Lecture 7 MICRO 329

Transformation Overview

• Transformation is when bacteria take up DNA from their environment, acquiring new characteristics like bioluminescence.
• Just because bacteria are able to take up DNA, does not guarantee they will, which shows the efficiency of the transformation.
• To get a bioluminescent colony, E. coli must take up the plasmid.
• The plasmid must have gDNA inserted, which should contain the lux operon.

Selection & Blue-White Screening

• The bacterial selection process involves growing transformed bacteria on ampicillin media.
• E. coli is not naturally resistant to ampicillin.
• Only E. coli cells that have taken up the plasmid will be resistant to ampicillin.
• The multiple cloning site (MCS) within the lacZ gene to determine if the plasmid contains an insertion.
• A successful insertion disrupts the lacZ gene, allowing us to screen for colonies with the disrupted gene.
• Screening involves using a blue/white screen where lacZ produces β-galactosidase enzyme, which is induced by IPTG.
• When lacZ is present, colonies appear blue, but when absent or interrupted, colonies appear white.

Blue / White Screening Details

• Blue/white screening is also known as α-complementation.
• Agar plates are prepared with X-gal (substrate) and IPTG (inducer).
• White colonies indicate a non-functional lacZ gene with a gDNA insert.
• Blue colonies indicate a functional lacZ gene without any insert.

Bioluminescence Screening & Next Steps

• The next step is to screen for bioluminescence.
• This involves selection by growing on Amp + media
• The blue/white screening uses IPTG + Xgal on media to find potential bioluminescent colonies.
• Next week will involve transformation procedures, moving plates from the incubator, analyzing transformation results, plating for bioluminescent clone screening.

Tuesday: Transformation Protocol & Controls

• Technical controls are necessary to confirm the protocol was executed correctly.
• A negative control uses buffer (TE) to check for contamination
• A positive control uses unmodified plasmid to ensure blue colonies appear
• Plasmid quality control uses digested plasmid to check for complete digestion.
• Blue colonies in this control indicate an incomplete digest.
• Tuesday's procedure includes reagent prep, cell preparation, performing the transformation, and plating the transformed cells.

Thursday: Analysis & Mutant Library

• The lab will calculate transformation efficiency and use those results to determine which transformants to use.
• Selected transformants will be plated to create a mutant library.
• The goal is to screen about 4,600 white colonies to identify a bioluminescent one, with about 200 colonies per plate.

General Advice for Transformations

• Keep items on ice to maintain cold temperatures for competent E. coli
• Handle competent E. coli gently, avoiding vortexing or centrifuging.
• Recovery media should not contain ampicillin.
• Let the cells stabilize by heat shocking them
• Allow some time for them to start producing ampicillin resistance protein.
• After overnight incubation, transfer plates for further incubation.
• A transformation efficiency calculation will happen next lab

Transformation Background & Competence

• Transformation was first demonstrated in 1928 by Frederick Griffiths woth Streptococcus pneumoniae strains, and finding virulence genes are transferrable.
• Transformation is dependent on bacterial competence, which is the ability to take up extracellular DNA
• Some bacteria are naturally competent and use DNA, and others aren't
• Examples that are naturally competent include; Bacillus subtilis, Pseudomonas aeruginosa, Streptococcus pneumoniae.
• E. coli is not naturally competent and therefore must be artificially made competent.

Making E. coli Competent & Selecting Transformed Cells

• Two methods to make E. coli artificially competent include chemical and electroporation.
• Chemical competence uses ice-cold CaCl2, heat-shock, and is less efficient.
• Electroporation uses an electric current to create pores in the cell membrane, and causes membrane damage
• E. coli is not naturally resistant to ampicillin.
• Use the antibiotic ampicillin to select the cells.
• Ampicillin is part of a class of antibiotics called penicillins, all based on a β-lactam ring.
• Lactam is a cyclic amide group useful against G+ cocci and some G- cocci & spirochetes.

α-Complementation Details in E. coli

• Wild-type E. coli contains lacZ
• E. coli JM109 is an E. coli missing an a-peptide region of lacZ
• Without a-peptide, LacZ always stays as a monomer and can't be a tetramer
• The a-peptide of lacZ can be found on the vector like pGEM-3ZF(+).
• E. coli can't produce functional lacZ unless the plasmid contains the a-peptide of lacZ
• Adding the a-peptide to the E. coli JM109 is called alpha-complementation.

Blue / White Theory

• If a plasmid does not have an insert has an intact alpha domain of lacZ.
• If a plasmid has an insert has an alpha domain of lacZ that is interrupted
• If the lux operon is successfully ligated into the LacZ locus, colonies will appear white.
• If a vector recircularized, then colonies will appear blue