genes. Blue color.
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